Mechanisms of Cell Polarity Establishment

描述（由申请人提供）：本项目的长期目标是了解哺乳动物细胞极化的信号转导途径的时空调控。极性的建立对后生动物发育的各个方面都至关重要。例如，将细胞命运决定子分离到干细胞的相反两极，再加上定向细胞分裂，允许特异性的子细胞表型。细胞极化也是形态发生、定向运动、抗原呈递和轴突引导所必需的;细胞极性的丧失是癌症进展中的关键步骤。值得注意的是，参与极化的新信号通路在后生动物进化过程中高度保守。Cdc 42与称为Par 6的蛋白质结合，Par 6与非典型蛋白激酶C（aPKC）结合。这些蛋白质形成一种复合物，调节许多类型的细胞极化。我们认为Par 6作为aPKC的靶向亚基，为磷酸化募集底物。已经发现了Par 6的三种推定效应子：Par 3、Lgl和Palsl。这3种蛋白质中的每一种都是上皮细胞极化所必需的，并且每一种都与也参与极化的其他几种蛋白质相互作用。这三组蛋白质（Par、帕尔斯和Lgl）在遗传和物理上相互作用，以产生定义极化上皮细胞的不同膜结构域，并且它们还参与其他类型的细胞极性。他们如何完成这些任务？需要解决的核心问题是：这些蛋白质如何相互关联和调节？它们如何在极化过程中定位到适当的位置（它们的目标线索是什么）？哪些蛋白质需要首先到达细胞连接处？他们是如何执行极化程序的？X射线晶体学将用于确定极性蛋白质复合物的结构。对于细胞生物学试验，MDCK细胞将用作已确立的、公认的模型系统。这些细胞形成高度极化的汇合单层，但响应于散射因子或创伤，它们失去其顶端/基底极性，并变得能动，具有前/后极性轴。将开发表达极性蛋白的荧光融合物的稳定的诱导型细胞系，并用于量化细胞连接组装和拆卸的动力学。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the spatial and temporal regulation of signal transduction pathways that underlie mammalian cell polarization. Polarity establishment is vital for every aspect of metazoan development. For example, the segregation of cell fate determinants to the opposite poles of a stem cell, coupled with oriented cell division, permits the specification of distinct daughter cell phenotypes. Cell polarization is also required for morphogenesis, directional motility, antigen presentation, and axon guidance; and the loss of cell polarity is a critical step in cancer progression. Remarkably, the novel signaling pathways involved in polarization have been highly conserved throughout metazoan evolution. Cdc42 binds to a protein called Par6, and Par6 binds to atypical protein kinases C (aPKC). These proteins form a complex that regulates many types of cell polarization. We propose that Par6 behaves as a targeting subunit for aPKC, recruiting substrates for phosphorylation. Three putative effectors for Par6 have been found: Par3, Lgl, and Pals1. Each of these 3 proteins is essential for epithelial cell polarization, and each interacts with several other proteins that have also been implicated in polarization. These three sets of proteins (Par, Pals, and Lgl) interact both genetically and physically to create the distinct membrane domains that define a polarized epithelial cell, and they also participate in other types of cell polarity. How do they accomplish these tasks? The central questions to be addressed are: how do these proteins associate with and regulate each other? How do they localize to their appropriate positions during polarization (what are their targeting cues)? Which proteins need to arrive first at the cell junctions? And how do they execute the polarization program? X-ray crystallography will be used to determine the structures of polarity protein complexes. For cell biological assays, MDCK cells will be used as an established, well-accepted model system. These cells form highly polarized confluent monolayers, but in response to scatter factor, or wounding, they lose their apical/basal polarity, and become motile, with an anterior/posterior polarity axis Stable, inducible cell lines will be developed that express fluorescent fusions of polarity proteins, and used to quantify the dynamics of cell junction assembly and disassembly.