Klingauf, J.; Glyvuk, N.; Duan, J.; Krishnan, S.; Tsytsyura, Y.; Keller, U.; Sunil, L. P.; Nosov, G.; Hueve, J.; Selenschik, P.; Reissner, C.; Missler, M.; Kahms, M.

The primary unit of exocytosis, the synaptic vesicle, is replete with proteins which enable vesicle fusion. For maintaining synaptic transmission the vesicle proteins have to be rapidly cleared from the active zone. The immediate fate of the cargo post-fusion and the precise mechanism of its resorting into a readily retrievable pool (RRetP) for endocytosis remains unclear. To address this, we developed a purely presynaptic preparation, the xenapse, presynaptic boutons formed en face directly onto a glass coverslip enabling optical single vesicle recording by total internal reflection microscopy (TIRFM). Electron microscopy showed xenapses contain a few hundred synaptic vesicles (SVs), of which ~40 SVs are primed (readily releasable pool, RRP), as revealed by single vesicle recordings using the pH-sensitive fluorescent protein pHluorin. Single fusion events synchronous with action potentials could be localized with ~20 nm precision and rapid post-fusion diffusional dispersion of vesicular proteins observed with diffusion constants in the order of 0.1 m2/s. Unroofing of xenapses revealed numerous Clathrin-coated structures, enriched for vesicle cargo and numerically equivalent to about twice the measured RRP size. In this way SV proteins are rapidly cleared from the release site into this RRetP of pre-assembled pits to allow for rapid re-docking and re-priming at vacated release sites in synapses tuned for high-fidelity fast signaling.