Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network.
Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network.
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DOI：
10.1083/jcb.111.2.553
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发表时间：
1990-08
期刊：
影响因子：
--
通讯作者：
Evans RM
中科院分区：
文献类型：
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作者：
Sarria AJ;Nordeen SK;Evans RM
用含有小鼠乳腺肿瘤病毒激素反应元件的小鼠波形蛋白cDNA表达载体转染人细胞。用地塞米松诱导后小鼠波形蛋白的分布通过用对小鼠或人波形蛋白特异性的抗波形蛋白抗体的间接免疫荧光来检查。在稳定转染的HeLa细胞，其中含有波形蛋白细丝，除了地塞米松导致小鼠波形蛋白在离散的区域，通常是核周，总是对应于人类的细丝网络的区域与最强的荧光最初出现。在加入地塞米松后20 h内，小鼠和人波形蛋白免疫荧光模式相同。然而，在稳定转染的MCF-7细胞中，缺乏波形蛋白丝，诱导小鼠波形蛋白合成导致波形蛋白丝组装在整个细胞质中，没有任何明显的局部浓度。瞬时表达实验SW-13细胞亚克隆，无论是缺乏或含有内源性波形蛋白丝产生类似的结果与MCF-7和HeLa转染子，分别获得。用HeLa转染子进行进一步的实验，以跟踪在地塞米松撤除后合成下降后小鼠蛋白质的命运。小鼠波形蛋白特异性荧光最初从细胞的外周区域丢失，而最后可检测到的小鼠波形蛋白总是对应于具有最强荧光的人类细丝网络。这些研究是一致的波形蛋白丝在整个细胞质中的均匀组装，并表明，以前的观察极化或矢量组装从核周区的细胞中的更外围的地区可能是由于波形蛋白丝在免疫荧光显微镜的不均匀分布的外观。
Human cells were transfected with a mouse vimentin cDNA expression vector containing the hormone response element of mouse mammary tumor virus. The distribution of mouse vimentin after induction with dexamethasone was examined by indirect immunofluorescence with antivimentin antibodies specific for either mouse or human vimentin. In stably transfected HeLa cells, which contain vimentin filaments, addition of dexamethasone resulted in the initial appearance of mouse vimentin in discrete areas, usually perinuclear, that always corresponded to areas of the human filament network with the most intense fluorescence. Within 20 h after addition of dexamethasone, the mouse and human vimentin immunofluorescence patterns were identical. However, in stably transfected MCF-7 cells, which lack vimentin filaments, induction of mouse vimentin synthesis resulted in assembly of vimentin filaments throughout the cytoplasm without any obvious local concentrations. Transient expression experiments with SW-13 cell subclones that either lack or contain endogenous vimentin filaments yielded similar results to those obtained with MCF-7 and HeLa transfectants, respectively. Further experiments with HeLa transfectants were conducted to follow the fate of the mouse protein after synthesis had dropped after withdrawal of dexamethasone. The mouse vimentin-specific fluorescence was initially lost from peripheral areas of the cells while the last detectable mouse vimentin always corresponded to the human filament network with the most intense fluorescence. These studies are consistent with a uniform assembly of vimentin filaments throughout the cytoplasm and suggest that previous observations of polarized or vectorial assembly from a perinuclear area to more peripheral areas in cells may be attributable to the nonuniformly distributed appearance of vimentin filaments in immunofluorescence microscopy.