Dissection of APC-B actin nucleation activity and mechanism. (A) Schematic of APC-B truncated polypeptides analyzed. The actin nucleation sequences identified (ANS1 and ANS2) are shaded blue. MTBS, microtubule binding site; N/A, not applicable; N/D, not determined. (B) Deletion of ANS1 diminishes the actin nucleation activity of APC-B in cells. Serum-starved NIH3T3 cells microinjected with EGFP-APC-B, EGFP-APC-B-ΔN4, or EGFP-APC-B-ΔN5 plasmids (arrows) were fixed and imaged for GFP fluorescence and rhodamine-phalloidin staining. Bar, 10 µm. (C) Quantification of total cellular F-actin levels (n > 50 cells). (D) Dimerization of APC-C polypeptide. Stokes radius and sedimentation coefficient were determined for MBP–APC-C and used to calculate its native molecular weight (MW). Predicted MW of monomer and dimer for MBP–APC-C are listed for comparison. (E) Dominant-negative effects of truncated APC-B polypeptides on wild-type APC-B–induced actin assembly. Reactions contain actin (2 µM; 5% pyrene labeled) and 50 nM wild-type APC-B, with or without 50 nM APC-B–ΔN4 (top) or 50 nM APC-B–ΔC2 (bottom).