Ena/VASP clustering at microspike tips involves Lamellipodin but not I-BAR proteins, and absolutely requires unconventional Myosin-X

Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi are essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared to B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation.Significance StatementUnlike filopodia that protrude well beyond the cell periphery and are implicated in sensing, morphogenesis and cell-to-cell communication, the function of microspikes consisting of actin-filament bundles fully embedded within lamellipodia is less clear. Microspike formation involves specific clustering of Ena/VASP family members at filament-barbed ends to enable processive actin polymerization in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering have remained unknown. Here, we systematically analyzed these processes in genetic knockout mutants derived from B16-F1 cells and show that Ena/VASP clustering at microspike tips involves Lamellipodin, but not inverse BAR-domain proteins, and strictly requires unconventional Myosin-X. Complete loss of microspikes was confirmed with CRISPR/Cas9-mediated MyoX knockout in Rat2 fibroblasts, excluding cell type-specific effects.