CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP.
ABSTRACT Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high-density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53-dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.
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ABSTRACT: In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations.eLife Sciences 12/2014; 3. DOI:10.7554/eLife.03116 · 8.52 Impact Factor
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ABSTRACT: Membrane protrusions at the leading edge of cells, known as lamellipodia, drive cell migration in many normal and pathological situations. Lamellipodial protrusion is powered by actin polymerization, which is mediated by the actin-related protein 2/3 (ARP2/3)-induced nucleation of branched actin networks and the elongation of actin filaments. Recently, advances have been made in our understanding of positive and negative ARP2/3 regulators (such as the SCAR/WAVE (SCAR/WASP family verprolin-homologous protein) complex and Arpin, respectively) and of proteins that control actin branch stability (such as glial maturation factor (GMF)) or actin filament elongation (such as ENA/VASP proteins) in lamellipodium dynamics and cell migration. This Review highlights how the balance between actin filament branching and elongation, and between the positive and negative feedback loops that regulate these activities, determines lamellipodial persistence. Importantly, directional persistence, which results from lamellipodial persistence, emerges as a critical factor in steering cell migration.Nature Reviews Molecular Cell Biology 08/2014; 15(9):577-90. DOI:10.1038/nrm3861 · 36.46 Impact Factor
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ABSTRACT: Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing and axonal guidance. Cdc42 induces filopodia formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its SH3 domain binding partners. Here we show that Dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Forster resonance energy transfer (FRET) analysis, and explore its role in filopodia formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes and the leading edge just behind the anti-capping protein Mena. Dyn1 knockdown reduces filopodia formation, which can be rescued by over-expressing wild-type Dyn1 but not GTPase mutant Dyn1-K44A and the loss-of-function ABD mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodia number and increased their lifetime. Using rapid time-lapse TIRF microscopy we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodia formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodia tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena and Eps8 to regulate filopodial dynamics.Journal of Biological Chemistry 07/2014; 289(35). DOI:10.1074/jbc.M114.553883 · 4.60 Impact Factor