CROSSLINKING STUDIES MEMBRANE CYTOSKELETON INTERACTIONS

该项目的目标是识别和表征质膜 在培养的成纤维细胞中细胞骨架的锚定位点。 的方法 将使用HAHS，一种新的交联剂， 从最初标记的蛋白质到可及范围内的其他分子 一个反应性的群体。 相邻的分子可以被简单地识别出来 以及它们获得的放射性。 将使用两种互补的方法。 在一个，由内而外的血浆 膜将通过让细胞吞噬特定的 配体包被的珠子，然后重新分离珠子;然后HAHS标记的肌动蛋白， 胞衬蛋白和黏着斑蛋白将在膜上重构，光解， 并通过电泳分析以鉴定它们的锚定位点。 在 另一种，标记的蛋白质将被显微注射到活细胞中， 允许其并入细胞骨架结构中，然后光解， 分析了 实验将用正常细胞和转化细胞进行， 扩散与悬浮，快速分裂与生长停滞细胞， 鉴定参与致癌转化的蛋白质，锚定到 基质和生长控制。 细胞与纤连蛋白的相互作用也将通过标记进行研究 将纤连蛋白11.5k细胞结合片段与HAHS交联， 到细胞表面。 一旦确定，其位置、分布、调节、功能和 将研究这些蛋白质的结构。 特别有趣 蛋白质将被分离出来，并进一步研究其结构和功能。

The goal of this project is to identify and characterize plasma membrane anchorage sites for the cytoskeleton in cultured fibroblasts. The approach will use HAHS, a new crosslinking reagent, which transfers a radioactive moiety from an initially labelled protein to other molecules within reach of a reactive group. Neighboring molecules can then be identified simply and unambiguously by their acquired radioactivity. Two complementary methods will be used. In one, inside-out plasma membranes will be purified by letting cells phagocytose specific ligand-coated beads, then reisolating the beads; then HAHS-labelled actin, fodrin and vinculin will be reconstituted onto the membranes, photolyzed, and analyzed by electrophoresis to identify their sites of anchorage. In the other, labelled proteins will be microinjected into living cells, allowed to incorporate into cytoskeletal structures, then photolyzed and analyzed. Experiments will be done with normal vs. transformed cells, well spread vs. suspended, and rapidly dividing vs. growth arrested cells to identify proteins involved in oncogenic transformation, anchorage to the substratum and growth control. The interaction of cells with fibronectin will also be studied by labelling the fibronectin 11.5 k cell-binding fragment with HAHS, and crosslinking it to the cell surface. Once identified, the location, distribution, regulation, function and structure of these proteins will be investigated. Particularly interesting proteins will be isolated and their structure and function studied further.