Microtubule dynamics during cell polarity and migration

描述（由申请人提供）：微管是真核细胞中许多过程的核心动态聚合物，包括染色体分离、细胞内转运和细胞形状重塑。这些微管功能中的许多是由生长中的微管加端处的蛋白质相互作用介导的。末端结合蛋白（EBs）直接识别生长微管末端的结构特征。一组不同的蛋白质，称为+TIPs，通过内在无序的，带正电荷的蛋白质区域中的短的，保守的SxIP肽基序与EB结合。这些特征导致了三十多个结构高度的鉴定 异质性+TIP，但胞内+TIP功能仍然不完全理解。这个项目的长期目标是回答+TIP细胞生物学的基本的、未解决的问题：特异性+TIP的分子功能是什么？微管加末端缔合对这些功能有何重要性？+ TIP在空间和时间上是如何控制的，从而使不同的+TIP复合物介导特定的微管活动？基于含SxIP基序的+TIP的意外多样性和上一个资助期的发现，该项目的中心假设是许多+TIP作为衔接子，促进EB阳性生长微管末端在空间和时间上受控的捕获，以实现微管依赖性活动，其通过提供细胞内受体以促进与EB覆盖的生长微管末端的特异性相互作用来扩展经典的搜索和捕获理论。本申请集中于CLASP和与粘着斑（FA）相关的其他+TIP的功能，粘着斑是介导动态细胞与细胞外基质（ECM）相互作用的多层大分子组装体。基于CLASPs聚集在FA周围，将微管拴系到FA，并促进FA周转的初步数据，提出了一种新的机制，其中FA相关的+TIPs建立朝向FA的囊泡运输轨道，以促进局部ECM重塑并促进由外向内的FA拆卸。本申请中的实验将定义+TIPs和微管如何通过采用生物化学、细胞生物学和先进的活细胞成像方法来控制细胞-基质粘附重塑：目的1询问生长和成熟的FA如何产生将特异性+TIPs招募到邻近FA的区域的信号。目的2定义CLASPs捕获和连接MT与FA的分子机制，并研究EB调控的新机制。目的3询问局部胞吐作用如何促进FA周转，通过开发高度创新的光控细胞粘附底物来测试细胞基质释放是否足以触发FA分解，并分析在生理3D环境中上皮重塑期间+TIP介导的局部ECM重塑的后果。由于异常的细胞-基质相互作用有助于癌症转移，并且EB在癌细胞中过表达，表明+TIP活性增加，除了建立与+TIP功能相关的新范例之外，我们的研究与理解病理细胞行为高度相关。

DESCRIPTION (provided by applicant): Microtubules are dynamic polymers central to many processes in eukaryotic cells, including chromosome segregation, intracellular transport, and cell shape remodeling. Many of these microtubule functions are mediated by protein interactions at growing microtubule plus ends. End-binding proteins (EBs) directly recognize a structural feature of growing microtubule ends. A diverse group of proteins, called +TIPs, bind to EBs through short, conserved SxIP peptide motifs in intrinsically disordered, positively charged protein regions. These characteristics led to the identification of over thirty structurally highly heterogeneous +TIPs, but intracellular +TIP functions remain incompletely understood. A long-term goal of this project is to answer fundamental, unresolved questions of +TIP cell biology: What are molecular functions of specific +TIPs? How is microtubule plus-end-association important for these functions? How are +TIPs spatially and temporally controlled such that different +TIP complexes mediate specific microtubule activities? Based on the unexpected diversity of SxIP-motif-containing +TIPs and findings in the previous funding period, a central hypothesis of this project is that many +TIPs act as adaptors that promote spatially and temporally controlled capture of EB-positive growing microtubule ends to polarize microtubule-dependent activities, which extends the classic search-and-capture theory by providing intracellular receptors to facilitate specific interactions with EB-covered growing microtubule ends. The current application focuses on the function of CLASPs and other +TIPs that are associated with focal adhesions (FAs), multi-layered macromolecular assemblies that mediate dynamic cell interactions with the extracellular matrix (ECM). Based on preliminary data that CLASPs cluster around FAs, tether microtubules to FAs, and facilitate FA turnover, a novel mechanism is proposed in which FA-associated +TIPs establish vesicle transport tracks toward FAs to promote localized ECM remodeling and facilitate outside-in FA disassembly. Experiments in this application will define how +TIPs and microtubules control cell-matrix adhesion remodeling by employing biochemical, cell biological and advanced live cell imaging approaches: Aim 1 asks how growing and mature FAs generate a signal that recruits specific +TIPs to a zone adjacent to FAs. Aim 2 defines molecular mechanisms by which CLASPs capture and link MTs to FAs, and investigates a novel mechanism of EB regulation. Aim 3 asks how localized exocytosis promotes FA turnover, tests whether cell-matrix release is sufficient to trigger FA disassembly by developing a highly innovative light-controlled cell adhesion substrate, and analyzes the consequences of +TIP-mediated local ECM remodeling during epithelial remodeling in a physiological 3D environment. Because abnormal cell-matrix interactions contribute to cancer metastasis, and EBs are overexpressed in cancer cells indicating increased +TIP activity, in addition to establishing new paradigms relating to +TIP function, our studies are highly relevant to understanding pathological cell behavior.