The Discovery and Regulation of Modes of Exocytosis Through the Lens of Computer Vision

The formation of the nervous system involves establishing complex networks of synaptic connections between proper partners, which requires the rapid expansion of the plasma membrane surface area as neurons grow. Critical to the expansion of the plasma membrane is exocytic vesicle fusion, a regulated mechanism driven by soluble N-ethylmaleimide-sensitive factor attachment proteins receptors (SNAREs). Multiple modes of exocytosis have been proposed, with full-vesicle fusion (FVF) and kiss-and-run (KNR) being the best described. The basis of SNARE-mediated fusion, the opening of a fusion pore, and its contribution to plasma membrane expansion remains enigmatic, as vesicle fusion is spatially small and temporally fast. We exploited TIRF microscopy to image VAMP-pHluorin mediated exocytosis in murine embryonic cortical neurons and developed computer-vision software and statistical tools to perform unbiased, efficient identification of exocytic events and uncover spatiotemporal aspects of exocytosis during neuron development. We further developed novel classification algorithms to describe. and classify individual exocytic events. Vesicle fusion behavior differed between vesicle types, cell types, developmental stage, and extracellular environment. Spatial statistics uncovered distinct spatiotemporal regulation of exocytosis in the soma and neurites of neurons that was modulated by developmental stage, the guidance cue netrin-1, and the E3 ubiquitin ligase TRIM9. Four distinguishable fusion modes were uncovered in developing neurons: two FVF-like modes that insert membrane material (FVFi and FVFd) and two KNR-like modes that do not (KNRi and KNRd). Experiment-based mathematical calculations and experiments indicated that FVFi and FVFd VAMP2-mediated vesicle fusion supplied sufficient material for the plasma membrane expansion that occurred early in neuronal morphogenesis. The mode of exocytosis is dependent on the E3-ubiquitin ligase TRIM67. Neurons lacking Trim67 have increased KNRi and KNRd as well as smaller surface area, presumably due to lowered rates of FVFi and FVFd. Our data suggest this is accomplished in part by limiting incorporation of the Qb/Qc SNARE SNAP47 into SNARE complexes, and thus, SNAP47 involvement in exocytosis. SNAP47 levels are elevated in neurons lacking TRIM67, and overexpression of SNAP47 in neurons expressing wildtype levels of TRIM67 have increased KNRi and KNRd and decreased FVFi. Thus, we establish a regulatory mechanism of TRIM67, distinct from TRIM9.Doctor of Philosoph