Dynamics of rab5 activation in endocytosis and phagocytosis.
Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimulation requires activation of rab5. We utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macrophages. Control CHO cells that expressed GFP-rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesicles with a mean diameter of 0.3 +/- 0.1 microm. Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cells. Coexpression 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 +/- 0.2 microm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present. Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruffling and pinosome formation were commonly observed. Alterations in GFP-rab5a:wt endosome structure 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 mutant. Furthermore, cells co-expressing 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 GFP-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 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. Phagocytosis is also regulated by GFP-rab5a:wt. J774 macrophages that expressed GFP-rab5a:wt showed transiently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodamine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structure and activity of the early endosomal and early phagosomal elements.
Roberts
R L
RL
Department of Cell Biology and Physiology, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.
Barbieri
M A
MA
Ullrich
J
J
Stahl
P D
PD
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000502:physiology; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004926:Escherichia coli; D005347:Fibroblasts / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000502:physiology; D008164:Luminescent Proteins / Q000378:metabolism; D008264:Macrophages / Q000502:physiology; D018613:Microscopy, Confocal; D010587:Phagocytosis / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D018631:ras Proteins / Q000096:biosynthesis / Q000502:physiology
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D020696:rab5 GTP-Binding Proteins; D018631:ras Proteins