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

Abstract

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.