MECHANISMS OF MICROTUBULE AND MITOTIC SPINDLE ASSEMBLY

微管和有丝分裂纺锤体的组装机制

• 批准号: 2853632
• 负责人: LYNNE CASSIMERIS
• 金额: $ 31.14万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2853632
• 关键词: Chlamydomonas; HeLa cells; SDS polyacrylamide gel electrophoresis; Urodela; Xenopus oocyte; binding proteins; cell cycle; cell growth regulation; crosslink; electron microscopy; enzyme activity; guanosinetriphosphatases; immunofluorescence technique; microinjections; microtubules; mitotic spindle apparatus; molecular cloning; oncoproteins; polymerase chain reaction; protein biosynthesis; protein structure function; scintillation counter; thin layer chromatography; video microscopy; western blottings

Our long term goal is to understand how a cell builds the mitotic spindle necessary for chromosome movement. We have concentrated efforts on understanding how the cell regulates the microtubule assembly necessary for the construction of this structure. The proposed work focuses on several proteins which are likely key components regulating microtubule assembly: oncoprotein 18/stathmin (Op18), XMAP215, and a human protein related to XMAP215, called TOGp. Both Op18 and TOGp are over-expressed in human tumors but the consequences of over-expression are not known. Proteins related to XMAP215 and TOGp have been identified in yeasts and C. elegans; here mutations support the idea that this protein family functions to regulate both microtubule assembly and chromosome movement. Future studies of Op18, XMAP215 and TOGp will provide important basic information on microtubule assembly in vivo. Since many chemotherapies are based on halting or slowing cell division, a better understanding of mitotic mechanisms has considerable potential health benefits. The specific aim of the current proposal comprise structure/function studies to determine: (1) the domains in Op18 responsible for functional activities; (2) whether Op18 interacts directly with microtubules to promote microtubule turnover; (3) the structure of microtubules assembled with XMAP215 and the orientation of XMAP215 on the microtubule lattice; (4) the microtubule binding domains and assembly-promoting domains within TOGp; and (5) the contributions of XMAP215 and TOGp to rapid microtubule assembly in the cell. For aim 1, we will use in vitro assays to determine the effects of Op18 truncations on microtubule assembly, tubulin GTPase activity and binding to alpha or beta tubulin. We will also probe the activities of selected truncations in living cells. We will use probabilistic analysis to determine whether Op18 modifies the kinetic steps responsible for switches between microtubule growth and shortening and determine whether Op18 stimulates GTP hydrolysis within microtubules (Aim 2). Aim 3 will use microtubule co-pelleting assays, electron microscopy and domain specific antibodies to determine the structure of microtubules bound by XMAP215. For aim 4, fragments of TOGp will be used in in vitro assays to map functional domains. Lastly, for aim 5 we will probe XMAP215 and TOGp functions in vivo by either blocking protein function with antibodies or by increasing protein levels by overexpression. Changes in microtubule turnover will be measured by tubulin fluorescence photoactivation and redistribution.

我们的长期目标是了解细胞如何建立染色体运动所需的有丝分裂纺锤体。 我们集中精力理解细胞如何调节该结构构建所需的微管组装。 所提出的工作集中于几种可能是调节微管组装的关键成分：癌蛋白18/stathmin（OP18），XMAP215，以及与XMAP215相关的人蛋白，称为TOGP。 OP18和TOGP在人类肿瘤中均过表达，但过表达的后果尚不清楚。 与XMAP215和TOGP相关的蛋白质已在酵母和秀丽隐杆线虫中鉴定出来。这里的突变支持了这种蛋白质家族起作用以调节微管组装和染色体运动的想法。 OP18，XMAP215和TOGP的未来研究将提供有关体内微管组装的重要基本信息。 由于许多化学疗法是基于停止或放慢细胞分裂的基础，因此对有丝分裂机制的更好理解具有相当大的潜在健康益处。当前建议的具体目的包括结构/功能研究，以确定：（1）OP18中负责功能活动的域； （2）OP18是否与微管直接相互作用以促进微管周转； （3）与XMAP215组装的微管结构，以及在微管晶格上的XMAP215的方向； （4）togp中的微管结合域和组装促进域； （5）XMAP215和TOGP对细胞中快速微管组件的贡献。对于AIM 1，我们将使用体外测定来确定OP18截断对微管组件，微管蛋白GTPase活性以及与α或β微管蛋白的结合的影响。 我们还将探测活细胞中选定的截断的活性。 我们将使用概率分析来确定OP18是否修改了负责微管生长和缩短之间切换的动力学步骤，并确定OP18是否刺激微管内的GTP水解（AIM 2）。 AIM 3将使用微管的共插图测定，电子显微镜和特异性抗体来确定由XMAP215结合的微管的结构。 对于AIM 4，TOGP的片段将用于体外测定法以绘制功能域。 最后，对于AIM 5，我们将通过用抗体阻断蛋白质功能或通过过表达来探测体内的XMAP215和TOGP功能。 微管周转的变化将通过微管蛋白荧光光活化和重新分布来衡量。