High Level Expression of Transfected / 3-and 7-Actin Genes Differentially Impacts on Myoblast Cytoarchitecture

The impact of the human/3and 7-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human/~-actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human 3,-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The/3-actin transfectants displayed well-defined filamentous organization of actin; whereas the ,y-actin transfectants displayed a more diffuse organization of the actin cables. The role of the/3-actin protein in generating the enlarged cell phenotype was examined by transfecting a mutant form of the human/3-actin gene. Transfectant cells were shown to incorporate the aberrant actin protein into stress-fiber-like structures. High level expression of the mutant/3-actin produced decreases in cell surface area and disruption of the actin microfilament network similar to that seen with transfection of the ~/-actin gene. In contrast, transfection of another mutant form of the/~-actin gene which encodes an unstable protein had no impact on cell morphology or cytoarchitecture. These results strongly suggest that it is the nature of the encoded protein that determines the morphological response of the cell. We conclude that the relative gene expression of /3and 3,-actin is of relevance to the control of myoblast cytoarchitecture. In particular, we conclude that the /~and ~-actin genes encode functionally distinct cytoarchitectural information. TIN is one of the most abundant and highly conserved proteins in nature. Although its function is relatively well defined in the contractile apparatus of muscle cells, the functions of actin in nonmuscle cells has not yet been unequivocally established. Actin is implicated to play a fundamental role in a number of cellular activities such as maintaining cellular architecture, cell motility, and cytokinesis. However, it is only in yeast that a genetic demonstration of a role for actin in the organization, assembly, and function of the yeast cell surface has been provided (Shortle et al., 1984; Novick and Botstein, 1985). In mammals, at least six different actin isoforms are known, each encoded by a separate gene and they differ by <10% of their amino acid sequence (Erba et al., 1988; Gunning et al., 1983; Hamada et al., 1982; Ng et al., 1985; Vandekerckhove and Weber, 1978). The cytoplasmic/3and ~/-actin isoforms predominate in avian and mammalian nonmuscle cells. The two smooth muscle actins , and 7are found primarily in smooth muscle, whereas s-cardiac and or-skeletal actin are expressed in striated cardiac and skeletal muscle, respectively. Although the biological significance of multiple actin isoforms is not well understood, certain observations suggest functional differences among them (Vandekerckhove and Weber, 1978). Firstly, the expression of actins is tissue-specific and developmentally regulated (Bains et al., 1984; Erba et al., 1988; Garrels and Gibson, 1976; McHugh and Lessard, 1988; Paterson and Eldridge, i984; Shani et al., 1981; Sawtell and Lessard, 1989; EppenbergerEberhardt et al., 1990). Secondly, the existence of multiple actin isoforms within tissues and even within a single cell suggests functional differences (Bravo et al., 1981). Thirdly, the presence of a strict control of tissue-specific ratios of cytoplasmic fland ~-actins also suggests that/3and ~/-actin may have distinct biological roles (Otey et al., 1987; Erba et al., 1988). Even though the observations listed provide a compelling argument that different functions exist for these isoforms, there is still a considerable lack of experimental data to prove conclusively that the cell can distinguish between the actin isoforms. Recent experiments have shown subcellular sorting of actin isoforms (Craig and Pardo, 1983; Otey ct al., 1988; De Nofrio et al., 1989), which proves that at some level, a cell can discriminate between actin isoforms. However, the observation of preferential subcellular localization of actin isoforms does not necessarily prove that the isoforms are functionally distinct since two isoforms may be differentially localized and yet still be functionally equivalent. Indeed, mouse nonmuscle L cells were unable to discriminate between a-cardiac actin and their endogenous nonmuscle actins (Gunning et al., 1984). McKenna et al. (1985) similarly observed that both chicken fibroblasts and cardiac myocytes failed to discriminate between muscle and nonmuscle actins. O The Rockefeller University Press, 0021-9525/92/05/775/11 $2.00 The Journal of Cell Biology, Volume 117, Number 4, May 1992 775-785 775 on July 8, 2017 jcb.rress. org D ow nladed fom Thus, if the actin isoforms are functionally distinct, one must conclude that the differences are too subtle to be detected by previously used assays. The aim of this work was to establish a model system to enable us to evaluate the functional equivalence of nonmuscle actin genes. This system involved the transfection of human Band -y-actin genes into mouse C2 myoblasts. We succeeded in altering the relative expressions of/3and -y-actin which elicited different changes in myoblast morphology and architecture. Thus, we conclude that these two genes encode different architectural information. Materials and Methods Cell Culture and Transfection C2 cells originally isolated by Dr. D. Yaffe (Weizmann Institute, Israel) and subcloned in the laboratory of Dr. H. Blau (Stanford University, Stanford, CA) were grown in DMEM medium (Gibco Laboratories, Grand Island, NY) supplemented with 20% FCS (Commonwealth Serum Labs, Melbourne, Australia) and 0.5% chicken embryo extract (Flow Laboratories Australasia Pry. Ltd., North Ryde). DNA transfection and isolation of transfected clones was performed as described by Graham and van der Eb (1973). In brief, C2 cells (4-6 • 105 cells) seeded on 100-ram dishes were transfected with 10 ~g of cesium chloride-purified plasmid DNA by the calcium phosphate precipitation technique. Selection was started 24 h later with 450 ~tg G418/ml medium (G418-geneticin; Sigma Chemical Company, St. Louis, MO). As colonies were formed they were seeded and cultured in 24-well plates followed by 60-ram dishes and finally grown in 100-ram dishes. The constructs used for transfection included (a) the human wild type/~-actin gene; (b) the human single mutant form of the/~-actin gene; (c) the human triple mutant form of the/~-actin gene, all originally isolated as 14 kllobase EcoRl (Leavitt et ai., 1984) fragments and subcloned into pSV2-neo (Southern and Berg, 1982) which contain the recombinant gene conferring resistance to the neomycin analogue G418; (d) the human wild type ~-actin gene, a BamH1 fragment~ubcloned into pBR322 (Erha et al., 1988), which was cotransfected with 1 ~g of pSV2-neo; and (e) the plasmid