Mulligan, A. G.; Lehmann, Z. J.; Robinson, K. L.; Tyska, M. J.

Actin-based membrane protrusions such as filopodia, microvilli, and stereocilia support a range of cell functions, from nutrient absorption to mechanosensation. In each case, membrane deformation is supported by a core bundle of actin filaments, organized in a unipolar 'barbed-end out' manner. Although their structures and proteomes are well characterized, mechanisms governing the growth and stability of these protrusions remain less clear. Factors that localize to the distal tips of these structures are of particular interest, as they are well positioned to control actin assembly at filament barbed ends. One such factor, EPS8, localizes to distal tip puncta in multiple protrusion types. While early biochemical studies suggested a role in filament capping, loss of EPS8 in multiple models shortened microvilli and stereocilia, suggesting roles in elongation. More recent studies in differentiating epithelial cells suggested that EPS8 promotes protrusion growth and stability. To clarify EPS8's function in the distal tip compartment, we leveraged acute loss-of-function experiments and titrated gain-of-function approaches in combination with live imaging. Acute sequestration of EPS8 led to rapid depletion of filopodia. Conversely, increasing cellular EPS8 levels elevated EPS8 per distal tip punctum, increased F-actin content within individual filopodia, reduced filopodia elongation rates, increased protrusion lifetimes, and protected filopodia against cytochalasin D-induced collapse. These findings suggest that EPS8 binds filament barbed ends as a 'leaky capper,' slowing monomer addition while stabilizing bundles and preventing collapse. These activities are likely critical for building and maintaining the large arrays of protrusions that are assembled by diverse epithelial cell types.