Analysis of Ena/VASP Anti-Capping Activity

Ena/VASP 抗加帽活性分析

• 批准号: 6791602
• 负责人: CRAIG D FURMAN
• 金额: $ 4.73万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6791602
• 关键词: DNA binding protein; actin binding protein; actins; cell adhesion molecules; cell line; cell migration; cell motility; green fluorescent proteins; phosphoproteins; phosphorylation; polymerization; postdoctoral investigator; protein kinase A; protein localization; protein structure function; video microscopy

DESCRIPTION (provided by applicant): Cell migration plays an integral role in many processes important for human health, including embryonic development, immunological response and metastasis. Ena/VASP proteins are key regulators of cell migration. Ena/VASP proteins bind to the barbed ends of filamentous actin and induce changes in the geometry of the actin cytoskeleton that include increased actin filament length. The overall goal of this proposal is to determine if Ena/VASP functions as an "anti-capping" protein, causing alteration of the actin network by shielding the barbed ends of actin from capping protein. To test this hypothesis, levels of capping protein activity will be altered in cells to determine if reciprocal changes in actin cytoskeleton geometry can be achieved. In vitro actin polymerization experiments will be performed to directly measure antagonism between Ena/VASP and capping protein. Microscopy will be used to detect binding of Ena/VASP to barbed ends and to determine if the anti-capping mechanism involves exclusion of capping protein from the barbed ends. Finally, Ena/VASP mutants will be used to determine domains necessary for anti-capping function and to test if phosphorylation of Ena/VASP by PKA regulates anti-capping activity.

描述（由申请人提供）： 细胞迁移在胚胎发育、免疫应答和转移等对人类健康至关重要的许多过程中起着不可或缺的作用。Ena/VASP蛋白是细胞迁移的关键调节因子。Ena/VASP蛋白与丝状肌动蛋白的倒刺末端结合，并诱导肌动蛋白细胞骨架的几何形状发生变化，包括增加肌动蛋白丝长度。该提案的总体目标是确定Ena/VASP是否作为“抗加帽”蛋白发挥功能，通过屏蔽肌动蛋白的倒刺末端使其不受加帽蛋白的影响而引起肌动蛋白网络的改变。为了验证这一假设，将改变细胞中的加帽蛋白活性水平，以确定是否可以实现肌动蛋白细胞骨架几何形状的相互变化。将进行体外肌动蛋白聚合实验以直接测量Ena/VASP和加帽蛋白之间的拮抗作用。显微镜检查将用于检测Ena/VASP与倒刺末端的结合，并确定抗加帽机制是否涉及从倒刺末端排除加帽蛋白。最后，Ena/VASP突变体将用于确定抗加帽功能所需的结构域，并测试PKA对Ena/VASP的磷酸化是否调节抗加帽活性。