Endosome fusion in living cells overexpressing GFP-rab5

Abstract

CHO and BHK cells which overexpress either wild-type rab5 or rab5:Q79L, a constitutively active rab5 mutant, develop enlarged cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab5 and transferrin receptor. Time-lapse video microscopy shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These fusion events occur mostly by a ‘bridge’ fusion mechanism in which the initial opening between vesicles does not expand; instead, membrane flows slowly and continuously from the smaller to the larger endosome in the fusing pair, through a narrow, barely perceptible membranous ‘bridge’ between them. The unique aspect of rab5 mediated ‘bridge’ fusion is the persistence of a tight constriction at the site where vesicles merge and we hypothesize that this constriction results from the relatively slow disassembly of a putative docking/fusion complex. To determine the relation of rab5 to the fusion ‘bridge’, we used confocal fluorescence microscopy to monitor endosome fusion in cells overexpressing GFP-rab5 fusion proteins. Vesicle docking in these cells is accompanied by recruitment of the GFP-rab5 into a brightly fluorescent spot in the ‘bridge’ region between fusing vesicles that persists throughout the entire length of the fusion event and which often persist for minutes following endosome fusion. Other endosomal membrane markers, including FM4-64, are not concentrated in fusion ‘bridges’. These results support the idea that the GFP-rab5 spots represent the localized accumulation of GFP-rab5 between fusing endosomes and not simply overlap of adjacent membranes. The idea that the GFP-rab5 spots do not represent membrane overlap is further supported by experiments using photobleaching techniques and confocal imaging which show that GFP-rab5 localized in spots between fusion couplets is resistant to diffusion while GFP-rab5 on endosomal membranes away from these spots rapidly diffuses with a rate constant of about 1.0 (+/-0.3) x10(-)(9)cm(2)/second.