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

项目摘要 胚胎细胞发育为形态发生和分化所需的专门化。对称性破坏通过改变RhoGT 3信号转导途径来影响许多细胞，从而触发PAR极性蛋白的不对称皮质定位。连接极性线索，RhoGT 3信号和PAR不对称性的分子联系知之甚少。利用C.作为一个简单的模型，该项目的长期目标是确定细胞接触信号如何调节RhoGTP酶，以诱导PAR不对称性，使细胞凋亡。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。鉴于细胞极性通路和调节剂的深度保守性，我们的研究将为人类关键接触介导的极化事件的机制提供新的见解。这些包括在压实过程中发生的胚胎卵裂球的极化，并且是内细胞团（其产生胚胎本身并且是胚胎干细胞的来源）的特化所需的;以及上皮细胞的极化，其是器官发生所需的并且是抑制肿瘤形成和侵袭所必需的。 在前一个奖项期间，我们定义了一个介导接触诱导的 通过在空间上改变RhoGTCDC 42的活性来极化卵裂球，RhoGTCDC 42是一种在细胞极性中具有古老和广泛保守作用的信号蛋白。我们发现，细胞接触招募保守的RhoGAP蛋白PAC-1/ARHGAP 10，其在接触位点使CDC-42失活。CDC-42在非接触表面保持活性，在那里它招募PAR蛋白PAR-3、PAR-6和PKC-3/aPKC，然后覆盖每个卵裂球。此外，我们已经获得的初步数据表明，CDC-42控制PAR-6和PAR-3的本地化通过不同的机制，CDC-42本地化PAR-3通过调节膜运输。该提案的目标是确定细胞间接触、PAC-1募集和CDC-42活性之间导致PAR-3不对称性的分子联系。我们的具体目标是：（1）确定卵裂球之间的细胞接触如何招募PAC-1来局部激活CDC-42;（2）确定在无接触表面激活CDC-42的机制;（3）检验CDC-42通过调节膜运输来控制PAR-3不对称性的假设。我们的实验将使我们能够建立一个分子途径，将细胞与细胞接触与CDC-42活性及其极性调节联系起来。我们期望我们的发现将为细胞极化的基本机制提供一般性的见解，并为细胞如何接触人类卵裂球和上皮细胞以促进胚胎发育和抑制肿瘤发生提供特定的分子见解

PROJECT SUMMARY Embryonic cells polarize to develop specializations needed for morphogenesis and differentiation. Symmetrybreaking cues polarize many cells by altering RhoGTPase signaling, which triggers the asymmetric cortical localization of PAR polarity proteins. The molecular links that connect polarity cues, RhoGTPase signaling, and PAR asymmetries are poorly understood. Using the C. elegans embryo as a simple model, the long-term goal of this project is to determine how cell contact cues regulate RhoGTPases to induce the PAR asymmetries that polarize 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. Given the deep conservation of cell polarity pathways and regulators, our studies will provide new insights into mechanisms of critical contact-mediated polarization events in humans. These include the polarization of embryonic blastomeres that occurs during compaction and is required for specification of the inner cell mass (which gives rise to the embryo proper and is the source of embryonic stem cells); and the polarization of epithelial cells that is needed for organogenesis and is essential for inhibiting tumor formation and invasion. In the prior award period, we defined a molecular pathway that mediates the contact-induced polarization of blastomeres by spatially altering the activity of the RhoGTPase CDC-42 ¿ a signaling protein with an ancient and broadly conserved role in cell polarity. We showed that cell contacts recruit the conserved RhoGAP protein PAC-1/ARHGAP10, which inactivates CDC-42 at contact sites. CDC-42 remains active at contact-free surfaces, where it recruits the PAR proteins PAR-3, PAR-6, and PKC-3/aPKC that then polarize each blastomere. In addition, we have obtained preliminary data suggesting that CDC-42 controls the localization of PAR-6 and PAR-3 through distinct mechanisms, and that CDC-42 localizes PAR-3 by regulating membrane trafficking. The goal of this proposal is to identify the molecular links between cell-cell contact, PAC-1 recruitment, and CDC-42 activity that lead to PAR-3 asymmetry. Our specific goals are (1) to determine how cell contacts between blastomeres recruit PAC-1 to locally inactivate CDC-42; (2) to identify the mechanisms that activate CDC-42 at contact-free surfaces; and (3) to test the hypothesis that CDC-42 controls PAR-3 asymmetry by regulating membrane trafficking. Our experiments will allow us to build a molecular pathway that connects cell-cell contact to CDC-42 activity and its regulation of polarity. We anticipate that our findings will provide general insights into the fundamental mechanisms of cell polarization, and specific molecular insights into how cell contacts polarize human blastomeres and epithelial cells to promote embryonic development and inhibit tumorigenesis