mDia1 and APC-B synergize to promote actin assembly in the combined presence of profilin and capping protein. (A) Reactions contain 2 µM actin monomers (5% pyrene labeled), 3 µM profilin, 2 nM CapZ, with or without 20 nM APC-B and/or 2 nM C-mDia1. (B) Quantification of elongation rates from the slopes of curves as in A. Rates are averages for reactions performed in triplicate in each of three separate experiments, where error bars represent standard error (n = 3). (C) Rhodamine-phalloidin staining of GAPDH or mDia1-depleted cells microinjected with EGFP-APC-B plasmid. Arrowheads point to injected cells. Insets show GFP signal for the same area. Bar, 10 µm. Right panel is an immunoblot showing expression levels 72 h after siRNA transfection for GAPDH and mDia1, with vinculin as a loading control. (D) Quantification of F-actin levels induced by EGFP-APC-B in GAPDH or mDia1-depleted cells (n > 40). (E) Model for synergy between APC and mDia1 in promoting actin assembly, where APC efficiently seeds polymer formation by recruiting actin monomers from a pool of profilin-actin to form a prenucleation complex. The barbed end of the seed is captured by the FH2 domain of mDia1, which processively moves with the growing barbed end, protecting it from capping proteins while accelerating elongation through FH1–profilin–actin interactions.