Morphological and cell biological studies on the interaction between membrane protein and membrane skeletal proteins of the mature skeletal muscle with special reference to integrin.

首先，我们开发了一种针对整合素β-1D的多克隆抗体，该抗体在成熟骨骼肌中特异性表达。利用该抗体，我们研究了整合素和肌营养不良蛋白-肌营养不良聚糖复合物之间的空间关系，它们被认为是骨骼肌细胞外基板和细胞内膜骨架之间的两个主要螺栓。利用胶体金标记抗体的免疫电镜显示，代表整合素和营养不良蛋白的两种金颗粒的距离分布峰分别为20-40nm。考虑到抗体的大小，数据意味着这两个分子的位置非常接近。此外，抗整合素和抗歧糖聚糖（细胞外表位）的双标记实验表明，两种抗原在质膜上夹心贴壁。在神经肌肉连接处，整合素和肌营养不良蛋白，而不是肌营养不良蛋白，与培养成纤维细胞局灶接触处发现的蛋白一起共定位。这些蛋白包括talin， vinculin， paxillin和focal adhesion kinase。除了这些蛋白质外，spectrin还具有共定位。在共聚焦激光扫描显微镜下观察，它们均出现在交界区波谷的质膜上，而不在交界区波峰处。为了了解两者之间是否存在直接相互作用，我们使用洗涤剂溶解大鼠骨骼肌的轻微粒体部分进行了免疫沉淀实验。当我们用抗整合素沉淀时，检测到的是肌动蛋白，而不是肌营养不良蛋白。当我们用抗肌营养不良蛋白沉淀时，检测到的是肌动蛋白，而不是整合素。当与抗血管蛋白沉淀时，发现的是肌营养不良蛋白，而不是整合素。综上所述，整合素螺栓和糖酐-肌营养不良蛋白复合物可能通过肌动蛋白相互作用。

First we developed a polyclonal antibody against integrin β-1D which specifically expresses in mature skeletal muscles. Using this antibody, we studied spatial relationship between integrin and dystrophin-dystroglycan complex which are supposed to two major bolts between the extracellular basal lamina and the intracellular membrane skeleton in the skeletal muscle. Immunoelectron microscopy in which we utilized colloidal gold-labeled antibody revealed that the distribution peak of the distance of the two kinds of gold particles which represent integrin and dystrophin respectively were 20-40nm. Taking the size of the antibody into consideration, the data mean that the two molecules locate quite near to each other. In addition, double labeling experiment of anti-integrin and anti-dystroglycan (extracellular epitope) showed that two antigens apposed sandwiching the plasma membrane. In neuromuscular junction, integrin and dystrophin, not utrophin, colocalized together with proteins which are found at focal contact in cultured fibroblasts. Among these proteins are talin, vinculin, paxillin and focal adhesion kinase. In addition to these proteins, spectrin also colocalized. They were all found at the plasma membrane of the trough of the junctional regions and not at the crest, when observed by confocal laser scanning microscope. To know whether direct interaction exists between the two bolts, we performed immunoprecipitation experiments using detergent solubilized light microsome fraction of rat skeletal muscle. Actin, not dystrophin, was detected when we precipitate it with anti-integrin. When we precipitate it with anti-dystrophin, actin, not integrin, was detected. When precipitated with anti-vinculin, dystrophin, not integrin, was found. Taking all results into consideration, integrin bolt and dystroglycan-dystrophin complex may interact through actin.