Identifying mechanisms of contact-mediated cell polarization - Resubmission

项目摘要 极性的建立对发育至关重要，从不对称的细胞分裂到组织的形成。 PAR极性蛋白的径向极化发生在早期哺乳动物胚胎中，并且是分离所必需的 胚胎和胚胎外的谱系。早期哺乳动物胚胎和上皮细胞使用细胞接触作为 建立极性的线索，但细胞接触线索和皮质PAR极性之间的分子联系 对细胞增殖的蛋白质仍知之甚少。梭当RhoGAP， PAC-1/ARHGAP 21被募集到细胞接触（作为对称性破坏线索）并使CDC-42失活， a Rho-GTdR，导致PAR蛋白从接触位点丢失。E-钙粘蛋白，一种嗜同性粘附 在许多不同的细胞类型中，接触介导的极性需要蛋白质。In C. E-cadherin在elegans中起作用， 通过帮助招募PAC-1与研究中心联系，在建立极性方面发挥指导作用。E-cadherin与 钙粘蛋白-连环蛋白复合物（CCC）的组成部分，以驱动极化，并与 未鉴定的钙粘蛋白非依赖性途径定位PAC-1。参与极性建立的蛋白质在 C.秀丽线虫是高度保守的，因此了解它们在这个遗传上易于处理的系统中的作用将提供 深入了解哺乳动物胚胎和上皮细胞中接触介导的极化。这个项目的首要目标是 建议是确定机制，细胞接触与顶基底极性内的细胞。到 为了实现这一点，我将确定E-cadherin和PAC-1之间的生物化学联系，将其招募到细胞 接触（目标1）。我假设保守的极性蛋白afadin（AFD-1）通过以下方式帮助PAC-1定位： 作为α-连环蛋白/HMP-1（E-钙粘蛋白的下游）和PAC-1之间的接头发挥作用。我来测试一下 假设使用CCC的遗传操作和免疫沉淀来鉴定蛋白质相互作用。我会 还使用质谱鉴定与AFD-1相互作用的蛋白质，以驱动PAC-1定位和潜在功能。 我将确定E-cadherin独立的途径，也定位PAC-1（目标2）。我假设另一个 极性蛋白直接（蛋白-蛋白相互作用）或间接（通过磷酸化）募集PAC-1 或膜曲率的变化）。我将检查通过酵母二号鉴定的候选蛋白质 杂交体与PAC-1的PH结构域相互作用（已知该区域对于没有E- 钙粘蛋白），因为它们在缺乏E-钙粘蛋白的情况下定位PAC-1的能力。我还会用质谱仪来鉴定 与PAC-1相互作用的蛋白质，有和没有E-钙粘蛋白。沿着磷酸化分析， 我们了解PAC-1如何在没有E-cadherin的帮助下定位。无偏EMS诱变筛选 E-钙粘蛋白空背景将有助于确定在缺乏E-钙粘蛋白的情况下PAC-1如何定位。这些 这些发现将为细胞接触信号如何影响胚胎和上皮细胞提供基本的见解。

PROJECT SUMMARY Polarity establishment is essential for development, from asymmetric cell division to the formation of tissues. Radial polarization of PAR polarity proteins occurs in early mammalian embryos and is required for separation of embryonic and extra-embryonic lineages. Early mammalian embryos and epithelial cells use cell contacts as cues for establishing polarity, but the molecular links between cell contact cues and the cortical PAR polarity proteins that polarize cells remain poorly understood. The C. elegans early embryo polarizes when the RhoGAP, PAC-1/ARHGAP21, is recruited to cell contacts (acting as the symmetry breaking cue) and inactivates CDC-42, a Rho-GTPase, resulting in the loss of PAR proteins from contact sites. E-cadherin, a homophilic adhesion protein, is required for contact-mediated polarity in many different cell types. In C. elegans, E-cadherin plays an instructive role in establishing polarity by helping to recruit PAC-1 to contact sites. E-cadherin acts with components of the cadherin-catenin complex (CCC) to drive polarization, and functions redundantly with an unidentified cadherin-independent pathway to localize PAC-1. The proteins involved in polarity establishment in C. elegans are highly conserved, so understanding their role in this genetically tractable system will provide insights into contact-mediated polarization in mammalian embryos and epithelia. The overarching goal of this proposal is to identify mechanisms that link cell contact with apicobasal polarity within the cell. To achieve this, I will identify the biochemical connections between E-cadherin and PAC-1 that recruit it to cell contacts (Aim 1). I hypothesize that the conserved polarity protein, afadin (AFD-1), aids in PAC-1 localization by functioning as a linker between a-catenin/HMP-1 (downstream of E-cadherin) and PAC-1. I will test this hypothesis using genetic manipulation of the CCC and immunoprecipitation to identify protein interactions. I will also use mass spec to identify proteins interacting with AFD-1 to drive PAC-1 localization and potentially function. I will identify the E-cadherin-independent pathway that also localizes PAC-1 (Aim 2). I hypothesize that another polarity protein recruits PAC-1, either directly (protein-protein interaction) or indirectly (through phosphorylation or changes in membrane curvature). I will examine candidate proteins that were identified through yeast two hybrid to interact with the PH domain of PAC-1 (the region known to be important for localization without E- cadherin) for their ability to localize PAC-1 in the absence of E-cadherin. I will also use mass spec to identify the proteins interacting with PAC-1 with and without E-cadherin. This, along with phosphorylation analysis, will allow us to understand how PAC-1 localizes without the aid of E-cadherin. An unbiased EMS mutagenesis screen in an E-cadherin null background will aid in determining how PAC-1 localizes in the absence of E-cadherin. These findings will provide fundamental insights into how cell contact cues can polarize embryos and epithelial cells.