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 rab4 and rab5, two small GTPases known to regulate distinct steps of membrane traffic in 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 rab4. Subsequently, membrane fission consumes this larger fusion compartment. This fission process is stimulated by rab4 and by the GTP hydrolysis-defective mutant rab4Q67L.
Chavrier
P
P
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Marseille, France.
van der Sluijs
P
P
Mishal
Z
Z
Nagelkerken
B
B
Gorvel
J P
JP
D000818:Animals; D002459:Cell Fusion / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D004705:Endocytosis; D011992:Endosomes / Q000648:ultrastructure*; D005434:Flow Cytometry / Q000379:methods; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry*; D007668:Kidney / Q000166:cytology; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins
D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins