Vesicle Clustering in a Living Synapse Depends on a Synapsin Region that Mediates Phase Separation

Abstract

Summary: Liquid-liquid phase separation is an increasingly recognized mechanism for compartmentalization in cells. Recent in vitro studies suggest that this organizational principle may apply to synaptic vesicle clusters. Here we test this possibility by performing microinjections at the living lamprey giant reticulospinal synapse. Axons are maintained at rest to examine whether reagents introduced into the cytosol enter a putative liquid phase to disrupt critical protein-protein interactions. Compounds that perturb the intrinsically disordered region of synapsin, which is critical for liquid phase organization in vitro, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions with synapsin are ineffective at rest. Our results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin. : Pechstein et al. report that presynaptic microinjection of reagents that bind to the intrinsically disordered region (IDR) of synapsin cause dispersal of synaptic vesicle clusters. Inhibition of SH3 domain interactions with synapsin do not cause vesicle dispersal. Synaptic vesicle clustering in vivo can be explained by liquid-liquid phase separation. Keywords: synapse, synaptic vesicle, synapsin, accessory proteins, phase separation, SH3 domain interaction