MECHANISM OF SEGREGATION OF MODIFIED TUBULIN IN VIVO

这项研究的长期目标是理解 细胞骨架分化的机制，即 改变其在细胞活动中的作用的细胞骨架。特别是，一个 微管蛋白的可逆翻译后修饰涉及 在C末端添加或去除酪氨酸残基。上一首 实验室的研究已经确定酪氨酸化和 非酪氨酸化微管蛋白存在于不同的人群中 培养细胞中的大管。这个项目的目标有三个方面： (1)确定非酪氨酸(Glu)的分布是否不对称 微管中酪氨酸(Tyr)微管蛋白广泛存在； (2)确定谷氨酸和酪氨酸微管蛋白是否分离 进入单独的微管似乎与有丝分裂或细胞有关 转变；以及(3)确定这两者的机制 翻译后修饰的微管蛋白在 活着。几种类型的培养细胞将用多肽进行检测 针对Tyr和Glu微管蛋白的抗体。参与有丝分裂的细胞 将进行光镜和电子显微镜免疫定位检查 为了确定哪组有丝分裂微管包含哪种 α-微管蛋白的种类。SV40病毒在两种细胞系中的转化 将被用作细胞骨架异常的例子 转型。在Tyr和Glu微管蛋白不对称的每种情况下 微管的观察、定位和任何可能的功能 包含每一个都会被注意到。要了解它的意义和 获得观测到的轮胎和轮胎差异性分布的机理 谷氨酸微管蛋白、微管在体内和裂解细胞中会解聚 模特们。在几种条件下重新聚合将导致 Glu和Tyr微管蛋白的分布不同或相当。几个 谷氨酸和酪氨酸微管蛋白在体内分离的可能机制 因此，可以测试单独的微管。 由于微管功能异常是癌症不可或缺的一部分 和其他疾病，试图发现其本地化的控制 在细胞分裂、分化和转化中的作用是 显然很重要。(L)

The long-range goal of this research is to understand the purpose and mechanism of a cytoskeletal differentiation, that is, modifications in the cytoskeleton which alter its role in cellular events. In particular, a reversible post-translational modification of a tubulin involves the addition or removal of a tyrosine residue at the C terminus. Previous research in the laboratory has determined that tyrosinated and nontyrosinated tubulin are contained in different populations of macrotubules in cultured cells. The aims of this project are three-fold: (1) to determine whether asymmetry of distribution of nontyrosinated (Glu) and tyrosinated (Tyr) tubulins in microtubules is of widespread occurrence; (2)\to ascertain whether or not the segregation of the Glu and Tyr tubulins into separate microtubules appears to be involved in mitosis or cell transformation; and (3) to determine the mechanism by which these two post-translationally modified tubulins become differentially distributed in vivo. Several types of cultured cells will be examined using peptide antibodies specific for Tyr and for Glu tubulin. Cells engaged in mitosis will be examined with light and electron microscopic immunolocalization in order to determine which population of mitotic microtubules contain which species of alpha-tubulin. Transformation by SV40 virus in two cell lines will be used as an example of cytoskeletal abnormalities induced by transformation. In each case in which asymmetry of Tyr and Glu tubulin is observed, the location and any putative functions of microtubules containing each will be noted. To learn about the significance and the mechanism of obtaining the observed differential distribution of Tyr and Glu tubulin, microtubules will be depolymerized in vivo and in lysed cell models. Repolymerization under several conditions will result in differential or equivalent distributions of Glu and Tyr tubulin. Several possible mechanisms for the segregation of Glu and Tyr tubulins into separate microtubules can thus be tested. Since the abnormal function of microtubules is an integral part of cancer and other diseases, attempts to discover the control of their localization and function in cell division, differentiation, and transformation is of obvious importance. (L)