Regulation of Adherens Junction Trafficking by Polarity Proteins

描述（由申请人提供）：极性蛋白对粘附体连接动力学的调节。细胞连接的关键成分，如e -钙粘蛋白和claudin，是主要通过囊泡运输传递和调节的跨膜蛋白。在极化上皮细胞中，通过运输调节连接复合物的动态可能是建立顶基极性的关键。我们认为极性蛋白Stardust （Sdt）、crumb （Crb）和Lethal giant幼虫（Lgl）对粘附体连接（AJ）动力学的控制是形成顶基极性的重要细胞机制。为了验证这一假设，我们在果蝇身上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饰成任何所需的突变等位基因。通过这种基因组工程方法，我们已经生成了多个基因验证的AJ标记，如E-Cadherin::GFP，用于定量分析果蝇上皮细胞体内AJ动态。在本研究中，我们将首先描述在顶基极化过程中AJ动力学（如生物合成、降解和局部（亚细胞）更新的速率）是如何受到差异调节的，以及极性蛋白Sdt、Crb和Lgl是如何在这种AJ动力学中发挥主要调节作用的。通过顺式和反式调节机制操纵AJ动力学，我们将确定在顶基极性发展过程中调节AJ动力学是否对极性的建立至关重要。然后，我们将探索Sdt/Crb复合物控制AJ动力学和极性的潜在分子机制。我们将重点关注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在细胞极化中的作用机制，利用基因组工程产生的基因验证的GFP和高亲和力表位敲入等位基因来鉴定它们的相互作用伙伴。完成本研究的具体目标，将确立AJ动力学调控是Sdt、Crb和Lgl调控上皮细胞顶基极化的关键分子和细胞机制。

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.