Microtubule dynamics during cell polarity and migration

细胞极性和迁移过程中的微管动力学

• 批准号: 7614315
• 负责人: Torsten Wittmann
• 金额: $ 28.51万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7614315
• 关键词: Actins; Affect; Affinity; Basement membrane; Binding; Biochemical; Biological Assay; Carcinoma; Cell Culture Techniques; Cell Polarity; Cell-Matrix Junction; Cells; Centrosome; Chemotaxis; Complex; Confocal Microscopy; Cytoskeleton; Dominant-Negative Mutation; Epithelial; Epithelial Cells; Family; Glycogen Synthase Kinase 3; Human; In Vitro; Injury; Intracellular Transport; Kinetochores; Label; Lateral; Life; Maintenance; Mediating; Metabolic; Methods; Microscopy; Microtubule Polymerization; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Modeling; Molecular; Movement; Mutate; Neoplasm Metastasis; Pathway interactions; Phosphorylation; Phosphorylation Site; Play; Plus End of the Microtubule; Process; Proteins; RNA Interference; Regulation; Resolution; Role; Subcellular structure; Testing; Wound Healing; cell behavior; cell cortex; cell motility; improved; in vitro Assay; keratinocyte; migration; monolayer; neuronal cell body; public health relevance; research study; skin regeneration; spatiotemporal; tumor

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.

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，包括在伤口愈合和趋化过程中定向迁移。微管聚合动力学在平面极化，迁移细胞中进行时空调节，并且细胞是迁移方向迁移所必需的。该提案的总体目的是确定微管在平面极性建立和维持过程中的功能以及上皮细胞的定向板迁移，并着重于 +尖端的clasp家族的功能和动力学。 +尖端是一组异质的蛋白质，该蛋白质由它们的动态定位定义为生长的微管加上细胞中的末端。 +尖端是在上皮片迁移过程中介导微管功能的主要候选者，因为它们可能会为微管与皮质细胞骨架结束的调节相互作用提供界面。当前的研究重点是扣子，因为它们与其他 +尖端通过它们与微管在迁移的上皮细胞中的空间临时调节结合而区别。尽管clasps跟踪微管加上细胞体的结束，但与其他 +TIPS clasps沿着微管的其他 +TIPS链球菌相比，迁移的上皮细胞的片段中的微管。通过糖原合酶激酶3的磷酸化，这种沿着薄片微管的链球菌沿着薄片微管的关联降低了？ （GSK3？），一种新兴的细胞极性途径积分器。因为GSK3？人们认为在迁移细胞的正面被局部灭活，可以预测细胞体中的链磷酸化降低了clasp-微管的亲和力，而非磷酸化clasps与Lamella微管的缔合可以使这些微管稳定这些微管。该提议的特定假设是，扣式介导的薄片微管稳定性以及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性极性至关重要。该假设将通过生化方法和克隆人角质形成细胞中的晚期活细胞显微镜进行检验。在AIM 1中，将在细胞和体外分析GSK3？依赖性抗磷酸化，并且将测试是否通过磷酸化调节了与微管或相关蛋白的扣发相互作用。在AIM 2中，将在平面极化，迁移细胞和纯化成分的体外分析微管聚合动力学的扣紧介导的时空调节。它将测试clasps是直接还是间接调节微管动力学，以及这是否受GSK3调节？磷酸化。在AIM 3中，通过RNA干扰将抑制cLASP功能在迁移的上皮细胞中，并且将通过分析使用活细胞旋转磁盘共磁盘式显微镜来分析薄片的突出，细胞基质粘附和极性动力学来确定clasps如何通过分析薄片延伸，细胞基质粘附和极性动力学来调节定向细胞的迁移。 公共卫生相关性：集体细胞迁移对于许多形态发生运动和受伤后角质形成细胞在地下室膜上的薄片迁移至关重要。此外，细胞迁移的异常调节有助于肿瘤细胞的转移，肿瘤细胞的集体迁移在肿瘤侵袭性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发展改善的角质形成细胞或其他癌以及受伤后皮肤再生至关重要。