MICRO-INJECTION AND LOCALIZATION OF A 130K-PROTEIN IN LIVING FIBROBLASTS - RELATIONSHIP TO ACTIN AND FIBRONECTIN
MICRO-INJECTION AND LOCALIZATION OF A 130K-PROTEIN IN LIVING FIBROBLASTS - RELATIONSHIP TO ACTIN AND FIBRONECTIN
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DOI：
10.1016/s0092-8674(80)80035-3
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发表时间：
1980-01-01
期刊：
影响因子：
64.5
通讯作者：
FERAMISCO, JR
中科院分区：
文献类型：
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作者：
BURRIDGE, K;FERAMISCO, JR
通过将荧光染料异硫氰酸荧光素 (FITC) 共价标记的蛋白质显微注射到活的培养哺乳动物细胞中，研究了从平滑肌中分离出的 130,000 道尔顿蛋白质（130K 蛋白质），其功能尚不清楚。还通过几种不同的方法研究了 130K 蛋白的分布，包括对显微注射 FITC-130K 蛋白的细胞进行双标记荧光显微镜检查，然后使用异硫氰酸罗丹明 (RITC) 标记的抗体通过间接免疫荧光对 130K 蛋白、肌动蛋白或纤连蛋白进行染色。显微注射 FITC-130K 蛋白 2-4 小时后，活成纤维细胞中的荧光可视化显示荧光蛋白的 2 个主要分布。其中之一在细胞下表面表现为明亮的焦点斑块，与盖革最近使用免疫荧光定位发现的分布相似。注射 FITC-130K 蛋白并在 2 小时后通过间接免疫荧光（RITC 抗体）对肌动蛋白染色的细胞进行双标记荧光显微镜检查，结果表明该焦点分布位于肌动蛋白微丝束的末端。观察到的注射蛋白质的第二个主要分布具有条纹或纤维状外观，更常见（但不总是）位于细胞上表面下方。使用抗纤连蛋白的抗体和双标记荧光显微镜，注射蛋白质的第二分布与这些细胞上表面纤连蛋白的分布密切相关。当纤连蛋白延伸到细胞之外时，130K 蛋白仍严格保留在细胞内，并且通常看起来集中在锚定点上。这些发现表明 130K 蛋白在膜附着位点的肌动蛋白丝组织中可能发挥作用，并证明可以通过将荧光标记的缀合物显微注射到活细胞中来研究功能未知的蛋白。
A 130,000 dalton protein (130K protein), isolated from smooth muscle and having an as yet unknown function, was studied through the use of microinjection of the protein covalently labeled with the fluorescent dye fluorescein isothiocyanate (FITC) into living cultured mammalian cells. The distribution of the 130K protein was also investigated by several different methods including double label fluorescence microscopy of cells microinjected with FITC-130K protein and subsequently stained for 130K protein, actin or fibronectin by indirect immunofluorescence with rhodamine isothiocyanate (RITC)-labeled antibodies. Visualization of fluorescence in living fibroblasts 2-4 h after microinjection of FITC-130K protein showed 2 major distributions of the fluorescent protein. One of these appeared as bright focal patches on the lower surface of the cells, and was similar to the distribution recently found by Geiger using immunofluorescent localization. Double label fluorescent microscopy of cells injected with FITC-130K protein and stained for actin 2 h later by indirect immunofluorescence (RITC-antibodies) demonstrated that this focal distribution was at the ends of actin microfilament bundles. A 2nd major distribution observed for the injected protein had a streaky or fibrillar appearance more commonly (but not always) located beneath the upper surface of the cell. Using antibodies against fibronectin and double label fluorescence microscopy, this 2nd distribution of the injected protein correlated closely with the distribution of surface fibronectin on these cells. Where the fibronectin extended beyond a cell, the 130K protein remained strictly cellular and often appeared concentrated as if in an anchorage point. These findings suggest a possible role for the 130K protein in the organization of actin filaments at membrane attachment sites, and demonstrate that proteins of unknown function can be studied by the microinjection of their fluorescently labeled conjugates into living cells.