Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation.

BACKGROUND

Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed.

METHODS

Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number.

RESULTS

Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells.

CONCLUSIONS

Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.