Microtubule dynamics during cell polarity and migration

描述(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、细胞内运输和细胞形状重塑。这些微管功能中的许多是由生长中的微管+末端的蛋白质相互作用所介导的。末端结合蛋白(EBS)直接识别生长中的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、带正电的蛋白质区域中的短的、保守的Sxip多肽基序与EBS结合。这些特征导致了30多个结构高度的 异质性+TIP，但细胞内+TIP功能仍不完全清楚。这个项目的一个长期目标是回答+TIP细胞生物学的基本的、尚未解决的问题：特定的+TIPS的分子功能是什么？微管+末端联合对这些功能的重要性是什么？不同的+TIP复合体如何在空间和时间上控制+TIPS介导特定的微管活动？基于包含Sxip基序的+TIPS的出人意料的多样性和在前一个资金时期的发现，本项目的一个中心假设是许多+TIPS作为适配器促进对EB阳性生长微管末端的空间和时间控制捕获，以极化微管依赖的活动，这通过提供细胞内受体来促进与EB覆盖的生长微管末端的特定相互作用，扩展了经典的搜索-捕获理论。目前的应用主要集中在与焦点粘连(FA)相关的卡环和其他+尖端的功能上，FA是一种多层大分子组装，介导细胞与细胞外基质(ECM)的动态相互作用。基于初步数据，卡环聚集在FA周围，将微管拴在FA上，促进FA周转，提出了一种新的机制，即FA相关的+TIP建立向FA的囊泡运输轨迹，促进局部ECM重塑，促进FA由外向内分解。这项应用中的实验将定义+TIPS和微管如何通过使用生化、细胞生物学和先进的活细胞成像方法来控制细胞-基质黏附重塑：目标1询问成长和成熟的FA如何产生信号，将特定的+TIPS招募到FA邻近的区域。目的2确定CLASPS捕获MTS并将其与FAs连接的分子机制，并研究一种新的EB调控机制。目的3研究局部胞吐如何促进FA的周转，通过开发一种高度创新的光控制细胞黏附底物，测试细胞基质释放是否足以触发FA分解，并分析在生理性3D环境中，+TIP介导的局部ECM重塑的后果。由于异常的细胞-基质相互作用导致肿瘤转移，而EBS在癌细胞中过表达表明+TIP活性增加，除了建立与+TIP功能相关的新范式外，我们的研究对理解病理细胞行为具有高度相关性。

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.