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. 2010 Feb 12;285(7):4337-47.
doi: 10.1074/jbc.M109.059881. Epub 2009 Nov 21.

Dominant negative mutant actins identified in flightless Drosophila can be classified into three classes

Taro Q P Noguchi  1 , Yuki Gomibuchi, Kenji Murakami, Hironori Ueno, Keiko Hirose, Takeyuki Wakabayashi, Taro Q P Uyeda
Affiliations

Dominant negative mutant actins identified in flightless Drosophila can be classified into three classes

Taro Q P Noguchi et al. J Biol Chem. .

Abstract

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin.gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca(2+). Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca(2+) sensitivity in the presence of tropomyosin-troponin.

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Figures

FIGURE 1.
FIGURE 1.
Mutated residues in the actins characterized in this study. The Dictyostelium actin structure (58) (Protein Data Bank code 1c0f) is depicted using Chimera software. Mutated residues are highlighted by space-filling representation. The numbers indicate the subdomains.
FIGURE 2.
FIGURE 2.
Expression of GFP-mutant actins and mutant ARTs in Dictyostelium cells. A, fluorescence from GFP-actins in Dictyostelium cells observed using a confocal laser-scanning microscope. For GFP-G156D, -G156S, -Q353@, and -W356@ mutant actins, the majority of cells were too dark for observation, and the cells shown here have atypically high expression. Regions of cortical GFP fluorescence are indicated by arrows. Scale bar, 10 μm. B, Western blot (top) and Coomassie Brilliant Blue staining (bottom) following SDS-PAGE of total cell lysates from Dictyostelium cells expressing GFP-mutant actins. Polyclonal anti-GFP antibodies were used for Western blotting. C and D, levels of ART protein and mRNA were examined by Western blot analysis using a monoclonal anti-actin antibody (C) and RT-PCR using ART-specific primers (D).
FIGURE 3.
FIGURE 3.
Sedimentation assay of mutant actins. Each purified mutant actin was allowed to polymerize under three conditions and then ultracentrifuged. Condition 1, polymerization of actin alone. Condition 2, polymerization with phalloidin. Condition 3, polymerization with additional equimolar concentration of WT actin. The resulting supernatant (S) and pellet (P) fractions were analyzed using 10% PAGE and stained with Coomassie Brilliant Blue (A). The band intensities were then quantified using ImageJ software (B). White, gray, and black bars represent the data obtained under conditions 1, 2 and 3, respectively. Error bars indicate S.D. (n = 3).
FIGURE 4.
FIGURE 4.
Polymerization of mutant actins at different temperatures. WT, G156S, G156D, and G268D actins were incubated in F-buffer at 4, 15, 25, and 37 °C and ultracentrifuged. The resulting supernatant and pellet fractions were subjected to SDS-PAGE and quantified (A). B, electron micrographs of G156D, G156S, and G268D actins incubated in F-buffer at 4 or 37 °C. Scale bar, 100 nm.
FIGURE 5.
FIGURE 5.
Actins (5 μm) or G-buffer (control) were incubated in solution including 10 mm HEPES (pH 7.4), 2 mm MgCl2, 100 mm KCl, 1 mm EGTA, 0.25 mm DTT, and 0.5 mm ATP at 37 °C for 5 min or 1 h. Following acid quenching, the amounts of total Pi were determined by the malachite green method. Values shown are after subtracting Pi concentration at 0 min. Error bars indicate S.D. (n = 4–7).
FIGURE 6.
FIGURE 6.
Effects of mutant actin on the polymerization kinetics of WT actin. 1 μm WT actin (15% pyrene-labeled) was mixed with 1 μm WT or mutant actin, after which polymerization was initiated by adding 100 mm KCl and 2 mm MgCl2 at 23 °C. Polymerization was followed for 60–90 min by monitoring the pyrene fluorescence intensity.
FIGURE 7.
FIGURE 7.
Interaction between G63D actin and gelsolin and the effect of the gelsolin·G63D complex on polymerization of WT actin. A, gelsolin·WT actin complex (Gel·WT complex), gelsolin·G63D actin complex (Gel·G63D complex), or gelsolin alone was added to 1 μm RhPh-labeled rabbit skeletal muscle actin filaments (SK actin). The mixture was then diluted and visualized on HMM-coated coverslips using a fluorescence microscope. Scale bar, 10 μm. B, rates of WT actin elongation in the presence of Gel·G63D or Gel·WT complex were measured to examine the interaction between the barbed ends of WT actin and the pointed ends of G63D actin. 1 μm WT actin (15% pyrene-labeled) was allowed to polymerize in the presence of the following additives: gelsolin·G63D complex (filled triangles), gelsolin·WT actin complex (open circles), and G-buffer (open squares). Polymerization was followed by monitoring the pyrene fluorescence intensity for 60 min. The boxed area is enlarged in C.
FIGURE 8.
FIGURE 8.
Effects of R95C mutation on Tm·Tn function. Velocities of moving filaments (A) and percentage of filaments moving at uniform speeds (B) of RhPh-labeled WT, R95C filaments, and cofilaments over HMM surfaces were measured in the presence of Tm·Tn and various Ca2+ concentrations (pCa) at 25 °C. The Hill coefficient, pCa50, maximum velocity (Vmax (μm/s)), and maximum percentages of filaments moving at uniform speeds (Fmax (%)) were obtained by fitting each data set to the Hill equation (the parameters used are given in Table 2). Error bars indicate S.D. The differences of velocities and percentages of filaments moving at uniform speeds at pCa 6.7 between each pair of WT filaments and R95C filaments or cofilaments were significant (p < 0.05, t test). A significant fraction of filaments was not moving at intermediate pCa, and to avoid very large S.D., those stalled filaments were excluded from velocity analysis (30). However, this procedure resulted in excessive high velocity at high pCa if a very small number of filaments moved at velocities above the cutoff, even though the vast majority of the filaments had stalled. Therefore, those data points were excluded from fitting to the Hill equation, and zero values were used instead at those data points, which are shown in parentheses. Shown here is a representative of three independent experiments.
FIGURE 9.
FIGURE 9.
Effects of E226K mutation on Tm·Tn function. Velocities of moving filaments (A) and percentage of filaments moving at uniform speeds (B) of RhPh-labeled WT, E226K filaments, and cofilaments over HMM surfaces were measured in the presence of Tm·Tn and analyzed as in Fig. 8. The differences of the velocities and percentages of filaments moving at uniform speeds between cofilaments and WT filaments at pCa 6.7 were significant (p < 0.05, t test). The velocities (C) and percentages of filaments moving at uniform speeds (D) in the presence or absence of regulatory proteins shown in the bar graphs are those at pCa 5, except that the data in the presence of Tm alone was obtained in the absence of Ca2+.
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