Biology subjects

Gormal, R. S.

Publications and source records attributed to Gormal, R. S..

4 recordsLinked to original sources

bioRxiv2025

Transmitted alpha-synuclein extracellular vesicles downregulate axonal flux of retrograde carriers in recipient neurons

-Synuclein (-syn) is a cytosolic protein located in nerve terminals and is involved in several neurodegenerative diseases such as Parkinsons disease. Recent studies have demonstrated that -syn can be transmitted from neuron to neuron via exosomal release, thereby contributing to the propagation of -syn pathology. However, the mechanism by which -syn-containing exosomes perturb the function of recipient neurons is currently unknown. Retrograde axonal transport of carriers emanating from the presynapse is essential for neuronal survival. To determine the effect of transmitted -syn on neuronal retrograde trafficking, we used conditioned medium from -syn transfected "donor" hippocampal neurons, applied to naive (non-transfected) "recipient" neurons cultured in microfluidic chambers. Time-lapse imaging of retrograde carriers containing cholera toxin-B subunit (CTB) was then performed in these recipient neurons. Here, we show that conditioned medium from -synWT-GFP transfected donor neurons significantly downregulated the frequency of retrograde CTB carriers when applied to recipient neurons. This effect was abolished by (i) inhibiting endocytosis in recipient neurons, (ii) by blocking exosomal release from donor cells via an Hsp90-dependent mechanism, or (iii) by using conditioned medium from neurons transfected with a Parkinsons mutant (-synA30P-GFP). Whilst transmitted -syn-mEos2, -synA30P-mEos2 and mEos2 alone were detected in recipient neurons using single molecule imaging, interestingly, -synWT-mEos2 exhibited lower mobility, and periodic (190 nm) immobilisations along the axon. Our data suggest that the downregulation of vesicular trafficking by transmitted -syn does not rely on the specific release nor uptake of exosomal -syn but probably depends on its interaction with endogenous -syn following endocytosis in recipient neurons. The transmitted -syn containing extracellular vesicles therefore controls essential axonal trafficking in recipient neurons, an effect lost with the Parkinsons disease mutant -synA30P.

neuroscience↗

bioRxiv2023

Location, Location, Location: Protein kinase nanoclustering for optimized signalling output

Protein kinases (PKs) are proteins at the core of cellular signalling and are thereby responsible for most of the cellular physiological processes and their regulations. As for all cellular proteins, they are subjected to Brownian thermal energy that tends to homogenise their distribution throughout the volume of the cell. To access their substrates and perform their critical functions, PKs localisation is therefore tightly regulated in space and time, relying upon a range of clustering mechanisms. These include post-translational modifications, protein-protein and protein-lipid interactions as well as liquid-liquid phase separation, allowing spatial restriction and ultimately regulating access to their substrates. In this review, we will mainly focus on key mechanisms mediating PK nanoclustering in physiological and pathophysiological processes. We propose that PK nanoclusters act as a cellular unit of signalling output capable of integration and regulation in space and time. We will specifically outline the various super-resolution microscopy approaches currently used to elucidate the mechanisms driving PK nanoscale clustering and explore the pathological consequences of altered kinase clustering in the context of neurodegenerative disorders, inflammation, and cancer.

neuroscience↗

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↗

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↗