Abstract
Morphological elongation of developing neurons requires plasmalemma expansion, hypothesized to occur primarily via exocytosis. We posited that exocytosis in developing neurons and non-neuronal cells exhibit distinct spatiotemporal organization. We exploited TIRF microscopy to image VAMP-pHluorin mediated exocytosis in murine embryonic cortical neurons and interphase melanoma cells, and developed computer-vision software and statistical tools to uncover spatiotemporal aspects of exocytosis. Vesicle fusion behavior differed between vesicle types, cell types, developmental stage, and extracellular environment. Experiment-based mathematical calculations indicated that VAMP2-mediated vesicle fusion supplied excess material for the plasma membrane expansion that occurred early in neuronal morphogenesis, which was balanced in part by clathrin-mediated endocytosis. Spatial statistics uncovered distinct spatiotemporal regulation of exocytosis in the soma and neurites of developing neurons that was modulated by developmental stage, exposure to the guidance cue netrin-1, and the brain-enriched ubiquitin ligase TRIM9. In melanoma cells, exocytosis occurred less frequently with distinct spatial clustering patterns.
