Abstract
Filopodia are slender cellular protrusions that dynamically extend and retract to facilitate directional cell migration, pathogen sensing, and cell-cell adhesion [1–4]. Each filopodium contains a rigid and organized bundle of parallel actin filaments, which are elongated at filopodial tips by formins and Ena/VASP proteins [5–10]. However, relatively little is known about how the actin filaments in the filopodial shaft are spatially organized to form a bundle with appropriate dimensions and mechanical properties. Here, we report that the mammalian formin Daam1 (Disheveled-associated activator of morphogenesis 1) is a potent actin-bundling protein and localizes all along the filopodial shaft, which differs from other formins that localize specifically to the tips. Silencing of Daam1 led to severe defects in filopodial number, integrity, and architecture, similar to silencing of the bundling protein fascin. This led us to investigate the potential relationship between Daam1 and fascin. Fascin and Daam1 coimmunoprecipitated from cell extracts, and silencing of fascin led to a striking loss of Daam1 localization to filopodial shafts, but not tips. Furthermore, purified fascin bound directly to Daam1, and multicolor single-molecule TIRF imaging revealed that fascin recruited Daam1 to and stabilized Daam1 on actin bundles in vitro. Our results reveal an unanticipated and direct collaboration between Daam1 and fascin in bundling actin, which is required for proper filopodial formation.