Analysis of Ena/VASP Anti-Capping Activity

描述(由申请人提供)： 细胞迁移在许多对人类健康至关重要的过程中发挥着不可或缺的作用，包括胚胎发育、免疫反应和转移。Ena/Vasp蛋白是细胞迁移的关键调节因子。EnA/Vasp蛋白结合到丝状肌动蛋白的带刺末端，导致肌动蛋白细胞骨架的几何形状发生变化，包括肌动蛋白细丝长度的增加。这项提议的总体目标是确定EnA/Vasp是否起到“抗封顶”蛋白的作用，通过保护肌动蛋白的带刺末端不受封顶蛋白的影响而导致肌动蛋白网络的改变。为了验证这一假说，我们将在细胞中改变封顶蛋白的活性水平，以确定是否可以实现肌动蛋白细胞骨架几何形状的互惠变化。体外肌动蛋白聚合实验将直接测定Ena/Vasp和Capping蛋白之间的拮抗作用。显微镜将被用来检测Ena/Vasp与带刺末端的结合，并确定抗封顶机制是否涉及从带刺末端排除封顶蛋白。最后，Ena/Vasp突变体将被用来确定抗封顶功能所需的结构域，并测试PKA对Ena/Vasp的磷酸化是否调节抗封顶活性。

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.