TROPOMYOSINS IN NORMAL AND TRANSFORMED CELLS

该建议旨在了解 原肌球蛋白的多种异构体在重组 原代培养的细胞发生致癌性转化后的微丝 细胞 我们已经证明，具有高Mr值的原肌球蛋白 被具有低Mr值的原肌球蛋白取代， 与各种培养的 大鼠细胞转化。 此外，我们还发现了两个新的 肌动蛋白捆绑蛋白控制低Mr 原肌球蛋白，看看原肌球蛋白结合的变化如何调节 细胞转化时微丝的组装。 使用 有限的蛋白水解，我们计划解剖肌动蛋白捆绑蛋白， 以及原肌球蛋白的那些。 我们将微量注射 新的肌动蛋白捆绑蛋白，高和低Mr原肌球蛋白，以及 单克隆抗体，进入活细胞，看看这两个肌动蛋白- 成束蛋白刺激或抑制低分子量的 原肌球蛋白到应力纤维的肌动蛋白束，以及 确定原肌球蛋白结合的变化是否改变了组织 微丝和微管。 最后，我们建议 对结构和分子进行深入分析 微丝和微管的组合，以确定是否 它们在细胞转化时发生变化。 我们将研究是否有这样的 这些变化在功能上与原肌球蛋白的变化有关 模式. 我们的长期目标是了解 原肌球蛋白的表达影响细胞形态的变化， 为什么它是转化表型的一个非常敏感的指标。

This proposal is aimed at understanding the molecular functions of the multiple isoforms of tropomyosin in the reorganization of microfilaments following oncogenic transformation of cultured cells. We have demonstrated that tropomyosins with high Mr values are replaced with tropomyosins with low Mr values concomitant with the morphological alterations associated with a variety of cultured rat cell transformations. Furthermore, we have found two new actin-bundling proteins control the binding of the low Mr tropomyosins, and see how changes in tropomyosin-binding regulate the assembly of microfilaments upon cell transformation. Using limited proteolysis we plan to dissect the actin-bundling proteins, as well as those of the tropomyosins. We will microinject the two new actin-bundling proteins, high and low Mr tropomyosins, and the monoclonal antibodies, into living cells to see if the two actin- bundling proteins stimulate or inhibit the binding of low Mr tropomyosins to the actin bundles of stress fibers, and to determine if changes in tropomyosin-binding alter the organization of microfilaments and microtubules. Finally, we propose to undertake an in depth analysis of the structures and molecular compasitions of microfilaments and microtubules to determine if they change upon cell transformation. We will examine if such changes are functionally related to the changes in tropomyosin patterns. Our long-term goal is to understand how differential expression of tropomyosin effects changes in cell morphology, and why it is a very sensitive indicator of the transformed phenotype.