Infection by Listeria monocytogenes involves escape from its phagocytic compartment prior to fusion with the lysosome. Previous studies show that small GTPase Rab5a plays a crucial role in Lm ability to escape from its compartment and that Lm is able to modulate Rab5a activity, promoting GDP exchange of Rab5a. Rab5a regulates the homo- and heterotypic fusion of membranous organelles during the early stages of endocytosis. The extent to which molecules that regulate Rab5a coordinate its cycling on membranes to affect the behavior of individual organelles has not been determined. This study used novel Förster Resonance Energy Transfer (FRET) microscopic methods to analyze the Rab5a cycle on macropinosomes and phagosomes, two large endocytic vesicles that form in ruffled regions of cell membranes. In Cos-7 cells and mouse macrophages stimulated with growth factors, Rab5a activation followed immediately after its recruitment to newly formed macropinosomes. Rab5a activity increased continuously and uniformly over macropinosome membranes then decreased continuously, with Rab5a deactivation preceding dissociation by 1-12 min. Maximal levels of Rab5a activity were independent of organelle size, but Rab5a cycles were longer on larger macropinosomes, consistent with an integrative activity governing Rab5a dynamics on individual organelles. The Rab5a cycle was destabilized by microtubule depolymerization and by bafilomycin A1. Overexpression of activating and inhibitory proteins indicated that active Rab5a stabilized macropinosomes. Thus, overall Rab5a activity on macropinosomes is coordinated by macropinosome structure and physiology. In macrophages Rab5a showed different levels of FRET on Lm-containing vacuoles. Macropinosomes that formed secondary to the phagocytic events showed higher FRET levels than during RBC and Lm phagocytosis. During uptake of hly- and heat-killed Lm, Rab5a localized to the vacuole with low FRET levels, which points to a possible role of LLO. During vesicle fusion events Rab5a FRET was higher at the point of contact of the vacuoles and increased over the whole organelle after fusion implying two distinct regulatory events. Thus, as on macropinosomes Rab5a plays a role in phagocytosis but requires lower levels of activation. Lm affects Rab5a activation cycling although currently we are not able to elucidate if Lm vacuoles with low FRET levels have higher survival rates
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