Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.

Abstract

Two different mutant human j-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 ,-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 I(-actin in excess of normal ,-actin were morphologically altered. In contrast, a second mutant ,-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 ,-and y-actin decreased in response to high-level expression of the stable mutant I-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous I8-and y-actins were equal, the ratio of net 0-actin (mutant plus normal) to y-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant j-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 P-actin/y-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 j-actin mutations.Actin is a ubiquitous, highly abundant protein that is responsible for a variety of cellular activities, including motility and the structural properties of the cytoplasm. This protein, highly conserved in evolution, forms complex structures in all eucaryotic cells by a combination of selfpolymerization and interactions with a host of binding proteins (reviewed in reference 37). These complex interactions have been the subject of intense biochemical and structural study but have not yet been amenable to genetic analysis. Few structural mutations in actin are known, as might be expected for a protein whose actions are so central to normal cell morphology and function. The recessive flightless mutants of Drosophila melanogaster show a disordered structure of the sarcomeres (18, 31) and induction of stress proteins (15,18,31) in the indirect flight muscles of homozygotes in which mutant alleles of a tissue-specific actin are expressed (18,31).Human fibroblasts which express mutant P-actins have been generated by the neoplastic transformation of diploid KD fibroblasts in vitro and the isolation of stable focusderived, neoplast...

Autoregulation of Actin Synthesis in Hepatocytes by Transcriptional and Posttranscriptional Mechanisms

“…Alternatively, G-actin could activate or inactivate a regulatory protein which binds to a 5' regulatory element of the actin gene complex [13]. In line with the present study, down regulation of the endogenous actin synthesis was observed in human fibroblasts after transfection and expression of a mutant p-actin gene [36]. In contrast, transfection of mouse myoblasts with human P-actin and y-actin genes and consecutive expression resulted in a transcriptional down regulation of the endogenous actin synthesis in y-actin transfectants but not in p-actin transfectants [37].…”

Autoregulation of Actin Synthesis by Physiological Alterations of the G-Actin Level in Hepatocytes

“…ADP-ribosylated Gteins, appears to be under autoregulatory control on the act i n acts as a F-actin capping protein (12), thereby basis of the equilibrium between the monomeric and the increasing the cellular pool of monomeric G-actin (13). polymeric form (4)(5)(6)(7)(8). Under experimental conditions, In cultured rat hepatocytes, the stabilizing of F-actin by ! )…”

Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype.

“…As a major constituent of the cytoskeleton or myofilaments, it is essential for the maintenance of cell polarity and motility (Bretcher, 1991;Cooper, 1991;Levitt et al, 1987;Pollard and Goldman, 1993), intracellular transport (Kuznetsov et al, 1992), protein synthesis (Hesketh and Pryme, 1991;Negrutskii and Deutscher, 1991;Yang et al, 1990), enzymatic processes (Farwell et al, 1990;Hunt et al, 1990;Knull and Walsh, 1992;Sarndahl et al, 1989), and mRNA localization (Singer, 1992;Sundell and Singer, 1991;Yisraeli et al, 1990). In chicken embryonic fibroblasts and myoblasts, actin mRNA is highly localized at the leading lamellae, quite different from the distribution of mRNAs coding for either vimentin or tubulin (Lawrence and Singer, 1986).…”

“…As a major constituent of the cytoskeleton or myofilaments, it is essential for the maintenance of cell polarity and motility (Bretcher, 1991;Cooper, 1991;Levitt et al…”