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丝和血浆 膜在各种类型的细胞。 拟议研究的目标是 来分析这些假定的连接分子的结构， 阐明它们在膜-微丝相互作用中的作用。 我们以前的 工作表明存在许多不同的血影蛋白样分子。 拟议的研究将解决有多少不同的 血影蛋白相关的分子 异同 这类分子的不同成员之间的关系将使用 生物化学、免疫学和电子显微镜工具。 化学 这些蛋白质的“结构域”将通过有限的胰蛋白酶消化产生， 胰蛋白酶片段通过二维肽图谱分析为 以及通过与单克隆和常规抗体的反应性。 结合抗体的旋转金属遮蔽电子显微镜 血影蛋白样蛋白将允许功能结构域被“映射”在 双链的细长结构。 组织细胞和亚细胞 这类蛋白质的单个成员的分布将是 通过免疫荧光显微镜和免疫印迹研究 技术. 血影蛋白样蛋白质附着在细胞表面的方式 质膜将被研究，除了可能的调节， 它们的结构或功能通过钙、钙调蛋白或磷酸化来实现。 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。 本研究 应该产生重要的见解膜微丝连接， 迄今为止，该领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 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、粘附和胞质分裂以及细胞内 流动性，这项研究应该帮助我们了解这些功能是如何 在正常和恶性细胞中介导。

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.