Effects of phorbol ester on vesicle dynamics as revealed by total internal reflection fluorescence microscopy

Exocytosis of neurotransmitter or hormone-filled vesicles is a highly dynamic process regulated by various proteins and lipids. As mainly revealed indirectly by the electrophysiological methods, exocytosis is believed to involve multiple kinetic steps in which vesicles transit from one state to another. Using total internal reflection fluorescence microscopy which enables direct visualization of individual vesicles, we developed an analytical framework to track and analyze vesicle dynamics. We demonstrated that all subplasmalemmal vesicles generally undergo constant and caged Brownian motion. And they can be classified into three populations that differ in their motion characteristics and fusion competence. Furthermore, we showed that these vesicle pools are differentially modulated by phorbol-12-myristate-13-acetate, a phorbol ester analog to endogenous diacylglycerol, through both protein-kinase-C-dependent and -independent pathways.