Mechanisms of Contact-Mediated Cell Polarizatioin in the C. elegans Embryo

描述（由申请方提供）：概述胚胎细胞分化为形态发生和分化所需的特化。利用C.作为一个简单的模型，我们的长期目标是了解早期胚胎细胞如何获得一个内外极性，调节原肠胚形成的细胞运动。许多类型的细胞在保守的PAR蛋白质形成不对称的皮质定位和调节下游极性效应物时发生突变。一个重要的和很大程度上尚未解决的问题是极性线索如何导致PAR不对称。这个建议的具体目标是了解细胞接触如何诱导PAR蛋白的内外不对称性，使早期胚胎细胞极化。我们将联合收割机胚胎学操作与细胞生物学和遗传学工具相结合的能力为在活胚胎中识别和表征这些机制提供了独特的机会。我们的研究应该提供洞察关键的接触介导的极性事件在人类，如压实所需的胚胎发育和器官发生所需的上皮细胞的apicobasal极化的早期胚胎细胞。我们已经确定了pac-1基因作为一个关键的调节器的内部-外部PAR不对称性。PAC-1含有一个RhoGAP结构域，预计可抑制RHO蛋白的活性，RHO蛋白是一个信号分子家族，包括细胞极性的重要调节因子。我们已经表明，皮质定位的RHO蛋白CDC- 42是所需的内部-外部PAR不对称性和GFP标记的PAC-1定位到内部，但不是外部皮质的细胞。由于这些发现，在这里，我们测试的假设，PAC-1控制内-外PAR不对称的空间调节RHO活动。具体来说，我们将描述PAC-1蛋白，确定PAC-1控制PAR本地化通过调节RHO蛋白的活性，并测试假设，即两个预测的相互作用的蛋白质-α-连环蛋白和Arf小G蛋白-需要PAC- 1的本地化或功能。最后，为了扩大我们对PAC-1极性通路的分子理解，我们将筛选PAR蛋白在早期胚胎细胞中不对称定位所需的新基因。总之，拟议的实验将帮助我们建立一个分子途径，从细胞接触到极化细胞的PAR蛋白的不对称定位。相关性：我们正在使用一种遗传模式生物来了解细胞接触如何导致胚胎细胞产生对其功能至关重要的极性。我们预计，我们的发现将有助于解释人类胚胎中的细胞如何发展对胚胎发育至关重要的极性。

DESCRIPTION (provided by applicant): Summary Embryonic cells polarize to develop specializations needed for morphogenesis and differentiation. Using the C. elegans embryo as a simple model, our long-term goal is to understand how early embryonic cells acquire an inner-outer polarity that regulates the cell movements of gastrulation. Many types of cells polarize when conserved PAR proteins develop asymmetric cortical localizations and regulate downstream polarity effectors. An important and largely unresolved question is how polarity cues lead to PAR asymmetry. The specific goal of this proposal is to understand how cell contacts induce an inner-outer asymmetry in PAR proteins that polarizes early embryonic cells. Our ability to combine embryological manipulations with cell biological and genetic tools provides a unique opportunity to identify and characterize these mechanisms in living embryos. Our studies should provide insights into critical contact-mediated polarity events in humans, such as the compaction of early embryonic cells required for embryonic development and the apicobasal polarization of epithelial cells needed for organogenesis. We have identified the pac-1 gene as a key regulator of inner-outer PAR asymmetry. PAC-1 contains a RhoGAP domain predicted to inhibit the activity of RHO proteins, a family of signaling molecules that includes important regulators of cell polarity. We have shown that the cortically localized RHO protein CDC- 42 is required for inner-outer PAR asymmetry and that GFP-tagged PAC-1 localizes to the inner but not outer cortex of cells. Because of these findings, here we test the hypothesis that PAC-1 controls inner- outer PAR asymmetry by spatially regulating RHO activity. Specifically, we will characterize the PAC-1 protein, determine if PAC-1 controls PAR localization by regulating the activity of RHO proteins, and test the hypothesis that two predicted interacting proteins-a-catenin and Arf small G proteins-are needed for PAC- 1 localization or function. Finally, to expand our molecular understanding of the PAC-1 polarity pathway, we will perform screens for new genes required for the asymmetric localization of PAR proteins in early embryonic cells. Together, the proposed experiments will help us build a molecular pathway from cell contacts to the asymmetric localization of PAR proteins that polarizes cells. Relevance: We are using a genetic model organism to learn how cell contacts cause embryonic cells to develop polarities essential for their function. We anticipate that our findings will help explain how cells in the human embryo develop polarities that are vital for embryonic development.