MECHANISMS OF MICROTUBULE AND MITOTIC SPINDLE ASSEMBLY

我们的长期目标是了解细胞如何构建染色体运动所必需的有丝分裂纺锤体。我们已经集中精力了解细胞如何调节这种结构所必需的微管组装。这项工作的重点是几种可能是调节微管组装的关键成分的蛋白质：癌蛋白18/stathmin (Op18), XMAP215，以及与XMAP215相关的人类蛋白质TOGp。Op18和TOGp在人类肿瘤中都过表达，但过表达的后果尚不清楚。与XMAP215和TOGp相关的蛋白已在酵母和秀丽隐杆线虫中发现；这里的突变支持了这个蛋白质家族调节微管组装和染色体运动的观点。未来对Op18、XMAP215和TOGp的研究将为体内微管组装提供重要的基础信息。由于许多化疗是基于停止或减缓细胞分裂，因此更好地了解有丝分裂机制具有相当大的潜在健康益处。当前提案的具体目标包括结构/功能研究，以确定：(1)Op18中负责功能活动的域；(2) Op18是否直接与微管相互作用促进微管周转；(3) XMAP215组装微管的结构和XMAP215在微管晶格上的取向；(4) TOGp内的微管结合域和组装促进域；(5) XMAP215和TOGp对细胞内微管快速组装的贡献。对于目的1，我们将使用体外实验来确定Op18截断对微管组装、微管蛋白GTPase活性以及与α或β微管蛋白结合的影响。我们还将探索活细胞中选定截断的活性。我们将使用概率分析来确定Op18是否改变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GTP水解（目的2）。Aim 3将使用微管共制粒试验、电子显微镜和结构域特异性抗体来确定XMAP215结合的微管结构。对于目标4，TOGp的片段将用于体外分析来绘制功能域。最后，对于目标5，我们将通过抗体阻断蛋白质功能或通过过表达增加蛋白质水平来探测XMAP215和TOGp在体内的功能。微管周转的变化将通过微管蛋白荧光光激活和再分配来测量。

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.