Regulation of Actin Dynamics

肌动蛋白细胞骨架的持续动态重塑，对内在和外在的 信号，是执行许多真核细胞功能，包括细胞周期的关键 进展、细胞运动、分泌和从细胞/环境应激中恢复。这些 动态重排由一个大的，但尚未完全定义或理解的， 一组肌动蛋白结合蛋白。这项建议旨在继续调查这些职能， 三种新的肌动蛋白动力学调节剂：NADPH氧化还原酶老黄酶 （Oye 2 p; Aim#1）、MAPKKK Ssk 2 p（Aim#2）和cofilin激活剂Aip 1 p（Aim#3）。我们 以前对Oye 2 p的研究表明，它控制着C285-C374二硫键的氧化还原状态 在肌动蛋白中，其可以反过来影响F-肌动蛋白稳定性、对氧化应激的敏感性和细胞死亡/衰老。 我们建议将这些研究扩展到研究肌动蛋白氧化如何改变肌动蛋白动力学， 细胞如何调节其F-肌动蛋白结构的组织在响应， 从氧化应激中恢复特别是，我们试图确定F-肌动蛋白的组成部分， 含有在严重氧化应激时形成的氧化肌动蛋白体。Ssk 2 p激酶 促进渗透胁迫后肌动蛋白细胞骨架的再极化。我们对此的研究 保守的蛋白质，以及对渗透胁迫的适应，将通过鉴定 激酶的相关底物驱动肌动蛋白细胞骨架的复极化， 候选蛋白质的方法，并通过质谱鉴定相关的蛋白质。Aip 1 p是 小肌动蛋白结合蛋白cofilin的保守辅因子。这两种蛋白质协同作用 在体外使肌动蛋白丝不稳定，并在体内驱动不同肌动蛋白网络中的肌动蛋白动力学。 Aip 1 p-cofilin复合物的结构/功能分析导致了复合物的模型;我们 我们将继续进行这些研究，以进一步完善这一模型， F-actin通过cofilin去稳定化的机制。这种方法将利用我们的 最近在cofilin中发现了功能突变体的获得， 晶体学数据

Continual dynamic remodeling of the actin cytoskeleton, in response to intrinsic and extrinsic signals, is critical for the execution of many eukaryotic cell functions including cell cycle progression, cell motility, secretion and recovery from cellular/environmental stress. These dynamic rearrangements are regulated by a large, and as yet incompletely defined or understood, battery of actin binding proteins. This proposal seeks to continue investigations on the functions of three novel regulators of actin dynamics: the NADPH oxidoreductase Old Yellow Enzyme (Oye2p; Aim #1), the MAPKKK Ssk2p (Aim #2), and the cofilin activator Aip1p (Aim #3). Our previous work on Oye2p suggests that it controls the redox state of a C285-C374 disulfide bond in actin that can in turn affect F-actin stability, sensitivity to oxidative stress and cell death/aging. We propose to extend those studies to investigate how actin oxidation alters actin dynamics and how the cell regulates the organization of its F-actin structures during the response to, and recovery from, oxidative stress. In particular we seek to identify the components of F-actin containing oxidized actrin bodies that form upon a severe oxidative stress. The Ssk2p kinase facilitates re-polarization of the actin cytoskeleton following osmotic stress. Our studies on this conserved protein, and the adaptation to osmotic stress, will be extended by identifying the relevant substrates of the kinase that drive re-polarization of the actin cytoskeleton employing a candidate protein approach and by identifying associated proteins by mass-spectrometry. Aip1p is a conserved cofactor of the small actin binding protein cofilin. These two proteins act in concert to destabilize actin filaments in vitro and drive actin dynamics in vivo in diverse actin networks. Structure/function analysis of the Aip1p-cofilin complex has led to a model for the complex; we seek to continue these studies in order to further refine this model to gain further insight into the mechanism of F-actin de-stabilization by cofilin. This approach will take advantage of our recently identified gain of function mutants in cofilin for which we have sub-two angstrom crystallography data.