Examining the interplay of complexin and synaptotagmin at the synapse - Project Summary Research into the molecules underlying synaptic exocytosis has revolutionized our understanding of synaptic transmission and connections to a variety of neurological and psychiatric disorders, but several fundamental challenges remain. In particular, decades of research have yet to resolve the precise function of a critical piece of the synaptic vesicle fusion apparatus: Complexin (CPX). CPX is a small cytoplasmic protein containing a highly conserved SNARE-binding motif known as the central helix (CH), that is surrounded by a poorly conserved accessory helix (AH) and a C-terminal amphipathic domain (CTD). Past studies suggest that CPX works in tandem with the vesicle-associated protein Synaptotagmin-1 (Syt1) to prepare and maintain synaptic vesicles (SVs) in a fusion-ready state, with all domains all playing crucial roles in CPX function. However, the precise mechanism remains unclear. Notably, perturbations of CPX function appear to have strikingly different impact on mammalian vs invertebrate model synapses, although few studies have directly compared CPX biochemical and functional properties together across widely divergent species. In this proposal, we describe a systematic and comprehensive approach to compare the proposed functions of worm and mouse CPX and its relationship to Syt1 using a combination of complementary in vitro, ex vivo and in vivo experimental systems, and computational modeling. Specifically, we aim to resolve whether AH domain function together with the CH and CTD domains or acts independently to determine the specificity and efficacy of the CPX inhibitory/facilitatory function. We compare the CTD-membrane interactions and curvature sensitivity to test the hypothesis that the CTD properly localizes CPX on vesicle membranes where it can efficiently bind assembling SNARE proteins. Additionally, we assess how variations in Syt1 inhibitory function influences CPX action across species. For in vitro analysis, we will deploy single-vesicle docking/fusion assay with high time resolution and precise control over protein identity/density. We will test worm/mouse CPX and Syt1 isoforms as well as variants/chimeras, with the species-specific SNARE proteins. These experiments will be complemented with physiological assays in both C. elegans and in cultured mouse neurons. We will utilize CRISPR/Cas9 gene editing and single-copy transgenics to explore the impact of the CPX/Syt1 variants on synaptic function in C. elegans. We will conduct correlative analysis in cultured mouse neurons by utilizing the fast fluorescent glutamate sensor iGluSnFR to image quantal glutamate release at individual presynaptic boutons, followed by post-hoc immunocytochemistry to correlate these release events with the expression levels of release machinery proteins. To close the loop, we will develop mechanistic models of synaptic transmission based on experimental data, incorporating the synergistic actions of CPX and Syt1, and test their feasibility through computational modelling. We expect this project will offer crucial insights into the molecular mechanisms underlying the regulation of neurotransmitter release and contribute to the development of a detailed mechanistic model.
