Fig. 1

CARMIL3 inhibits capping protein (CP) activity in actin polymerization assays, with pyrene-actin fluorescence (arbitrary units) plotted vs time. (A) Cartoon of the zebrafish Carmil3 protein illustrating its functional domains, and the positions of two zebrafish mutations studied here, carmil3^sa19830 and carmil3^stl413 and the location of the CBR mutations. PH, Pleckstrin Homology; L, linker; N, N-cap; LRR, Leucine Rich Repeat; C, C-cap; HD, helical / homodimerization domain; CBR, Capping protein Binding Region; CPI, capping protein interaction; CSI, CARMIL-specific interaction; MBD, membrane-binding domain; PRR, Proline Rich Region. Amino acid sequences for uman CARMIL1 and 3 and D. rerio Carmil3 for the CPI region are in a box, with and the location of the D. rerio mutant known as HVR->AVA denoted by asterisks. (B) Human CARMIL3 (C3) inhibited the ability of CP to cap barbed ends of actin filaments. Black points, labeled “Seeds,” are from a control with monomeric actin and filamentous actin seeds. Pink points, labeled “CP,” correspond to a sample to which CP was added. Red and blue curves contain CP plus the indicated concentrations of human CARMIL1 or CARMIL3 CBR fragment. (C) Zebrafish Carmil3 (C3, blue curves) CBR fragment inhibited capping by CP. In comparison to human CARMIL3 (C3, red curves), the inhibitory activity of zebrafish Carmil3 was slightly less than that of human CARMIL3, based on similar concentrations as indicated. A mutant form of zebrafish Carmil3 CBR fragment (C3-HVR->AVA, green curve) containing two point mutations at conserved residues of the CP-binding CPI motif (Fig. 1A), failed to block CP activity, as expected.