MEMBRANE-MICROFILAMENT CONNECTIONS

最近的研究表明，与红细胞高度相关的分子 血影蛋白可能参与连接肌动蛋白细丝和血浆 膜存在于各种类型的细胞中。拟议研究的目标是 分析这些假定的连接分子的结构，并 阐明它们在膜-微丝相互作用中的作用。我们以前的 研究表明，存在一些截然不同的类似幽灵蛋白的分子。 这项拟议的研究将解决以下问题： 可能存在与幽灵蛋白相关的分子。相同与不同之处 这类分子的不同成员之间的关系将使用 生化、免疫学和电子显微镜工具。化学制品 这些蛋白质的“结构域”将由有限的胰酶消化产生， 经二维肽图谱分析的胰酶片段为 以及与单抗和常规抗体的反应。 结合抗体的旋转金属阴影电子显微镜 像幽灵蛋白一样的蛋白质将允许功能结构域在 双链细长结构。组织、细胞和亚细胞 这类蛋白质的单个成员的分布将是 免疫荧光显微镜和免疫印迹法研究 技巧。像幽灵蛋白一样的蛋白质附着在 将研究质膜，除了可能的调节外 它们的结构或功能由钙、钙调蛋白或磷酸化决定。 这些蛋白质的数量、分布或功能的改变 细胞的恶性转化也将被探索。这项研究 应该对膜-微丝连接产生重要的见解，以及 到目前为止，该区域在分子和特定相互作用方面定义不清。 由于质膜与微丝的相互作用被认为是 对许多重要的细胞功能至关重要，如细胞形状和 变形性、细胞运动、黏附和胞质分裂以及细胞内 移动性，这项研究应该帮助我们了解这些功能是如何 在正常细胞和恶性肿瘤细胞中均有表达。

Recent research has suggested that molecules highly related to erythrocyte spectrin may be involved in connecting actin filaments to the plasma membrane in a variety of cell types. The goal of the proposed research is to analyze the structure of these putative connecting molecules and elucidate their role in membrane-microfilament interactions. Our previous work suggested that a number of distinct spectrin-like molecules existed. The proposed research will address the question of how many different spectrin -related molecules there may be. Similarities and differences between different members of this class of molecules will be analyzed using biochemical, immunological and electron microscopic tools. Chemical "domains" of these proteins will be generated by limited tryptic digestion, and the tryptic fragments analyzed by two dimensional peptide mapping as well as by reactivity with monoclonal and conventional antibodies. Rotary metal shadowing electron microcopy of antibodies bound to spectrin-like proteins will allow functional domains to be "mapped" on the double stranded, elongated structure. The tissue, cellular and subcellular distribution of individual members of this class of proteins will be investigated by immunofluorescence microscopy and immunoblotting techniques. The manner in which spectrin-like proteins are attached to the plasma membrane will be studied, in addition to possible regulation of their structure or function by calcium, calmodulin or phosphorylation. Alterations in the amount, distribution or function of these proteins when cells are malignantly transformed will be explored as well. This research should yield important insights into membrane-microfilament connections, an area thus far ill-defined in terms of molecules and specific interactions. Since interaction of the plasma membrane with microfilaments is thought to be crucial to many important cellular functions such as cell shape and deformability, cell movement, adhesion and cytokinesis and intracellular mobility, this research should help us understand how these functions are mediated in normal and malignant cells.