Biology subjects

Wallis, T. P.

Publications and source records attributed to Wallis, T. P..

3 recordsLinked to original sources

bioRxiv2023

VAMP2/Munc18-1 domain 3a interaction controls the nanoscale reorganization underpinning vesicular priming

SNARE-mediated secretory vesicle (SV) exocytosis underpins neuronal communication. Munc18-1 orchestrates SNARE complex formation by controlling the opening of syntaxin-1A. How the SV-plasma membrane interface becomes fusion-competent at the nanoscale level is poorly understood. Here, we propose that the interaction of Munc18-1 with VAMP2 during vesicular docking triggers nanoscale re-organization which renders the SV-plasma membrane interface fusion-competent. We identified and mutated key residues in Munc18-1 domain 3a (A297 and T304) hypothesised to impair its interaction with VAMP2. Munc18-1A297H, and to a lesser extent Munc18-1T304H, constrained SVs on the plasma membrane and reduced stimulated secretion, under re-expression conditions in Munc18-1/2 double knockout neurosecretory cells. Moreover, the de-clustering of Munc18-1 in response to activity was lost for both mutants. The interaction of VAMP2 with the Munc18-1 domain 3a therefore controls the re-organization of the nanoscale environment of the docked SV-plasma membrane interface, fostering syntaxin-1A opening and Munc18-1 release to ensure that SNARE assembly only occurs within the confinement of docked vesicles.

neuroscience↗

bioRxiv2022

Synaptotagmin 1 mediates toxicity of botulinum neurotoxin type A

The extreme neurotoxicity of botulinum neurotoxins (BoNTs), broadly used as a therapeutics, is mediated by their direct binding to two plasma membrane receptors: polysialogangliosides (PSGs) and serotype-dependently either synaptotagmin 1/2 (Syt1/2) or synaptic vesicle glycoprotein 2 (SV2). Here, we demonstrate that although BoNT/A serotype binds directly to PSG and SV2, its neurotoxicity depends on Syt1. Using single-molecule superresolution microscopy of pathological concentrations of BoNT/A holotoxins, we demonstrate that the toxin binds to a preassembled PSG-Syt1 complex that forms nanoclusters with SV2 on the plasma membrane of hippocampal neurons. This coincidental interaction controls the selective endocytic targeting of BoNT/A into synaptic vesicles, leading to incapacitation of neuronal communication. Our results suggest that Syt1-SV2 plasma membrane nanoclusters may acts as a common target for various BoNT serotypes. One-Sentence SummaryBoNT/A hijacks an intrinsic tripartite PSG-Syt1-SV2 endocytic sorting mechanism to incapacitate neuronal communication

neuroscience↗

bioRxiv2021

MOLECULAR VIDEOGAMING: SUPER-RESOLVED TRAJECTORY-BASED NANOCLUSTERING ANALYSIS USING SPATIO-TEMPORAL INDEXING

Single-molecule localization microscopy (SMLM) techniques are emerging as vital tools to unravel the nanoscale world of living cells. However, current analysis methods primarily focus on defining spatial nanoclusters based on detection density, but neglect important temporal information such as cluster lifetime and recurrence in "hotspots" on the plasma membrane. Spatial indexing is widely used in videogames to effectively detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to perform SMLM data analysis and determine whether the bounding boxes of individual molecular trajectories overlap, as a measure of their potential membership in nanoclusters. Extending the spatial indexing into the time dimension allows unique resolution of spatial nanoclusters into multiple spatiotemporal clusters. We have validated this approach using synthetic and SMLM-derived data. Quantitative characterization of recurring nanoclusters allowed us to demonstrate that both syntaxin1a and Munc18-1 molecules transiently cluster in hotspots on the neurosecretory plasma membrane, offering unprecedented insights into the dynamics of these protein which are essential to neuronal communication. This new analytical tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), has been implemented as a free and open-source Python graphic user interface.

biophysics↗