Interactions between CLIP-170 and tubulin

描述(由申请人提供)：细胞组织的过程是从极化生长到多细胞发育的基本生物学过程。古德森实验室的长期目标是了解这个组织的起源。由于细胞组织的许多方面依赖于微管细胞骨架，这个最初弥漫的问题的主要部分可以归结为两个问题：a)微管细胞骨架本身是如何形态定义的？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)动力学和调节货物-MT相互作用的蛋白质。这项提议的重点是CLIP-170，这是一种进化上保守的MT结合蛋白，参与了这两个过程。Clip-170还具有一个耐人寻味的特性，可以动态本地化到不断增长的MT Plus末端。这项建议的目标是定义CLIP-170和MT之间的相互作用，CLIP-170如何与MT关联，其动态正端跟踪行为的机制和意义，以及它对MT动力学的精确影响。这些知识将有助于建立CLIP-170的功能和机制，有助于理解其他MT+端跟踪蛋白，并有助于确定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共编辑、荧光各向异性和表面等离子体共振，确定CLIP-170与不同构象的微管蛋白/MTS的亲和力。这项工作将确定哪些相互作用是生理相关的，并测试加端跟踪机制。2.通过在纯化系统和非洲爪哇提取物中进行体外MT动态不稳定性分析，确定CLIP-170对MT动力学的影响。这将定义CLIP-170的活性，并为进一步研究CLIP-170的调控和蛋白质伙伴建立一个系统。3.通过将标记的CLIP-170片段瞬时导入组织培养细胞并在体内检测其对MT+末端的跟踪能力，确定最小的正端追踪域。这将使我们能够建立CLIP-170结构和末端追踪活动之间的关系，以及对MT动力学的影响，并将为剖析涉及末端追踪行为的调控提供一个简化的系统。

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.