Nanoscale Dynamics of Voltage-Gated Calcium Channels at Presynaptic Active Zones in Live C. Elegans

Abstract

At presynaptic active zones (AZ), voltage-gated calcium channels (VGCCs) and synaptic vesicles (SVs) are tightly coupled by evolutionarily conserved active zone cytomatrix (CAZ) proteins for fast neurotransmission. The number of activated VGCCs and their coupling distance to SVs are key determinants of the SV release probability. Because VGCCs diffuse laterally along the presynaptic membrane, their nanoscale diffusion could also tune the SV release probability by setting local channel densities and Ca2+ concentrations. How the lateral dynamics of VGCCs are modulated by CAZ proteins for precise, yet plastic synaptic transmissions remain undefined. To probe this question, we use tissue-specific single molecule imaging by Complementation Activated Light Microscopy (CALM) and CRISPR genetics to track endogenous VGCCs with nanometer precision on the presynaptic membrane in live C. elegans nematodes. We generated transgenic animals expressing split-fluorescent proteins (sfCherry2 or sGFP) fused to VGCCs, and characterized the heterogeneous diffusive behaviors and the nanoscale organizations of these neuronal calcium channels in vivo. We identified three sub-diffusive behaviors of VGCCs at the presynaptic membrane, including a dominant behavior with a diffusion coefficient similar to that observed for synaptic SVs, as previously determined by FRAP. By introducing mutants of the key CAZ protein RIM/UNC-10 to disrupt coupling between SV and VGCCs, this dominant diffusive behavior of VGCCs is significantly altered, both in terms of diffusion coefficient and membrane nanodomain confinement. Our results suggest that VGCCs coupling to SVs via CAZ proteins determine their nanoscale diffusion at the presynaptic membrane in vivo. Using single molecule CALM, co-immunoprecipation assays and CRISPR genetics, we are now attempting to further dissect the molecular mechanisms by which other CAZ proteins regulate the presynaptic membrane dynamics of VGCCs in order to guaranty precise neurotransmission in intact animals.