Thoracic aortic aneurysm (TAAD)-causing mutation in actin affects formin regulation of polymerization

Abstract

More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients.

FIGURE 1.

Model of residue R256H in the actin monomer and filament. A, back view of yeast actin monomer crystal structure (43), modified from Protein Data Bank code 1YAG using the PyMOL Molecular Graphics System (version 1.3 (Schrödinger, LLC)). The position of the R256H mutation studied is color-highlighted in red and labeled. The remaining known TAAD missense mutations are color-highlighted in blue. ATP is depicted in orange, and Mg²⁺ is shown as a yellow sphere. The numbers denote the actin subdomains, and N and C mark the respective termini. B, model of an actin sextmer based on the filament model by Fujii et al. (17) with the R256H mutation color-highlighted and labeled as described above. The filament model displays the location of the mutation along the interstrand interface. The numbers denote the actin subdomains of the individual monomer. Subunits of one strand are in varied shades of blue, and subunits of the opposing strand are in shades of gray.

FIGURE 2.

Effect of the R256H mutation on cytology. A, fluorescence microscopy of cells expressing wild type or R256H yeast actin. Cells assessed were those in which the bud was between one-half and one-third the size of the mother cell. The cytoskeleton was visualized after staining fixed cells with rhodamine-phalloidin. White arrows identify actin cables. Vacuoles were observed following exposure of the cells to the dye FM4-64. Mitochondria were visualized with GFP. Scale bar, 2 mm. B, quantified results are based on assessment of 100 cells for each sample. The bar height indicates the percentage of the cell population that exhibited abnormal morphology with error bars denoting the S.D. Differences were statistically significant compared with wild type actin (p < 0.05).

FIGURE 3.

Effect of the R256H mutation on sensitivity to cytoskeletal disassembly. A, sterile filtered discs (0.5 cm in diameter) were presoaked in either 2 μl of dimethyl sulfoxide (control) or 2 μl of 0.1, 0.5, or 1 mm latrunculin A and placed on YPD plates containing 100 μl of evenly spread wild type or mutant cells (A₆₀₀ = 0.1). The plates were incubated at 30 °C for 48 h. B, quantification of growth inhibition by latrunculin A. The bar height indicates the area of growth inhibition (cm²) for the four concentrations of latruncilin A (mm) with error bars denoting the S.D. The area of growth inhibition for R256H cells was 1.9-fold higher for the highest latrunculin A concentration. Differences were statistically significant compared with wild type actin for all concentrations (p < 0.05).

FIGURE 4.

Impact of the R256H mutation on actin cytoskeleton assembly in vivo. A scatter plot of the indexed percentage of cells with actin cables relative to time after treatment with latrunculin A. Cells were transiently treated with latrunculin A to disassemble the cytoskeleton, washed, and incubated. Aliquots of cells were removed at designated time points, fixed, stained with rhodamine-phalloidin, and imaged as described under “Experimental Procedures.” The percentage of cells with actin cables was quantified for each sample and indexed to the pretreatment fraction. At least 50 cells were analyzed per sample, and the experiments were repeated three times. The scatter plot graph displays the indexed percentage of cells with actin cables relative to time for all analyses. Logarithmic trend lines depict the differences in rate of actin cable reassembly between wild type and R256H actin.

FIGURE 5.

Polymerization kinetics of wild type and R256H actin. A, polymerization of 2.25 μm actin was initiated by the addition of magnesium and potassium chloride, and the change in light scattering was monitored as a function of time at 25 °C. Shown are representative plots of experiments performed at least three times with three independent actin preparations. B, critical concentration (C_c) of actin was determined from the net change in light scattering upon polymerization performed as in A as a function of increasing actin concentration. The critical concentration of actin was determined by drawing a line through the points and determining the intersection at the x axis. A.U., arbitrary units.

FIGURE 6.

Actin polymerization kinetics in the presence of the formin, Bni. A, actin polymerization was quantified in the presence of various concentrations of the Bni1 FH1-FH2 fragment. Ten percent of the actin was labeled with pyrene, and the polymerization-dependent increase in fluorescence was quantified over time. To account for the increased critical concentration of R256H mutant actin, actin concentrations with equivalent final extents of polymerization were selected to ensure the same amount of polymerizable actin was used. Accordingly, concentrations for wild type and R256H actin were 1.5 and 2.25 μm, respectively, as described under “Experimental Procedures.” Shown are representative plots of experiments performed at least three times with three independent actin preparations. A.U., arbitrary units.

FIGURE 7.

Effect of late addition of Bni1 on actin polymerization kinetics. A, polymerization of 10% pyrene-labeled wild type and R256H actin was fluorescently monitored over time as in Fig. 6 with the exception that 100 nm Bni was added to the reaction midway during elongation. Time of Bni addition is marked by an arrow. Concentrations for wild type and R256H actin were 1.5 and 2.25 μm, respectively. Shown is a representative plot from experiments performed at least three times with three independent actin preparations. A.U., arbitrary units.

FIGURE 8.

Actin polymerization kinetics in the presence of phalloidin. Polymerization of 10% pyrene-labeled actin was quantified by change in fluorescence over time with and without phalloidin and with and without Bni. Actin concentrations were 2.25 μm as was the concentration of phalloidin when included. 100 nm of the FH1-FH2 fragment of Bni1 was used when included. A, wild type actin. B, R256H mutant actin. Shown are representative plots from experiments performed at least two times with two independent actin preparations. A.U., arbitrary units.

FIGURE 9.

Polymerization kinetics of admixtures of wild type and mutant actin. A, ratios of wild type and R256H mutant actin were combined with a final concentration of 2.25 μm and induced to polymerize. The polymerization-dependent increase in fluorescence of the 10% pyrene-labeled actin was monitored over time. B, as in A, polymerization kinetics of ratios of wild type and R256H mutant actin was quantified in the presence 100 nm of Bni1. Shown are representative plots of experiments performed at least three times with three independent actin preparations. A.U., arbitrary units.

FIGURE 10.

Model of the actin trimer. A model of the actin trimer based on the filament model of Oda et al. (43). The actin monomer colors are blue, gray, and green. Residue Arg-256 is color-highlighted and labeled (red), residue Glu-195 is color-highlighted and labeled (blue), and residue Lys-113 is color-highlighted and labeled (orange). Models were modified using the PyMOL Molecular Graphics System (version 1.3; Schrödinger, LLC).

FIGURE 11.

Model of Bni interaction with the actin oligomer. A, model of the actin oligomer based on the filament model of Oda et al. (43) and the Bni1 FH1-FH2 fragment from Rosen et al. (49). The actin monomer colors are blue and gray. Residues are color highlighted: orange, Lys-113; marine blue, Lys-118; yellow, Glu-195; red, Arg-256; and pink, helix containing residues 113–125. The FH1-FH2 domain colors are gold and green. The red rectangle frames this unit of interaction. Models were generated using the PyMOL Molecular Graphics System (version 1.5; Schrödinger, LLC). The two models are a side view and a pointed end view of the structure. B, schematic drawing demonstrating the formin/actin complex colored as above. The post site of the green bridge is free and accessible to recruit an incoming actin subunit.