Phosphatidylinositol 4,5-biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck, WIP, and Grb2.
Wiskott-Aldrich syndrome protein (WASP)/Scar family proteins promote actin polymerization by stimulating the actin-nucleating activity of the Arp2/3 complex. While Scar/WAVE proteins are thought to be involved in lamellipodia protrusion, the hematopoietic WASP has been implicated in various actin-based processes such as chemotaxis, podosome formation, and phagocytosis. Here we show that the ubiquitously expressed N-WASP is essential for actin assembly at the surface of endomembranes induced as a consequence of increased phosphatidylinositol 4,5-biphosphate (PIP2) levels. This process resulting in the motility of intracellular vesicles at the tips of actin comets involved the recruitment of the Src homology 3 (SH3)-SH2 adaptor proteins Nck and Grb2 as well as of WASP interacting protein (WIP). Reconstitution of vesicle movement in N-WASP-defective cells by expression of various N-WASP mutant proteins revealed three independent domains capable of interaction with the vesicle surface, of which both the WH1 and the polyproline domains contributed significantly to N-WASP recruitment and/or activation. In contrast, the direct interaction of N-WASP with the Rho-GTPase Cdc42 was not required for reconstitution of vesicle motility. Our data reveal a distinct cellular phenotype for N-WASP loss of function, which adds to accumulating evidence that the proposed link between actin and membrane dynamics may, at least partially, be reflected by the actin-based movement of vesicles through the cytoplasm.
Benesch
Stefanie
S
Department of Cell Biology, Gesellschaft für Biotechnologische Forschung (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Lommel
Silvia
S
Steffen
Anika
A
Stradal
Theresia E B
TE
Scaplehorn
Niki
N
Way
Michael
M
Wehland
Juergen
J
Rottner
Klemens
K
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D051380:GRB2 Adaptor Protein; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009068:Movement / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014923:Wiskott-Aldrich Syndrome / Q000235:genetics; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; C078332:Nck protein; D009419:Nerve Tissue Proteins; D015513:Oncogene Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011506:Proteins; D011993:Recombinant Fusion Proteins; C495771:WASL protein, human; C109903:WIPF1 protein, human; C495772:Wasl protein, mouse; C491671:Waspip protein, mouse; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D049452:Green Fluorescent Proteins