Processive acceleration of actin barbed-end assembly by N-WASP.
Neuronal Wiskott-Aldrich syndrome protein (N-WASP)-activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3, N-WASP binds actin-filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We use nanofibers coated with N-WASP WWCA domains as model cell surfaces and single-actin-filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed-end assembly. Individual barbed ends captured by WWCA domains grow at or below their diffusion-limited assembly rate. At high filament densities, however, overlapping filaments form buckles between their nanofiber tethers and myosin attachment points. These buckles grew ∼3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native polyproline (PP) region show similar rate enhancements in the absence of profilin, but profilin slows barbed-end acceleration from constructs containing the PP region. Increasing Mg(2+) to enhance filament bundling increases the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed-end bundling. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion.
Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Nimisha
N
Khanduja
Jeffrey R
JR
Kuhn
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D055767:Immobilized Proteins / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D057139:Nanofibers / Q000737:chemistry; D051304:Profilins / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000737:chemistry*
D000199:Actins; D055767:Immobilized Proteins; D051304:Profilins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D008274:Magnesium