Optical analysis of synaptic vesicle protein molecules during exo- and endocytosis using pH-switchable fluorescent probes

Abstract

At conventional synapses of the central nervous system (CNS), fast synaptic transmission is mediated by the release of neurotransmitters (NTs) upon Ca2+-triggered synaptic vesicle (SV) exocytosis. Upon exocytosis SV proteins have to be resorted and retrieved from the surface by compensatory endocytosis in order to replenish the pool of NT-filled SVs. For my thesis I used pH-switchable dyes, both genetically encoded as well as new exogenous ones, for optically analysing SV protein molecules necessary for fusion as well as their retrieval. Exocytosis is mediated by the assembly of low-energy SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes formed by the coil-coiling of three SNARE proteins: Synaptosomal associated protein - 25 (SNAP-25), Syntaxin 1A (Syx1A), and Synaptobrevin 2 (Syb2). However, it is unknown how many SNARE complexes are minimally needed for SV priming and fusion at CNS synapses. To resolve this issue, single vesicle fusion events were optically measured in real time using the genetically encoded probe SynaptopHluorin (SpH), a pH-sensitive green fluorescent protein (GFP), pHluorin (pHl) fused to the luminal domain of the SV SNARE Syb2. Fluorescence responses upon fusion displayed a quantal distribution of SpH molecules into SVs. Quantitative single molecule experiments revealed that the quantal size corresponds to single SpH molecule fluorescence. Surprisingly, when overexpressed on a genetic null background, SpH could fully rescue evoked SV fusion. However, SVs expressing only one copy of SpH were unable to rapidly fuse upon stimulation. Taken together, the first part of the study demonstrates that two copies of SpH and hence two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.In order to maintain a steady-state rate of synaptic transmission the fused SV constituents are retrieved for further rounds of use by a compensatory process of endocytosis. Although clathrin-mediated endocytosis (CME) is thought to be the predominant mechanism of SV recycling, it seems too slow to account for fast recycling. Therefore, it has been suggested that a pre-sorted and pre-assembled pool of SV proteins on the presynaptic membrane might support the first phase of CME. In the second part of this study the spatial and temporal dynamics of such a readily retrievable pool (RRetP) of SV proteins at the presynaptic membrane of hippocampal neurons was monitored using a novel probe. By applying CypHer 5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies against luminal domains of SV proteins, the preferential retrieval of native SV constituents from the RRetP upon exocytosis was demonstrated. The functional size and capacity of this pool was found to closely resemble that of the readily releasable pool (RRP) of docked and primed SVs, suggesting that the RRetP can sustain SV recycling during moderate synaptic activity. Thus, the second part of the thesis demonstrates that small central synapses can avoid SV depletion in response to mild stimulation by having a preassembled pool of ready-to-go SV constituents (RRetP), which efficiently supports compensatory endocytosis to a significant degree without relying on freshly exocytosed SV constituents.