Trio/CARMIL Regulation of Epithelial Cell Rearrangement

项目摘要 上皮细胞的定向重排在人类发育过程中至关重要，包括 原肠形成和神经形成的事件。基底侧向突出活动是如何促成这一点的 人们对这一过程知之甚少。线虫胚胎表皮的背部嵌合是 一种用于研究上皮细胞中的基侧事件的优秀模型系统 单细胞水平的重排。我们已经证明，背侧插入依赖于一个 Rho家族鸟嘌呤核苷酸激活的系统发育保守的RAC/RhoG盒 交换因子，UNC-73/Trio，并受高度保守的肌动蛋白负调控 封端蛋白调节因子CRML-1/CARMIL(封端蛋白-、Arp2/3-和肌球蛋白I-连接物 蛋白质)。 这项新的提案将调查这个保守的RAC/RhoG盒式磁带是如何调节 通过在四个方面进行实验来进行细胞重排： (1)我们将使用胚胎学和结构-功能方法来确定CRML是如何- 1/CARMIL定位于间质细胞的后方； (2)我们将通过生化和体内救援实验来确定CRML-1 通过直接结合或间接通过其受体对Trio/Rac/RhoG途径进行负调控 对封顶蛋白募集的影响； (3)我们将确定Sit/Robo/srGAP盒是否通过局部排斥来调节RAC 或作为“细胞接触缓冲器”；以及 (4)我们将确定其他组件如何调节Trio/Rac/RhoG途径 候选人筛选和前进的遗传方法。 作为这些研究的结果，我们将阐明一种新的途径，通过 基底外侧突出活动，这是一个广泛的过程，对理解主要 出生缺陷和正常的人类胚胎发育。

Project Summary The directed rearrangement of epithelial cells is crucial during human development, including the events of gastrulation and neurulation. How basolateral protrusive activity contributes to this process is poorly understood. Dorsal intercalation in the epidermis of the C. elegans embryo is an outstanding model system for examining basolateral events during epithelial cell rearrangement at the level of single cells. We have shown that dorsal intercalation relies on a phylogenetically conserved Rac/RhoG cassette activated by the Rho family guanine nucleotide exchange factor (GEF), UNC-73/Trio, and is negatively regulated by the highly conserved actin capping protein regulator, CRML-1/CARMIL (capping protein-, Arp2/3- and myosin I-linker protein). This new proposal will investigate how this conserved Rac/RhoG cassette regulates directed cell rearrangement by performing experiments in four areas: (1) We will use embryological and structure-function approaches to determine how CRML- 1/CARMIL localizes to the rear of intercalating cells; (2) We will use biochemical and in vivo rescue experiments to determine whether CRML-1 negatively regulates the Trio/Rac/RhoG pathway via direct binding or indirectly through its effects on capping protein recruitment; (3) We will determine whether a Slit/Robo/srGAP cassette regulates Rac via localized repulsion or as a “cell contact buffer”; and (4) We will determine how additional components regulate the Trio/Rac/RhoG pathway using candidate screens and forward genetic approaches. As a result of these studies, we will elucidate a novel pathway regulating cell intercalation via basolateral protrusive activity, a widespread process with implications for understanding major birth defects and normal human embryogenesis.