Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.
Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.
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转染突变β-肌动蛋白基因的表达：细胞形态的改变和肌动蛋白池中自动调节的证据。
DOI：
10.1128/mcb.7.7.2457-2466.1987
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发表时间：
1987
影响因子：
5.3
通讯作者：
Gunning,P
中科院分区：
文献类型：
--
作者：
Leavitt,J;Ng,SY;Aebi,U;Varma,M;Latter,G;Burbeck,S;Kedes,L;Gunning,P
已将两种不同的突变型人β-肌动蛋白基因引入正常二倍体人（KD）成纤维细胞及其永生化衍生细胞系HuT-12中，以评估异常细胞骨架蛋白对细胞表型（如形态学、生长特征和与肿瘤表型相关的性质）的影响。含有一个突变（Gly-244→Asp-244）的突变体β-actin是稳定的，并被掺入细胞骨架应力纤维。表达稳定的突变体β-肌动蛋白超过正常β-肌动蛋白的转染KD细胞在形态上发生改变。相比之下，含有两个额外突变（Gly-36→Glu-36和Glu-83→Asp-83，以及Gly-244→Asp-244）的第二个突变β-肌动蛋白基因在转染细胞中高水平表达时不会改变细胞形态，但蛋白质不稳定，不会在应力纤维中积累。在KD和HuT-12细胞中，内源性β-和γ-肌动蛋白响应于稳定突变体β-肌动蛋白的高水平表达而降低，其方式与肌动蛋白浓度的自动调节反馈一致。由于内源性β-肌动蛋白和γ-肌动蛋白的减少百分比相等，因此转染细胞中净β-肌动蛋白（突变体加正常）与γ-肌动蛋白的比率显著增加。能够区分突变体和正常表位的抗血清显示，突变体β-肌动蛋白在应力纤维中积累，但不参与富含肌动蛋白的核周网络的形成。这些观察结果表明，不同的细胞内位置差异纳入肌动蛋白的细胞骨架微丝。对转染的二倍体KD细胞中的细胞形态和β-肌动蛋白/γ-肌动蛋白比率的显著影响可能与获得经历肿瘤转化事件的细胞的一些特征有关，所述肿瘤转化事件最初导致β-肌动蛋白突变的出现。
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.