Microtubule dynamics during cell polarity and migration

描述（由申请人提供）：细胞骨架的极性对于许多细胞行为至关重要，包括伤口愈合期间的定向迁移和趋化性。微管聚合动力学在平面极化的迁移细胞中受到时空调节，并且是细胞定向迁移所需的。本提案的总体目标是确定微管在建立和维持平面极性和上皮细胞定向片迁移过程中的功能机制，重点是CLASP家族+TIPs的功能和动力学。+TIPs是一组异质性蛋白质，由它们动态定位于细胞中生长的微管+末端来定义。+TIPs是在上皮片迁移过程中介导微管功能的主要候选者，因为它们可以为微管末端与皮质细胞骨架的调节性相互作用提供界面。目前的研究集中在CLASPs，因为它们与其他+TIPs的区别在于它们在迁移上皮细胞中与微管的时空调节关联。尽管CLASP跟踪细胞体中的微管+末端，但与其他+TIPs相反，CLASP在迁移上皮细胞的板层中沿着沿着微管缔合。这种协会的CLASPs沿着板层微管减少，通过磷酸化糖原合成酶激酶3？(GSK3?),一个新兴的细胞极性通路整合者。因为GSK 3？被认为在迁移细胞的前部局部失活，预测细胞体中的CLASP-磷酸化降低CLASP-微管亲和力，而非磷酸化的CLASP与板层微管的缔合稳定这些微管。该提议的具体假设是CLASP介导的板层微管稳定化和这些微管与皮质细胞骨架的相互作用对于迁移上皮细胞的持续平面极性是必不可少的。这一假设将通过生物化学方法和先进的活细胞显微镜在克隆人角质形成细胞中进行检验。在目标1中，GSK 3？将在细胞和体外分析依赖性CLASP磷酸化，并测试CLASP与微管或相关蛋白的相互作用是否受磷酸化调节。在目标2中，CLASP介导的时空调控微管聚合动力学将在平面极化，迁移细胞中进行分析，并在体外与纯化的成分。将测试CLASP是否直接或间接调节微管动力学，以及这是否受GSK 3调节？磷酸化在目标3中，CLASP功能将通过RNA干扰在迁移的上皮细胞中被抑制，并且将通过使用活细胞旋转盘共聚焦显微镜分析板层突出、细胞-基质粘附和极性动力学来确定CLASP如何调节定向细胞迁移。 公共卫生相关性：集体细胞迁移对于许多形态发生运动和损伤后角质形成细胞穿过基底膜的片状迁移是必不可少的。此外，细胞迁移的异常调节有助于肿瘤细胞的转移，并且肿瘤细胞的集体迁移在肿瘤侵袭中起着至关重要的作用。因此，了解平面上皮细胞极性和定向迁移的分子机制对于开发用于角质形成细胞或其他癌以及用于创伤后皮肤再生的改进疗法至关重要。

DESCRIPTION (provided by applicant): Polarity of the cytoskeleton is essential for many cell behaviors, including directed migration during wound healing and chemotaxis. Microtubule polymerization dynamics are spatiotemporally regulated in planar polarized, migrating cells, and are required for cells to migrate directionally. The overall objective of this proposal is to determine the mechanisms by which microtubules function during establishment and maintenance of planar polarity and directed sheet migration of epithelial cells with an emphasis on the function and dynamics of the CLASP family of +TIPs. +TIPs are a heterogeneous group of proteins defined by their dynamic localization to growing microtubule plus ends in cells. +TIPs are prime candidates to mediate microtubule function during epithelial sheet migration because they may provide interfaces for regulated interactions of microtubule ends with the cortical cytoskeleton. The current study focuses on CLASPs because they are distinguished from other +TIPs by their spatiotemporally regulated association with microtubules in migrating epithelial cells. Although CLASPs track microtubule plus ends in the cell body, in contrast to other +TIPs CLASPs associate along microtubules in the lamella of migrating epithelial cells. This association of CLASPs along lamella microtubules is decreased through phosphorylation by glycogen synthase kinase 3? (GSK3?), an emerging integrator of cell polarity pathways. Because GSK3? is thought to be locally inactivated in the front of migrating cells, it is predicted that CLASP-phosphorylation in the cell body decreases CLASP-microtubule affinity, while association of non-phosphorylated CLASPs with lamella microtubules stabilizes these microtubules. The specific hypothesis of this proposal is that CLASP- mediated lamella microtubule stabilization and interactions of these microtubules with the cortical cytoskeleton are essential for persistent planar polarity of migrating epithelial cells. This hypothesis will be tested by biochemical methods and advanced live cell microscopy in clonal human keratinocyte cells. In Aim 1, GSK3?-dependent CLASP phosphorylation will be analyzed in cells and in vitro, and it will be tested whether CLASP interactions with microtubules or associated proteins are regulated by phosphorylation. In Aim 2, CLASP-mediated spatiotemporal regulation of microtubule polymerization dynamics will be analyzed in planar polarized, migrating cells, and in vitro with purified components. It will be tested whether CLASPs directly or indirectly regulate microtubule dynamics and whether this is regulated by GSK3? phosphorylation. In Aim 3, CLASP function will be inhibited in migrating epithelial cells by RNA interference, and it will be determined how CLASPs regulate directed cell migration by analyzing lamella protrusion, cell- matrix adhesion, and polarity dynamics using live cell spinning disk confocal microscopy. PUBLIC HEALTH RELEVANCE: Collective cell migration is essential for many morphogenetic movements and the sheet migration of keratinocytes across the basement membrane after injury. In addition, abnormal regulation of cell migration contributes to metastasis of tumor cells, and collective migration of tumor cells plays a crucial role in tumor invasiveness. Understanding the molecular mechanisms underlying planar epithelial cell polarity and directed migration is thus crucial to developing improved therapies for keratinocyte or other carcinomas, and for skin regeneration after wounding.