Regulation of Adherens Junction Trafficking by Polarity Proteins

描述(申请人提供)：极性蛋白质对黏附连接动力学的调节。细胞连接的关键成分，如E-钙粘附素和Claudins，是主要通过囊泡运输运输和调节的跨膜蛋白。在极化上皮细胞中，通过运输调节连接复合体的动力学可能是建立顶端-基底端极性的关键。在这里，我们提出了由极性蛋白Stardust(SDT)、Crumbs(CRB)和致死巨型幼虫(LGL)控制附着连接(AJ)动力学是建立顶端-基底极性的一个基本的细胞机制。为了验证这一假设，我们在果蝇中开发了一种新的基因组工程方法，使我们能够将目标基因修改为任何所需的突变等位基因。通过这种基因组工程方法，我们已经产生了多个经过遗传验证的AJ标记，如E-钙粘附素：：GFP，用于定量分析果蝇上皮细胞体内的AJ动态。在这个提案中，我们将首先描述AJ动力学，如生物合成、降解和局部(亚细胞)周转的速率，如何在顶端-基础极化过程中受到不同的调节，以及极性蛋白SDT、CRB和LGL如何在这种AJ动力学中发挥主要调节作用。通过同时通过顺式和反式调节机制操纵AJ动力学，我们将确定在顶端-基础极性发育过程中调节的AJ动力学是否对建立极性是必要的。然后，我们将探索SDT/CRB络合物控制AJ动力学和极性的潜在分子机制。我们将重点讨论SDT/CRB通过调节aPKC介导的DE-Cad磷酸化和/或通过调节Moesin调节的肌动蛋白网络来控制AJ动力学的假设。最后，我们建议利用基因验证的GFP和基因组工程产生的高亲和力表位敲入等位基因，通过蛋白质组学方法系统地鉴定DE-Cad、CRB、LGL和SDT在细胞极化中作用的新机制。实现这一建议中的特定目标将建立AJ动力学的调节，作为SDT、CRB和LGL控制上皮细胞尖底极化的关键分子和细胞机制。 公共卫生相关性：果蝇是解决人类疾病中关键生物学问题的领先遗传模型系统。我们对细胞极性和E-钙粘附素/粘附素连接动力学的研究将为癌症的分析和治疗提供新的机制，为肿瘤细胞在肿瘤进展和转移过程中如何破坏细胞黏附和极性提供新的机制。

DESCRIPTION (provided by applicant): Regulation of Adherens Junction Dynamics by Polarity Proteins. Key components of cell junctions such as E-Cadherin and claudins are transmembrane proteins that are delivered and regulated primarily through vesicle trafficking. In polarizing epithelial cells, regulating the dynamics of junctional complex through trafficking may be crucial for establishing the apical- basal polarity. Here we propose that control of adherens junction (AJ) dynamics by polarity proteins Stardust (Sdt), Crumbs (Crb) and Lethal giant larvae (Lgl) is an essential cellular mechanism for establishing apical-basal polarity. To test this hypothesis, we have developed a novel genomic engineering approach in Drosophila that enables us to modify a target gene into any desired mutant alleles. By this genomic engineering approach we have generated multiple genetically validated AJ markers such as E-Cadherin::GFP for quantitatively assaying the AJ dynamics in vivo in Drosophila epithelial cells. In this proposal, we will first characterize how AJ dynamics, such as the rates of biosynthesis, degradation, and local (subcellular) turnover, may be differentially regulated during apical- basal polarization, and how polarity proteins Sdt, Crb and Lgl play major regulatory roles in such AJ dynamics. By manipulating the AJ dynamics through both cis- and trans-regulatory mechanisms, we will identify whether regulated AJ dynamics during apical-basal polarity development is essential for establishing the polarity. We will then explore the potential molecular mechanisms by which Sdt/Crb complex controls the AJ dynamics and polarity. We will focus on hypotheses that Sdt/Crb controls the AJ dynamics by regulating the aPKC-mediated DE-Cad phosphorylation and/or by regulating the Moesin- regulated actin-network. Finally, we propose to identify novel mechanisms of DE-Cad, Crb, Lgl, and Sdt function in cell polarization by systematically identifying their interacting partners through proteomics approaches, using genetically validated GFP and high-affinity epitope knock-in alleles generated by genomic engineering. Accomplishing the specific aims in this proposal will establish the regulation of AJ dynamics as a key molecular and cellular mechanism by which Sdt, Crb and Lgl to control the apical-basal polarization in epithelial cells. PUBLIC HEALTH RELEVANCE: Drosophila is a leading genetic model system for addressing crucial biological questions in human diseases. Our studies on cell polarity and E-Cadherin/adherens junction dynamics will greatly facilitate the analysis and treatment of cancer by providing novel mechanisms about how cell adhesions and polarity may be disrupted in cancer cells during tumor progression and metastatic transformation.