Expression of Transfected Mutant β-actin Genes: Alterations of Cell Morphology and Evidence for Autoregulation in Actin Pools

Abstract

Two different mutant human β-actin genes have been introduced into normal diploid human (KD) fibroblasts and their immortalized derivative cell line, HuT-12, to assess the impact of an abnormal cytoskeletal protein on cellular phenotypes such as morphology, growth characteristics, and properties relating to the neoplastic phenotype. A mutant β-actin containing a single mutation (Gly-244→Asp-244) was stable and was incorporated into cytoskeletal stress fibers. Transfected KD cells which expressed the stable mutant β-actin in excess of normal β-actin were morphologically altered. In contrast, a second mutant β-actin gene containing two additional mutations (Gly-36→Glu-36 and Glu-83→Asp-83, as well as Gly-244→Asp-244) did not alter cell morphology when expressed at high levels in transfected cells, but the protein was labile and did not accumulate in stress fibers. In both KD and HuT-12 cells, endogenous β- and γ-actin decreased in response to high-level expression of the stable mutant β-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous β- and γ-actins were equal, the ratio of net β-actin (mutant plus normal) to γ-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant β-actin accumulated in stress fibers but did not participate in the formation of the actin filament-rich perinuclear network. These observations suggest that different intracellular locations differentially incorporate actin into cytoskeletal microfilaments. The dramatic impact on cell morphology and on β-actin/γ-actin ratios in the transfected diploid KD cells may be related to the acquisition of some of the characteristics of cells that underwent the neoplastic transformation event that originally led to the appearance of the β-actin mutations.