Molecular mechanisms linking missense ACTG2 mutations to visceral myopathy

Abstract

Visceral myopathy is a life-threatening disease characterized by muscle weakness in the bowel, bladder, and uterus. Mutations in smooth muscle γ-actin (ACTG2) are the most common cause of the disease, but the mechanisms by which the mutations alter muscle function are unknown. Here, we examined four prevalent ACTG2 mutations (R40C, R148C, R178C, and R257C) that cause different disease severity and are spread throughout the actin fold. R178C displayed premature degradation, R148C disrupted interactions with actin-binding proteins, R40C inhibited polymerization, and R257C destabilized filaments. Because these mutations are heterozygous, we also analyzed 50/50 mixtures with wild-type (WT) ACTG2. The WT/R40C mixture impaired filament nucleation by leiomodin 1, and WT/R257C produced filaments that were easily fragmented by smooth muscle myosin. Smooth muscle tropomyosin isoform Tpm1.4 partially rescued the defects of R40C and R257C. Cryo-electron microscopy structures of filaments formed by R40C and R257C revealed disrupted intersubunit contacts. The biochemical and structural properties of the mutants correlate with their genotype-specific disease severity.

Fig. 1.. Choice and purification of ACTG2 mutations for study.

(A) Surface representation of F-actin [Protein Data Bank (PDB) code: 8F8P] showing a ribbon diagram of a middle subunit (marine blue) with the side chains of ACTG2 residues reported to be mutated in VM displayed in beige or maroon for the four mutations studied here (R40C, R148C, R178C, and R257C). (B) Close-up view of the middle subunit shown in (A), highlighting ACTG2 residues reported to be mutated in VM (see also fig. S1 and table S1). The location of the hydrophobic cleft and the D-loop is indicated. (C) Coomassie-stained SDS–polyacrylamide gel electrophoresis of purified WT ACTG2 and mutants R40C, R257C, R148C, and R178C (full gels in fig. S2). MW, molecular weight.

Fig. 2.. Mutants R40C and R257C alter actin polymerization in different ways.

(A to C) Time course of actin polymerization (2 μM total actin, including 6% pyrene-labeled α-actin) showing comparisons between WT ACTG2 and α-actin (A); WT, R40C, WT/R40C, and half-WT (1 μM WT ACTG2, 12% pyrene-labeled α-actin) (B); and WT, R257C, and WT/R257C (C). (D) Polymerization rates of actin variants calculated from 7–12 reactions using at least three independent actin preparations. (E) Example of pyrene-actin depolymerization experiment, showing WT and α-actin. F-actin (10% pyrene-labeled α-actin) is diluted to 0.1 μM (i.e., below the Cc). (F) Relative depolymerization rates of actin variants from 11 to 15 reactions using at least three independent protein preparations. Data in (D) and (F) were analyzed by one-way analysis of variance (ANOVA) with Welch’s test (P < 0.0001), and the P values shown are from Dunnett’s T3 multiple comparisons test. (G) Example of Cc plots, showing equilibrium fluorescence as a function of the actin concentration (5% pyrene-labeled α-actin) for WT ACTG2 and α-actin. Cc values were calculated from the x intercept of the graphs. a.u., arbitrary units. (H) Cc values of actin variants calculated from n = 3 independent experiments. Data in (H) were analyzed by one-way ANOVA (P < 0.0001), and P values are from Bonferroni’s multiple comparisons test. Graphs in (D), (F), (G), and (H) represent means ± SD, with “ns” indicating P values > 0.05.

Fig. 3.. R40C and R257C affect filament nucleation by Lmod1.

(A to C) Time course of actin polymerization with 20 nM Lmod1 (2 μM total actin, including 6% pyrene-labeled α-actin) showing comparisons between WT ACTG2 and α-actin (A); WT, R40C, WT/R40C, and half-WT (1 μM WT, 12% pyrene-labeled α-actin) (B); and WT, R257C, and WT/R257C (C). (D) The polymerization rates of actin variants were calculated from 8 to 11 experiments and at least three independent actin preparations. Data were analyzed by one-way ANOVA with Welch’s test (P < 0.0001). P values are provided from Dunnett’s T3 multiple comparisons test. Bar graphs represent means ± SD, with ns indicating P values > 0.05.

Fig. 4.. Mutants R40C and R257C affect filament length but not myosin motility.

(A) Representative epifluorescence images of rhodamine-phalloidin α-actin (gray), WT ACTG2 (blue), R40C (gold), R257C (red), WT/R40C (green), and WT/R257C (purple) gliding on SMM-S1–coated coverslips at different times (as indicated; scale bars, 10 μm). (B) Filament length quantification at time = 0 (n = 5 experiments). (C and D) Filament length quantification over time for WT, R40C, and WT/R40C (C) and WT, R257C, and WT/R257C (D) (n = 5 experiments from at least three independent actin preparations). (E) Quantification of SMM-S1–driven filament velocities. Filaments were manually tracked in ImageJ (n = 4 experiments). Data in (B) and (E) were analyzed by one-way ANOVA (P < 0.0001), with P values from Bonferroni’s multiple comparisons test. Data from (C) and (D) were analyzed by two-way repeated measures (RM) ANOVA. P values, displayed in tables below the graphs, are from Tukey’s multiple comparisons test. Data were pooled from three actin and two SMM-S1 independent preparations. Graphs show means ± SEM, with ns indicating P values > 0.05.

Fig. 5.. Tpm1.4 partially rescues the defects of R40C and R257C.

(A) Representative epifluorescence images of Tpm1.4-coated rhodamine-phalloidin α-actin (gray), WT ACTG2 (blue), R40C (gold), R257C (red), WT/R40C (green), and WT/R257C (purple) gliding on SMM-S1–coated coverslips at different times (as indicated; scale bars, 10 μm). (B) Filament length quantification with Tpm1.4 at time = 0 (n = 5 to 6 experiments). Data were analyzed by one-way ANOVA (P = 0.0007), and P values are from Bonferroni’s multiple comparisons test. (C) Quantification of SMM-S1–driven filament velocities. Filaments were manually tracked in ImageJ (n = 4 experiments). Quantification of filament velocity (n = 4 experiments). Data were analyzed by one-way ANOVA with Welch’s ANOVA tests (P = 0.7873), and P values are from Dunnett’s T3 multiple comparisons test. (D) Filament length quantification over time for WT, R257C, and R257C + Tpm1.4 (n = 5 experiments). Data were analyzed by two-way RM ANOVA. P values, displayed in the table below the graph, are from Tukey’s multiple comparisons test. Data were pooled from three actin and two SMM-S1 independent preparations. Graphs display means ± SEM, with ns indicating P values > 0.05.

Fig. 6.. R40C and R257C impair subunit-subunit contacts in F-actin.

(A to C) Cryo-EM maps of R40C (purple), WT (gray), and R257C (blue) with resolutions of 2.54, 2.45, and 2.72 Å, respectively. The polymerization of R40C is inefficient, requiring its structure to be determined with bound phalloidin (green). Close-up ribbon diagrams highlighting local differences between WT and the mutants and the disruption of inter- and intrasubunit contacts (dashed lines). Different actin subunits in the vicinity of the mutations are colored differently. The definition of the short-pitch (orange) and long-pitch (cyan) helices is shown on the WT map. PE and BE indicate pointed and barbed ends, respectively.