Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

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 actin⅐gelsolin 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.Actin filaments are major components of the cytoskeletons of all eukaryotic cells and play key roles in a variety of cellular functions, including cell migration, organelle transport, and muscle contraction. These diverse processes depend on the dynamic nature of actin filaments and their interactions with various actin-binding proteins (1), which are thought to involve conformational changes in the subunits that make up actin filaments. For instance, the stability of actin filaments is affected by the conformational difference between actin subunits carrying ATP or ADP and those that do not (2, 3), whereas cofilin, a major actin filament severing protein, alters the twist of actin filaments (4). In addition, modification of actin filaments through chemical cross-linking causes motility defects with myosin, suggesting a possible link between actin-myosin interaction and the conformational dynamics of actin (5, 6). Moreover, the myosin-induced conformational changes appear to be cooperative (7-9), e.g. Ca 2ϩ -triggered conformational changes in the skeletal muscle actin filaments are enhanced by cooperative conformational changes induced by myosin (10). In many cases, however, details of the molecular relationship between conformational changes in actin and its physiological function remain unclear.Actin is a highly conserved protein, so that Dictyostelium actin 15 shares 91% amino acid identity with rabbit skeletal muscle actin. This implies ...

A Periodic Pattern of Evolutionarily Conserved Basic and Acidic Residues Constitutes the Binding Interface of Actin-Tropomyosin

“…Pure fractions were combined, concentrated using an Amicon concentrator, and dialyzed into G buffer (5 mM Tris, pH 8.26 at 4°C, 0.2 mM CaCl 2 , 0.1 mM NaN 3 , 0.5 mM DTT, 0.2 mM Na 2 ATP, 1 g/ml leupeptin). The tag was cleaved off of the actin, and the tag was separated from actin on a MonoQ column, essentially as described in Noguchi et al (26). Pure actin was dialyzed against G-buffer for 2 days, clarified, and polymerized by the addition of 2 mM MgCl 2 and 0.1 mM KCl.…”

“…Human vascular smooth muscle actins (ACTA2) were tagged at the C terminus with a His 6 , using the method described in Noguchi et al (26). Infected Sf9 cells were harvested after 72 h and lysed in 1 M Tris-HCl, pH 7.5 at 4°C, 0.6 M KCl, 0.5 mM MgCl 2 , 4% Triton X-100, 1 mg/ml Tween 20, 0.5 mM Na 2 ATP, 1 mM DTT, 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 5 mM benzamidine, and 5 g/ml leupeptin for 2.5 h (40 ml/1 billion cells).…”

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

“…Dictyostelium cells expressing GFP-D11Q actin did not show noticeable defects in growth or cell morphology (data not shown). This is presumably because the relative content of GFP-D11Q actin was much less than that of endogenous actin, as was the case with other GFP-mutant actins (14).…”

“…Purification of Actin and Cofilin-Recombinant WT and mutant actins were purified as described previously (14). The concentration of actin was estimated using Advanced Protein Assay (Cytoskeleton, Denver, CO) using actin that was calibrated by absorption at 290 nm as the standard.…”

“…The fusion protein was purified and treated with chymotrypsin, which efficiently cleaved the protein immediately after the native final residue of actin, to separate the actin and thymosin moieties (13). This expression system was used to characterize dominant negative actin mutations (14) previously identified in Drosophila indirect flight muscle (2), as well as in our own genetic screens using yeast cells (3). These studies demonstrated the usefulness of dominant negative mutant actins and prompted us to characterize other dominant negative actin mutants.…”