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Interactions between CLIP-170 and tubulin

CLIP-170 和微管蛋白之间的相互作用

基本信息

批准号：
7266352
负责人：
HOLLY V GOODSON
金额：
$ 34.63万
依托单位：
UNIVERSITY OF NOTRE DAME
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2008-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The process of cell organization underlies fundamental biological processes ranging from polarized growth to multicellular development. The long-term goal of the Goodson laboratory is to understand the origins of this organization. Because many aspects of cell organization depend on the microtubule cytoskeleton, a major part of this initally diffuse problem can be reduced to two questions:: a) How is the microtubule cytoskeleton itself morphologically defined? b) How do other cellular components interact with microtubules? To answer these questions requires knowledge of the proteins that control microtubule (MT) dynamics and mediate cargo-MT interactions. This proposal focuses on CLIP-170, an evolutionarily conserved MT-binding protein that is involved in both processes. CLIP-170 also has the intriguing property of localizing dynamically to growing MT plus ends. The goal of this proposal is to define interactions between CLIP-170 and MTs how CLIP-170 associates with MTs, the mechanism and significance of its dynamic plus-end tracking behavior, and the precise effects it has on MT dynamics. This knowledge will establish CLIP-170 function and mechanism, aid in understanding other MT plus-end tracking proteins, and help define the regulation of MT growth. Our Specific Aims are to: 1. Determine the affinity of CLIP-170 for different conformational states of tubulin/MTs by biophysical techniques including MT cosedimentation, fluorescence anisotropy, and surface plasmon resonance. This work will establish which interactions are physiologically relevant and test plus-end tracking mechanisms. 2. Define the effect of CLIP-170 on MT dynamics by performing in vitro MT dynamic instability assays in purified systems and in Xenopus extracts. This will define CLIP-170 activity and establish a system for further study of CLIP-170 regulation and protein partners. 3. Define the minimal plus-end tracking domain by transiently transfecting tagged CLIP-170 fragments into tissue culture cells and assaying their ability to track MT plus ends in vivo. This will allow us to establish the relationships between CLIP-170 structure, plus end tracking activity, and effects on MT dynamics, and will provide a simplified system for dissecting regulation involved in plus end tracking behavior.

描述（由申请人提供）：细胞组织过程是从极化生长到多细胞发育等基本生物过程的基础。古德森实验室的长期目标是了解这个组织的起源。因为细胞组织的许多方面都依赖于微管细胞骨架，所以这个最初扩散的问题的主要部分可以简化为两个问题：a) 微管细胞骨架本身的形态如何定义？ b) 其他细胞成分如何与微管相互作用？要回答这些问题，需要了解控制微管 (MT) 动力学和介导货物与 MT 相互作用的蛋白质。该提案重点关注 CLIP-170，这是一种进化上保守的 MT 结合蛋白，参与这两个过程。 CLIP-170 还具有动态定位到不断增长的 MT plus 端的有趣特性。该提案的目标是定义 CLIP-170 和 MT 之间的交互，CLIP-170 如何与 MT 关联，其动态加端跟踪行为的机制和意义，以及它对 MT 动态的精确影响。这些知识将建立 CLIP-170 的功能和机制，帮助理解其他 MT 加端跟踪蛋白，并帮助定义 MT 生长的调节。我们的具体目标是： 1. 通过生物物理技术（包括 MT 共沉降、荧光各向异性和表面等离子共振）确定 CLIP-170 对微管蛋白/MT 不同构象状态的亲和力。这项工作将确定哪些相互作用具有生理相关性并测试加端跟踪机制。 2. 通过在纯化系统和非洲爪蟾提取物中进行体外 MT 动态不稳定性测定，确定 CLIP-170 对 MT 动态的影响。这将定义 CLIP-170 活性并建立一个系统来进一步研究 CLIP-170 调节和蛋白质伙伴。 3. 通过将标记的 CLIP-170 片段瞬时转染到组织培养细胞中并测定其在体内跟踪 MT 加端的能力来定义最小加端跟踪域。这将使我们能够建立 CLIP-170 结构、加上端跟踪活动和对 MT 动态的影响之间的关系，并将提供一个简化的系统来剖析涉及加上端跟踪行为的调节。