THE MOLECULAR-ORGANIZATION OF MYOSIN IN STRESS FIBERS OF CULTURED-CELLS
THE MOLECULAR-ORGANIZATION OF MYOSIN IN STRESS FIBERS OF CULTURED-CELLS
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DOI：
10.1083/jcb.102.1.200
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发表时间：
1986-01-01
影响因子：
7.8
通讯作者：
DEMEY, J
中科院分区：
文献类型：
--
作者：
LANGANGER, G;MOEREMANS, M;DEMEY, J
使用鸡砂囊肌球蛋白、亚片段 1、轻链 20 和轻链肌球蛋白的抗体来观察培养鸡细胞应力纤维中的肌球蛋白。使用蛋白质印迹上的免疫金银染色对纯化的砂囊蛋白和总细胞裂解物测试抗体特异性。培养的鸡成纤维细胞和上皮细胞的免疫荧光显示，总肌球蛋白、亚片段 1 和轻链 20 的抗体具有相似的染色模式，而轻肌球蛋白的抗体则显示出明显不同的反应。使用间接和直接免疫金染色法对透化和固定的细胞研究这些抗体的电子显微镜分布。间接方法使我们能够描述肌球蛋白在应力纤维中的一般分布。然而，直接双免疫金标记提供了肌球蛋白分子的方向及其相对于α-肌动蛋白的定位的更详细信息：用与10-nm金偶联的抗体鉴定的α-肌动蛋白集中在应力纤维的致密体或电子致密带中，而肌球蛋白局限于中间的电子透明区域。根据与α-肌动蛋白组合使用的抗体，介入区域揭示了不同的染色模式：针对肌球蛋白的抗体（与非肌肉肌球蛋白的头部反应）和针对轻链20的抗体（均与5-nm金偶联）标记了与α-肌动蛋白相邻的两个相对条带，并且针对轻肌球蛋白的抗体（与轻链肌球蛋白偶联） 5 nm 金）标记单个中心区域。基于这些结果，我们得出结论，应力纤维中的肌球蛋白被组织成双极丝。
Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence of cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information of the orientation of myosin molecules and their localization relative to .alpha.-actinin: .alpha.-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with .alpha.-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to .alpha.-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.