Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.
Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.
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培养的成纤维细胞中细胞肌肉和细胞 - 细胞接触部位的免疫电子显微镜研究。
DOI：
10.1083/jcb.95.1.205
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发表时间：
1982-10
期刊：
影响因子：
--
通讯作者：
Singer SJ
中科院分区：
文献类型：
--
作者：
Chen WT;Singer SJ
我们的目的是获得有关培养的成纤维细胞形成的细胞-基质和细胞-细胞接触位点的分子结构的信息。我们对切过这些接触位点的超薄冷冻切片进行了双重免疫电镜标记实验，以确定纤连蛋白、纽蛋白和α-肌动蛋白这三种蛋白质相对于这些位点的绝对和相对配置。 (a) 塑料切片中常见的三种类型的细胞-基质和细胞-细胞接触位点也可以在冷冻切片中仅通过形态学标准来区分，即两个表面之间的间隙距离和膜下密度的存在。这些类型是：（i）粘着斑（FA）； (ii) 密切接触者 (CC)； (iii) 细胞外基质接触 (ECM)。这种接触位点的形态学分型使我们能够识别三种蛋白质的独特免疫标记模式并将其分配给冷冻切片上每种类型的位点。 (b)在细胞内对纽蛋白和α-肌动蛋白的FA位点进行免疫标记，其中纽蛋白标记比α-肌动蛋白更靠近膜。纤连蛋白在细胞表面和基质之间或两个细胞之间的 FA 位点之间的狭窄间隙中没有被标记。对照实验表明，这不能归因于免疫标记试剂无法接近 FA 窄间隙，但表明这些位点纤连蛋白不存在或严重耗尽。 (c) CC位点在细胞内标记为α-辅肌动蛋白，但没有标记粘蛋白，并在细胞外标记为纤连蛋白。 (d) ECM位点的特征是细胞和基质之间或两个细胞之间的大间隔（通常大于100 nm），它们通过细胞外基质成分（包括纤连蛋白）的长电缆连接。在晚期（24-36 小时）培养中，ECM 接触比其他类型占主导地位。 ECM 位点似乎有两种，一种在细胞内标记 α-辅肌动蛋白和纽蛋白，另一种仅标记 α-辅肌动蛋白。 (e)根据这些和其他结果，为这些接触位点的分子超微结构提出了一个连贯但暂定的方案，并提出了纤连蛋白、纽蛋白和α-辅肌动蛋白在细胞粘附和细胞内微丝与不同类型接触位点的膜的连接中的特定功能作用。
Our object was to obtain information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured fibroblasts. We have carried out double immunoelectron- microscopic labeling experiments on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the three proteins fibronectin, vinculin, and alpha- actinin with respect to these sites. (a) Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the two surfaces, and the presence of submembranous densities. These types were: (i) focal adhesions (FA); (ii) close contacts (CC); and (iii) extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed us to recognize and assign distinctive immunolabeling patterns for the three proteins to each type of site on the frozen sections. (b) FA sites were immunolabeled intracellularly for vinculin and alpha- actinin, with vinculin labeling situated closer to the membrane than alpha-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum, or between two cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites. (c) CC sites were labeled intracellularly for alpha-actinin but not vinculin and were labeled extracellularly for fibronectin. (d) ECM sites were characterized by large separations (often greater than 100 nm) between the cell and substratum or between two cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of two kinds, one labeled intracellularly for both alpha-actinin and vinculin, the other for alpha-actinin alone. (e) From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin, and alpha-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.