Cofilin/ADF Regulation in Rho GTPase Signaling

描述（由申请人提供）：细胞运动有助于正常（如伤口愈合）和病理（如转移）细胞行为。细胞迁移能力的基础是前沿肌动蛋白的动态，受Rho GTPases的调节。其中一种由Rac gtpase发起的途径对细胞定向迁移至关重要，它涉及通过磷酸化调控周期对cofilin依赖性肌动蛋白解聚/切断活性的精确空间调控。在过去的资助期间，我们的实验室鉴定并表征了一种新的cofilin/ADF磷酸酶，我们将其命名为chronophin （CIN）。我们的数据显示CIN是cofilin磷酸化循环的重要调节因子，从而调节细胞分裂和运动过程中的肌动蛋白动力学。将研究上游信号调节rac依赖性肌动蛋白重塑和细胞运动对CIN活性的调节。我们将使用生化和分子生物学方法，以及基于显微镜的细胞成像，来确定影响CIN调节cofilin的信号通路、蛋白质和分子复合物。将研究CIN在协调rac依赖的肌动蛋白动力学和细胞运动中的作用。我们将使用定量荧光散斑显微镜（qFSM）分析在前沿通过cin调节的cofilin磷酸化循环对肌动蛋白动力学的调节。此外，我们首次证明我们可以应用FSM直接研究生长因子（EGF）调控的癌细胞系MTLn3的前沿肌动蛋白动力学。热休克和/或缺血/ATP耗竭引起的细胞骨架重排是心血管细胞、神经细胞和肾近端小管细胞损伤的主要原因。这些细胞骨架反应可能同时具有适应性（生存）和损伤性后果。我们发现Hsp90是CIN的内源性结合伙伴，并负向调节CIN活性。我们将研究Rho GTPase-Hsp90/CIN-cofilin信号在atp消耗介导的细胞骨架重塑中的作用。在这些条件下，我们将研究Hsp90作为CIN活性的直接调节剂的生化作用，并评估CIN依赖的cofilin循环如何参与应激介导的细胞骨架反应和细胞存活。与公共卫生相关：细胞运动在正常生理和许多疾病状态中起着重要作用。我们的研究将探讨这一重要过程的一种新的调节因子。此外，该蛋白可能在缺血性疾病和其他应激反应中发挥重要作用。

DESCRIPTION (provided by applicant): Cell motility contributes to both normal (e.g. wound healing) and pathological (e.g. metastasis) cell behavior. Underlying the migratory abilities of cells are the dynamics of actin at the leading edge, regulated in response to Rho GTPases. One such Rac GTPase-initiated pathway crucial to directed cell migration involves the precise spatial regulation of cofilin-dependent actin depolymerizing/severing activity through a phosphoregulatory cycle. During the past grant period, our laboratory identified and characterized a novel cofilin/ADF phosphatase which we termed chronophin (CIN). Our data show CIN to be an important regulator of cofilin phosphocycling, thereby modulating actin dynamics during cell division and motility. The regulation of CIN activity by upstream signals modulating Rac-dependent actin remodeling and cell motility will be investigated. We will use biochemical and molecular biological approaches, along with microscopy-based cell imaging, to determine the signaling pathways, proteins, and molecular complexes that influence cofilin regulation by CIN. The role(s) of CIN in coordinating Rac-dependent actin dynamics and cell motility will be studied. We will use quantitative fluorescent speckle microscopy (qFSM) to analyze the regulation of actin dynamics through CIN-modulated cofilin phosphocycling at the leading edge. In addition, we have shown for the first time that we can apply FSM to directly investigate growth factor (EGF)- regulated leading edge actin dynamics in the carcinoma cell line MTLn3. Cytoskeletal rearrangements induced by heat shock and/or ischemia/ATP depletion are a major cause of injury to cardiovascular cells, neuronal cells, and renal proximal tubule cells. These cytoskeletal responses may have both adaptive (survival) and injurious consequences. We have found that Hsp90 is an endogenous binding partner of CIN and negatively regulates CIN activity. We will examine the participation of Rho GTPase-Hsp90/CIN-cofilin signaling in ATP-depletion-mediated cytoskeletal remodeling. We will investigate the biochemical role of Hsp90 as a direct regulator of CIN activity under these conditions, and assess how the CIN-dependent cofilin cycle participates in stress-mediated cytoskeletal responses and cell survival. Relevance to public health: Cell motility plays important roles in normal physiology and in numerous disease states. Our studies will investigate a novel regulator of this important process. In addition, this protein is likely to play important roles in ischemic disorders and other stress responses.