Constitutively active mDia1 enables visualization of actin assembly in filopodia

Visualizing individual actin filaments in living cells is notoriously difficult. Formins, which track filament plus ends, are powerful probes of actin assembly, yet their behavior in cells is incompletely understood. We dissect the formin mDia1 to reveal how regulatory states control filament assembly. Using TIRF microscopy, we directly compare new regulatory mutants with canonical activity constructs (full-length, FH1-C, FH2-C, and ΔDAD). A two amino-acid substitution mutant, mDia1(CA), is constitutively active and drives strong actin nucleation and elongation. In cells, SNAP-mDia1(CA) is enriched in filopodia, where it forms bright, persistent signals at the tips and dim puncta in the shafts. SNAP-mDia1(CA) increases filopodia number but not elongation rate or length, regardless of its use as an overexpression or rescue construct. Although mDia1(CA) is not a universal actin plus-end tracker in this system, its selective enrichment at filopodial plus ends establishes it as a mechanistically distinct probe for interrogating filopodial actin dynamics.