Dynamics of rab5 activation in endocytosis and phagocytosis

Fluid-phase endocytosis is stimulated by H-ras-linked growth factor recepto rs and this stimulation requires activation of rab5. We utilized a GFP-rab5 a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblast s and in J774 macrophages. Control CH cells that expressed GFP-rab5a:wt wer e cultured in serum-free conditions and showed GFP-rab5a:wt localized to en dosomal vesicles with a mean diameter of 0.3 +/- 0.1 mum. Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cel ls. Coexpression of H-ras:G12V, a constitutively active H-ras mutant that a ctivates rab5a, in cells resulted in marked enlargement of labeled endosome s (mean diameter 0.7 +/- 0.2 mum) and large numbers of giant GFP-rab5a:wt-p ositive endosomes were present. Time-lapse recordings showed abundant fusio n among giant labeled endosomes, and membrane ruffling and pinosome formati on were commonly observed. Alterations in GFP-rab5a:wt endosome structure a nd activity in cells expressing H-ras:G12V were linked to rab5a activation because these changes were identical to those found in cells expressing GFP -rab5a:Q79L, a constitutively activated rab5a mutant. Furthermore, cell co- expressin H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant exhibited no evidence of H-ras:G12V-induced endosome enlargement. To observe changes in endosome structure and activity that directly followed activation of GF P-rab5a:wt, we performed time-lapse recordings of cells cultured overnight in serum-free media after addition of EGF. EGF caused a rapid increase in e ndosome fusion and in membrane ruffling activity. Membrane ruffling was oft en associated with GFP-rab5a:wt-positive vesicle (pinosome) formation at th e base of membrane ruffles. Endosome and pinosome fusion were common in EGF -stimulated cells. Phagocytosis is also regulated by GFP-rab5a:wt showed tr ansiently activation and recruitment of GFP-rab5a:wt to newly formed phagos omes that contained rhodamine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structur e and activity of the early endosomal and early phagosomal elements.