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

项目总结 胚胎细胞极化形成形态发生和分化所需的特化。对称性破坏信号通过改变RhoGTPase信号使许多细胞极化，从而触发PAR极性蛋白的不对称皮质定位。连接极性信号、RhoGTPase信号和PAR不对称性的分子链接还知之甚少。使用线虫胚胎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，以诱导导致细胞极化的PAR不对称。我们将胚胎学操作与细胞、生物和遗传工具相结合的能力，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因子的高度保守性，我们的研究将为人类接触介导的关键极化事件的机制提供新的见解。这些包括胚胎分裂球的极化，它发生在压实过程中，是指定内部细胞团所必需的(这会产生胚胎本身，是胚胎干细胞的来源)；以及上皮细胞的极化，这是器官发生所需的，对抑制肿瘤的形成和侵袭是必不可少的。 在之前的获奖期，我们定义了一种分子通路，它介导了接触诱导的 通过空间改变RhoGTP酶CDC-42的活性来极化卵裂球，RhoGTP酶CDC-42是一种信号蛋白，在细胞极性中具有古老而广泛保守的作用。我们发现细胞接触招募保守的RhoGAP蛋白PAC-1/ARHGAP10，该蛋白在接触部位失活CDC-42。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，然后极化每个卵裂球。此外，我们还获得了初步的数据，表明CDC-42通过不同的机制控制PAR-6和PAR-3的定位，CDC-42通过调节膜转运来定位PAR-3。该建议的目的是确定导致PAR-3不对称的细胞-细胞接触、PAC-1招募和CDC-42活性之间的分子联系。我们的具体目标是(1)确定裂球之间的细胞接触如何招募PAC-1来局部灭活CDC-42；(2)确定在非接触表面激活CDC-42的机制；以及(3)验证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条分子通路，将细胞间的接触与CDC-42的活性及其对极性的调节联系起来。我们预计，我们的发现将为细胞极化的基本机制提供一般性的见解，并为细胞接触如何极化人类卵裂球和上皮细胞以促进胚胎发育和抑制肿瘤的发生提供具体的分子见解。

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