The cellular filopodia mechanism in Shigella membrane protrusion formation

描述（由申请人提供）：志贺氏菌属是细胞内肠道人类病原体，其通过基于肌动蛋白的运动性移动到细胞外周，于是它们产生质膜突起，所述质膜突起穿透到相邻未感染细胞中并被其吸收。质膜突起的产生是志贺氏菌传播的关键，但所涉及的机制知之甚少。 我们已经表明，细胞透明的formins和IRTKS都需要有效形成质膜突起和有效传播志贺氏菌通过细胞单层。这两个透明的formins和IRTKS同系物IRSp 53的细胞fiolopodia尖端复合物的组成部分，都是需要有效地形成丝状伪足。IRTKS和IRSp 53是I-BAR结构域蛋白，其连接质膜与细胞骨架，同时诱导膜的凹陷变形。基于这些结果，我们推测志贺氏菌产生质膜突起利用细胞丝状伪足形成机制。在这个应用程序中，我们提出了一套探索性的调查来测试我们的假设。我们的具体目标如下： 目标1。IRTKS在志贺菌突起形成中的功能分析 目标二。测试IRTKS的Tyr磷酸化在S.弗氏突形成 目标3。确定I-BAR结构域蛋白的其他相互作用物是否是S.弗氏杆菌属 本申请中提出的研究很可能会产生新的见解 志贺氏菌通过肠上皮传播的机制，以及进入涉及细胞膜突起的基本过程，特别是丝状伪足形成。

DESCRIPTION (provided by applicant): Shigella are intracellular enteric human pathogens that move to the cell periphery via actin-based motility, whereupon they generate protrusions of the plasma membrane that penetrate into and are taken up by adjacent uninfected cells. The generation of plasma membrane protrusions is critical for Shigella spread, but the mechanisms involved are poorly understood. We have shown that cellular diaphanous formins and IRTKS are each required for both efficient formation of plasma membrane protrusions and efficient spread of Shigella through cell monolayers. Both diaphanous formins and the IRTKS homolog IRSp53 are components of the cellular fiolopodia tip complex and both are required for efficient formation of filopodia. IRTKS and IRSp53 are I-BAR domain proteins that link the plasma membrane with the cytoskeleton while inducing concave deformations of the membrane. Based on these results, we hypothesize that Shigella generation of plasma membrane protrusions utilizes the cellular filopodia formation machinery. In this application, we propose a set of exploratory investigations to test our hypothesis. Our specific aims are as follows: Aim 1. Analysis of IRTKS functions involved in Shigella protrusion formation. Aim 2. Test the role of Tyr phosphorylation of IRTKS in S. flexneri protrusion formation. Aim 3. Determine whether other interactors of I-BAR domain proteins are required for efficient generation of plasma membrane protrusions by S. flexneri. The investigations proposed in this application are highly likely to generate new insights into the mechanisms of Shigella spread through the intestinal epithelium, as well as into the fundamental processes involved in cellular membrane protrusion, particularly filopodia formation.