[Seminar] "Direct measurement of coupling distance by Flash & Freeze-fracture replica labeling" by Prof. Ryuichi Shigemoto, ISTA

Prof. Ryuichi Shigemoto, Institute of Science and Technology Austria (ISTA)

Title: Direct measurement of coupling distance by Flash & Freeze-fracture replica labeling

Abstract:
The coupling distance between voltage-gated calcium channels and calcium sensors on synaptic vesicles (SVs) is critical for regulating probability and kinetics of SV release at the presynaptic active zone (AZ). The mean coupling distances in different types of synapses have been estimated by electrophysiological recording using fast and slow calcium buffers. Here, we tried to estimate the coupling distance of individual vesicles directly by Flash & Freeze-fracture replica labeling. Our method successfully visualizes fusion profiles of SVs within the AZ together with high sensitivity detection of Cav2.1 subunit of P/Q-type channels. Nearest neighbor distances (NNDs) between fusion profiles and Cav2.1 were significantly shorter than those between munc13-1/syntaxin1B cluster centers and Cav2.1. The former may be useful to estimate probability of fusion of individual release ready vesicles, whereas the munc13-1/Syntaxin1B clusters may include fusion sites without release ready vesicles. We compared the NNDs between fusion profiles and Cav2.1 in the AZs of cerebellar parallel fiber (PF)-Purkinje cells (PC) and basket cell (BC)-PC synapses. Frequencies of fusion profiles and multiple fusion profiles in single AZs were higher in the BC-PC than PF-PC synapses, and the NNDs were significantly shorter in BC-PC than PF-PC AZs, consistent with the higher release probability in the BC-PC synapses. These results indicate that Flash & Freeze-fracture replica labeling is useful to analyze directly the coupling distance of individual release ready vesicles.