Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.
BACKGROUND
Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane.


RESULTS
We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology.


CONCLUSIONS
This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.
Castellano
F
F
Centre d'Immunologie, INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France.
Montcourrier
P
P
Guillemot
J C
JC
Gouin
E
E
Machesky
L
L
Cossart
P
P
Chavrier
P
P
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D003598:Cytoskeletal Proteins; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism; D008840:Microfilament Proteins; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011554:Pseudopodia / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D000098662:Vasodilator-Stimulated Phosphoprotein; D000099149:Ezrin
D000199:Actins; D000903:Antibiotics, Antineoplastic; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D019204:GTP-Binding Proteins; D008667:Metalloproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011506:Proteins; D011956:Receptors, Cell Surface; D020123:Sirolimus; D051316:Wiskott-Aldrich Syndrome Protein; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; D000098662:Vasodilator-Stimulated Phosphoprotein; D000099149:Ezrin