Regulation of Adherens Junction Trafficking by Polarity Proteins

描述（由申请人提供）：极性蛋白对粘附连接动力学的调节。细胞连接的关键组分如E-钙粘蛋白和密蛋白是主要通过囊泡运输递送和调节的跨膜蛋白。在极化上皮细胞中，通过运输调节连接复合物的动力学可能对建立顶-基底极性至关重要。在这里，我们提出，极性蛋白星尘（Sdt），Crumbs（Crb）和致命的巨大幼虫（Lgl）的粘附连接（AJ）的动态控制是一个重要的细胞机制，建立顶部-基底极性。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，使我们能够将靶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，我们已经产生了多种遗传验证的AJ标记，如E-Cadherin：：GFP，用于定量测定果蝇上皮细胞中体内AJ动力学。在这个建议中，我们将首先表征AJ动态，如生物合成，降解和局部（亚细胞）营业额的速率，可能会在顶-基底极化差异调节，以及极性蛋白Sdt，Crb和Lgl如何发挥主要的调节作用，在这样的AJ动态。通过操纵顺式和反式调节机制的AJ动态，我们将确定是否调节AJ动态在顶部-基底极性发展是必不可少的建立极性。然后，我们将探讨潜在的分子机制，Sdt/Crb复合物控制AJ的动力学和极性。我们将重点放在假设Sdt/Crb控制AJ动力学通过调节aPKC介导的DE-Cad磷酸化和/或通过调节Moesin调节肌动蛋白网络。最后，我们建议通过蛋白质组学方法系统地识别它们的相互作用伙伴，使用基因工程产生的基因验证的GFP和高亲和力表位敲入等位基因，来识别DE-Cad，Crb，Lgl和Sdt在细胞极化中的功能的新机制。实现本提案中的具体目标将建立AJ动力学的调节作为Sdt、Crb和Lgl控制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.