The small GTPase R-Ras regulates organization of actin and drives membrane protrusions through the activity of PLCepsilon.
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 Cepsilon (PLCepsilon) 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 PLCepsilon and increased its activity. Knockdown of PLCepsilon 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 PLCepsilon.
Department of Pharmacology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Aude S
AS
Ada-Nguema
Harry
H
Xenias
Jake M
JM
Hofman
Chris H
CH
Wiggins
Michael P
MP
Sheetz
Patricia J
PJ
Keely
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002614:Chelating Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D054801:Phosphoinositide Phospholipase C; D011233:Precipitin Tests; D011554:Pseudopodia / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D010738:Type C Phospholipases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D018631:ras Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium