Troponin and a Myopathy-Linked Mutation in TPM3 Inhibit Cofilin-2-Induced Thin Filament Depolymerization

Figures

• 

  Figure 1. Binding of cofilin-2 to F-actin regulated either by Tpm3.12 or Tpm3.12-R91C in the absence (A) or presence of Tn ± Ca2+ (B). The experimental points were fit to the Hill model. Conditions: 3.0 μM F-actin saturated with 2 μM Tpm3.12 or Tpm3.12-R91C ± 3 μM Tn, and cofilin-2 at concentrations ranging from 0 to 10 μM in 5.0 mM Tris–HCl, pH 7.5, 2 mM MgCl2, 100 mM NaCl, ±0.1 mM CaCl2. The data were collected from two independent experiments.

• 

  Figure 2. Electrophoretic separation of the proteins collected in pellets after ultracentrifugation of the regulated thin filaments incubated with increasing concentrations of cofilin-2. (A) F-actin coated with Tpm3.12 (upper panel) or Tpm3.12-R91C (lower panel). (B) F-actin coated with Tpm3.12 (upper panel) or Tpm3.12-R91C (lower panel) in the presence of Tn + Ca2+. (C) F-actin coated with Tpm3.12 (upper panel) or Tpm3.12-R91C (lower panel) in the presence of Tn − Ca2+. Conditions: 3 μM F-actin, 2 μM Tpm3.12 or Tpm3.12-R91C, 3.0 μM Tn, 0.1 mM CaCl2 or 0.2 mM EGTA, and cofilin-2 at concentrations ranging from 0 to 10 μM in 5.0 mM Tris–HCl, pH 7.5, 2 mM MgCl2, 100 mM NaCl.

• 

  Figure 3. Cofilin-induced dissociation of Tpm3.12 and Tpm3.12-R91C from F-actin in the absence and presence of Tn ± Ca2+. In the absence of Tn, the averaged experimental points were fit to decay regression. In the presence of Tn ± Ca2+, the averaged experimental points were fit to linear regression. Conditions: 3.0 μM F-actin saturated with 2 μM Tpm variants, 3 μM Tn ± Ca2+, 5.0 mM Tris–HCl, pH 7.5, 2 mM MgCl2, 100 mM NaCl, and 0.1mM CaCl2 or 0.2 mM EGTA were titrated with cofilin-2 at concentrations ranging from 0 to 10 μM. The points are means of three independent experiments ± SE.

• 

  Figure 4. The effects of Tpm3.12, Tpm312-R91C, and Tn on cofilin-2-induced filament severing and depolymerization. Snapshots of F-actin alone and F-actin regulated by Tpm3.12 or Tpm3.12-R91C in the presence of Tn and Ca2+ before (0 s) and after (22 s) the addition of cofilin-2 (A). The mean length of F-actin, F-actin regulated by Tpm3.12 or Tpm3.12-R91C (left panel), and the fraction of filaments with breaks (right panel) in the absence of Tn (B,C), in the presence of Tn + Ca2+ (D,E), and the presence of Tn +Ca2+ (F,G). Conditions: 25 mM Tris-HCl, pH 7.5, 25 mM KCl, 2 mM MgCl2, and 10 mg/mL of BSA supplemented with 0.1 mg/mL of glucose oxidase, 0.01 mg/mL of catalase, and 3 mg/mL of glucose. Error bars are standard error (SE) calculated from 50 filaments. A comparison of the differences between the experimental groups showed that the differences were significant in: (B) at the time range from 11 to 27.5 s (p < 0.05); (C) within groups with different numbers of breaks (p < 0.05); (D,F) at the time range from 16 to 27.5 sec (p < 0.05); (E,G) within groups with different numbers of breaks (p < 0.05).

• 

  Figure 5. The rate of actin filament depolymerization in the presence of Tpm variants and Tn ± Ca2+. The lengths of the filaments at different time points are shown as the percentage of the lengths obtained before adding cofilin-2. The conditions were the same as in Figure 4. The differences were statistically significant between F-actin and F-actin-Tpm3.12 or F-actin-Tpm3.12-R91C within the whole time range (p < 0.05). The difference between F-actin-Tpm3.12 and F-actin-Tpm3.12-R91C was significant at the time range of 16.5 to 27.5 s (p < 0.05). The differences between F-actin-Tpm3.12 and Tpm3.12-R91C in the presence of Tn (±Ca2+) were significant at the time range of 16.5 to 27.5 s (p < 0.05).