Early endosome membrane dynamics characterized by flow cytometry.

Early endosomes are very dynamic intracellular membrane organelles that undergo multiple fusion and fission events. In this study, we developed a novel assay based on multiparametric flow cytometric analyses and early endosome sorting to characterize better the mechanisms of early endosome membrane dynamics in vitro. In particular, we have investigated the role of rabi and rab5, two small GTPases known to regulate distinct steps of membrane traffic ire the endocytic pathway. We show that early endosomes undergo homotypic fusion reactions, which lead to the formation of fusion intermediates with increased size, Fusion is efficiently stimulated by recombinant rab5 but not by recombinant rabi. Subsequently, membrane fission consumes this larger fusion compartment. This fission process is stimulated by rabi and by the GTP hydrolysis-defective mutant rab4Q67L. (C) 1997 Wiley-Liss, Inc.