Mechanisms of Cell Polarity Establishment

描述（由申请人提供）：该项目的长期目标是了解哺乳动物细胞极化信号转导通路的时空调节。极性的建立对后生动物发育的各个方面都至关重要。例如，细胞命运决定因素分离到干细胞的相反两极，再加上定向细胞分裂，允许明确的子细胞表型。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；而细胞极性的丧失是癌症发展的关键一步。值得注意的是，在后生动物的进化过程中，涉及极化的新信号通路一直高度保守。Cdc42与一种叫做Par6的蛋白质结合，而Par6与非典型蛋白激酶C （aPKC）结合。这些蛋白质形成一种复合物，调节多种类型的细胞极化。我们提出Par6作为aPKC的靶向亚基，招募底物进行磷酸化。已经发现了三种可能的Par6效应物：Par3、Lgl和pal1。这3种蛋白质中的每一种都是上皮细胞极化所必需的，并且每种蛋白质都与其他几种也与极化有关的蛋白质相互作用。这三组蛋白（Par、Pals和Lgl）在遗传和物理上相互作用，形成不同的膜结构域，定义极化上皮细胞，它们也参与其他类型的细胞极性。他们是如何完成这些任务的？要解决的核心问题是：这些蛋白质是如何相互联系和调节的？它们如何在极化过程中定位到合适的位置（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计划的？x射线晶体学将用于确定极性蛋白质复合物的结构。对于细胞生物学分析，MDCK细胞将被用作一个已建立的、被广泛接受的模型系统。这些细胞形成高度极化的融合单层，但在散射因子或损伤的作用下，它们失去了顶端/基部的极性，并变得具有前/后极性轴的运动性，稳定的、可诱导的细胞系将被开发出来，表达极性蛋白的荧光融合，并用于量化细胞连接组装和拆除的动力学。

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.