Dynamics of rab5 activation in endocytosis and phagocytosis.

Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimu- lation requires activation of rab5. We utilized a GFP- rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macro- phages. Control CHO cells that expressed GFP- rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesi- cles with a mean diameter of 0.3 6 0.1 mm. Endo- some fusion, membrane ruffling, and pinosome for- mation were rarely detected in these cells. Coexpres- sion of H-ras:G12V, a constitutively active H-ras mutant that activates rab5a, in cells resulted in marked enlargement of labeled endosomes (mean diameter 0.7 6 0.2 mm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present. Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruf- fling and pinosome formation were commonly ob- served. Alterations in GFP-rab5a:wt endosome struc- ture and 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 mu- tant. Furthermore, cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant, ex- hibited no evidence of H-ras:G12V-induced endo- some enlargement. To observe changes in endosome structure and activity that directly followed activation of GFP-rab5a:wt, we performed time-lapse record- ings of cells cultured overnight in serum-free media after addition of EGF. EGF caused a rapid increase in endosome fusion and in membrane ruffling activity. Membrane ruffling was often associated with GFP- rab5a:wt-positive vesicle (pinosome) formation at the base of membrane ruffles. Endosome and pinosome fusion were common in EGF-stimulated cells. Phago- cytosis is also regulated by GFP-rab5a:wt. J774 mac- rophages that expressed GFP-rab5a:wt showed tran- siently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodam- ine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alter- ations in the structure and activity of the early endo- somal and early phagosomal elements. J. Leukoc. Biol. 68: 627-632; 2000.