The cellular filopodia mechanism in Shigella membrane protrusion formation

描述(申请人提供)：志贺氏菌是细胞内的肠道人类病原体，通过基于肌动蛋白的运动移动到细胞外围，在那里它们产生质膜的突起，穿透并被邻近的未感染细胞占据。质膜突起的产生是志贺氏菌传播的关键，但其中涉及的机制尚不清楚。 我们已经证明，细胞透明福尔马林和IRTKS都是有效形成质膜突起和志贺氏菌通过细胞单层有效传播所必需的。透明福尔马林和IRTKS同源基因IRSP53都是细胞纤足顶端复合体的组成部分，两者都是有效形成丝状足突所必需的。IRTKS和IRSP53是连接质膜和细胞骨架的I-bar结构域蛋白，同时诱导质膜的凹陷变形。基于这些结果，我们假设志贺氏菌产生的质膜突起利用了细胞丝足形成机制。在这一应用中，我们提出了一系列探索性调查来验证我们的假设。我们的具体目标如下： 目的1.分析IRTKS在志贺氏菌突起形成中的作用。 目的2.检测IRTKS酪氨酸磷酸化在福氏志贺氏菌突起形成中的作用。 目的3.确定福氏志贺氏菌是否需要I-bar结构域蛋白的其他相互作用元件才能有效地产生质膜突起。 本申请书中提议的调查极有可能对 志贺氏菌的传播机制通过肠道上皮，以及涉及细胞膜突起的基本过程，特别是丝状足的形成。

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.