Figure 1.
Figure 1. Specific point mutations in the APC Basic domain disrupt actin nucleation. (A) Schematic of FL-APC domains and their interactions. Highlighted below the schematic is the 17–amino acid sequence (2,526–2,542) identified as required for actin nucleation (Fig. S1) and the four alanine substitution mutants that were generated (m1–m4). (B) Cartoon of WT and m4 FL-APC molecules as dimers, color coded as in A and with G-actin in yellow and sites of m4 mutations indicated by red stars. (C) Representative images from TIRF assays 300 s after initiation of actin assembly. Reactions contain 1 µM G-actin (10% OG labeled and 0.2% biotin labeled) and 5 µM profilin, with or without 5 nM APC-B (WT or mutant). (D) Mean number of actin filaments assembled per FOV in TIRF assays as in B. Data averaged from three FOVs in each of three independent experiments (n = 200 actin filaments per condition). Ordinary one-way Student’s t test, Dunnett´s multiple comparisons test. (E) Quantification of OG-actin fluorescence intensity over time in TIRF assays; conditions as in B. Data averaged from fluorescence intensity in ≥3 FOVs. ****, P < 0.0001 (F) Bulk actin assembly assays containing 2 µM G-actin (5% pyrene labeled), 5 µM profilin, 3 nM capping protein, and 10 nM APC-B (WT or m4) and/or 1 nM C-Daam1. (G) Initial rates of actin assembly from bulk assays as in E, averaged from three independent experiments. (H) Quantification of single-molecule TIRF colocalization of surface-adsorbed SNAP-649–APC-C (WT or m4) with soluble, Latrunculin B–bound OG-labeled actin monomers. Data averaged from three independent experiments (n ≥ 215 spots per condition). Two-tailed Student’s t test. (I) Fluorescence assay measuring binding of APC-B (WT, m4, and ΔN6; 0–100 nM) to 100 nM Latrunculin B–bound pyrene-labeled actin monomers. (J) Representative step photobleaching of single molecules of SNAP-649–APC-C (WT and m4). Insets show raw images corresponding to the traces. (K) Distribution of number of observed bleaching events for SNAP-649–APC-C (WT) and SNAP-649–APC-C (m4) single molecules, and comparison to predicted number of bleaching events (based on calculated SNAP-649 labeling efficiency; Breitsprecher et al., 2012) for different oligomeric states (color-coded squares). Lines connect squares for the best fit to the observed data. n.s., not significant. Error bars, SD.

Specific point mutations in the APC Basic domain disrupt actin nucleation. (A) Schematic of FL-APC domains and their interactions. Highlighted below the schematic is the 17–amino acid sequence (2,526–2,542) identified as required for actin nucleation (Fig. S1) and the four alanine substitution mutants that were generated (m1–m4). (B) Cartoon of WT and m4 FL-APC molecules as dimers, color coded as in A and with G-actin in yellow and sites of m4 mutations indicated by red stars. (C) Representative images from TIRF assays 300 s after initiation of actin assembly. Reactions contain 1 µM G-actin (10% OG labeled and 0.2% biotin labeled) and 5 µM profilin, with or without 5 nM APC-B (WT or mutant). (D) Mean number of actin filaments assembled per FOV in TIRF assays as in B. Data averaged from three FOVs in each of three independent experiments (n = 200 actin filaments per condition). Ordinary one-way Student’s t test, Dunnett´s multiple comparisons test. (E) Quantification of OG-actin fluorescence intensity over time in TIRF assays; conditions as in B. Data averaged from fluorescence intensity in ≥3 FOVs. ****, P < 0.0001 (F) Bulk actin assembly assays containing 2 µM G-actin (5% pyrene labeled), 5 µM profilin, 3 nM capping protein, and 10 nM APC-B (WT or m4) and/or 1 nM C-Daam1. (G) Initial rates of actin assembly from bulk assays as in E, averaged from three independent experiments. (H) Quantification of single-molecule TIRF colocalization of surface-adsorbed SNAP-649–APC-C (WT or m4) with soluble, Latrunculin B–bound OG-labeled actin monomers. Data averaged from three independent experiments (n ≥ 215 spots per condition). Two-tailed Student’s t test. (I) Fluorescence assay measuring binding of APC-B (WT, m4, and ΔN6; 0–100 nM) to 100 nM Latrunculin B–bound pyrene-labeled actin monomers. (J) Representative step photobleaching of single molecules of SNAP-649–APC-C (WT and m4). Insets show raw images corresponding to the traces. (K) Distribution of number of observed bleaching events for SNAP-649–APC-C (WT) and SNAP-649–APC-C (m4) single molecules, and comparison to predicted number of bleaching events (based on calculated SNAP-649 labeling efficiency; Breitsprecher et al., 2012) for different oligomeric states (color-coded squares). Lines connect squares for the best fit to the observed data. n.s., not significant. Error bars, SD.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal