| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
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.
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