EARLY ENDOSOME MEMBRANE DYNAMICS CHARACTERIZED BY FLOW-CYTOMETRY

Early endosomes are very dynamic intracellular membrane organelles tha t undergo multiple fusion and fission events. In this study, we develo ped a novel assay based on multiparametric flow cytometric analyses an d early endosome sorting to characterize better the mechanisms of earl y endosome membrane dynamics in vitro. In particular, we have investig ated the role of rabi and rab5, two small GTPases known to regulate di stinct steps of membrane traffic ire the endocytic pathway. We show th at early endosomes undergo homotypic fusion reactions, which lead to t he formation of fusion intermediates with increased size, Fusion is ef ficiently 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.