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.

对成熟骨骼肌膜蛋白和膜骨骼蛋白之间相互作用的形态学和细胞生物学研究，特别是整合素。

• 批准号: 16590134
• 负责人: YORIFUJI Hiroshi
• 金额: $ 2.43万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590134/
• 关键词: integrin β1D; skeletal muscle; immunoelectron microscopy; immunoprecipitation; インテグリン β1D; 免疫組織化学; 免疫電顕; ジストロフィン; α-ジストログリカン

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.

首先，我们研制了一种针对整合素β-1D的多克隆抗体，该抗体在成熟骨骼肌中特异表达。利用这种抗体，我们研究了整合素和dystrophin-dystroglan复合体之间的空间关系，这两个复合体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。用胶体金标记抗体的免疫电子显微镜显示，分别代表整合素和抗肌营养不良蛋白的两种金粒子的距离分布峰在20-40 nm。考虑到抗体的大小，这些数据意味着这两个分子彼此非常接近。此外，抗整合素和抗肌营养不良蛋白多糖(胞外表位)的双重标记实验表明，两种抗原相对夹在质膜上。在神经肌肉交界处，整合素和dystrophin，而不是utroin，与培养的成纤维细胞中的局灶性接触蛋白共同定位。在这些蛋白质中有talin、vinculin、paxlin和粘着斑激酶。除了这些蛋白质外，幽灵蛋白也是共定位的。激光共聚焦扫描显微镜观察发现，它们均位于交界区谷部的质膜上，而不在顶端。为了了解两个螺栓之间是否存在直接相互作用，我们进行了免疫沉淀实验，使用洗涤剂增溶的大鼠骨骼肌轻微体部分。当我们用抗整合素沉淀肌动蛋白时，检测到的是肌动蛋白，而不是dystrophin。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。当用抗纽蛋白沉淀时，发现的是营养不良蛋白，而不是整合素。综上所述，整合素Bolt和dystrocan-dystrophin复合体可能通过肌动蛋白相互作用。