The small GTPase R-Ras regulates organization of actin and drives membrane protrusions through the activity of PLCϵ
Author:
Ada-Nguema Aude S.1, Xenias Harry2, Sheetz Michael P.2, Keely Patricia J.1
Affiliation:
1. Department of Pharmacology, University of Wisconsin-Madison, Madison, WI 53706, USA 2. Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Abstract
R-Ras, an atypical member of the Ras subfamily of small GTPases, enhances integrin-mediated adhesion and signaling through a poorly understood mechanism. Dynamic analysis of cell spreading by total internal reflection fluorescence (TIRF) microscopy demonstrated that active R-Ras lengthened the duration of initial membrane protrusion, and promoted the formation of a ruffling lamellipod, rich in branched actin structures and devoid of filopodia. By contrast, dominant-negative R-Ras enhanced filopodia formation. Moreover, RNA interference (RNAi) approaches demonstrated that endogenous R-Ras contributed to cell spreading. These observations suggest that R-Ras regulates membrane protrusions through organization of the actin cytoskeleton. Our results suggest that phospholipase Cϵ (PLCϵ) is a novel R-Ras effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion, because R-Ras was co-precipitated with PLCϵ and increased its activity. Knockdown of PLCϵ with siRNA reduced the formation of the ruffling lamellipod in R-Ras cells. Consistent with this pathway, inhibitors of PLC activity, or chelating intracellular Ca2+ abolished the ability of R-Ras to promote membrane protrusions and spreading. Overall, these data suggest that R-Ras signaling regulates the organization of the actin cytoskeleton to sustain membrane protrusion through the activity of PLCϵ.
Publisher
The Company of Biologists
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