Abstract
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 Statement Unlike 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Additional data and amendments of the revised manuscript include: - Validation of key findings in independently isolated clonal knockout (KO) cell lines - Validation of key findings by rescue experiments - Quantification of filopodia in B16-F1 and NIH 3T3-derived cells lacking I-BAR proteins - Validation of the MyoX-KO phenotype regarding loss of microspikes and VASP clustering in a different cell type (Rat2 cells) - Analyses of microspike formation and VASP clustering in reconstituted MyoX-KO cells expressing different MyoX variants - Quantification of microspikes and VASP clusters in each cell line, now expressed per unit length in lamellipodia - Magnified insets now provided in respective images for better clarity - Validation of BioID probes and localization of biotinylated proteins in transfected cells now shown - Previous Figures 1 and 3 have been moved to the Supplemental information - Amended Supplemental information including new data now encompasses 21 Figures
