Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains.
Actin filament networks exert protrusive and attachment forces on membranes and thereby drive membrane deformation and movement. Here, we show that N-WASP WH2 domains play a previously unanticipated role in vesicle movement by transiently attaching actin filament barbed ends to the membrane. To dissect the attachment mechanism, we reconstituted the propulsive motility of lipid-coated glass beads, using purified soluble proteins. N-WASP WH2 mutants assembled actin comet tails and initiated movement, but the comet tails catastrophically detached from the membrane. When presented on the surface of a lipid-coated bead, WH2 domains were sufficient to maintain comet tail attachment. In v-Src-transformed fibroblasts, N-WASP WH2 mutants were severely defective in the formation of circular podosome arrays. In addition to creating an attachment force, interactions between WH2 domains and barbed ends may locally amplify signals for dendritic actin nucleation.
Co
Carl
C
Department of Cellular and Molecular Pharmacology, UCSF/UCB Cell Propulsion Lab, University of California-San Francisco, San Francisco, CA 94158, USA.
Wong
Derek T
DT
Gierke
Sarah
S
Chang
Vicky
V
Taunton
Jack
J
D008841:Actin Cytoskeleton / Q000378:metabolism*; D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology*; D002472:Cell Transformation, Viral; D005347:Fibroblasts; D016391:Genes, src; D008563:Membrane Lipids; D008840:Microfilament Proteins / Q000378:metabolism; D008863:Microspheres; D009068:Movement; D009154:Mutation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D008563:Membrane Lipids; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C495770:Wasl protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal