Regulation of Vascular Smooth Muscle Cytoskeleton

描述(申请人提供)：肌动蛋白细胞骨架在分化的血管平滑肌(VSM)细胞中起重要作用，在生理和病理条件下需要肌动蛋白和肌动蛋白结合蛋白(ABPs)之间的密切合作。我们的总体目标是确定通过与ABPs的相互作用来调节VSM细胞的肌动蛋白结构的机制。近期的目标是确定钙调蛋白(CAD)和皮质素在收缩和迁移过程中的生理作用以及它们在控制细胞骨架中的相互作用。具体地说，我们建议：(1)检验假设，在VSM细胞中CAD与Cortactin相互作用，其中肌动蛋白细胞骨架经历动态重组，并且这种相互作用需要对两种蛋白质进行特定的修饰。我们将在激动剂刺激下对A7r5细胞和完整的小鼠VSM组织进行下拉试验和亲和力结合，并通过免疫显微镜进行原位共定位研究。这些蛋白质的潜在翻译后修饰(例如，磷酸化)随后将通过质谱学进行检测。(2)明确CAD-Cortactin相互作用和磷酸化对肌动蛋白调控的结构基础。我们将通过研究在肌动蛋白不存在和存在的情况下，皮质肌蛋白和CAD之间的结合来测试磷酸化对CAD的影响是如何通过与皮质肌动蛋白的相互作用来体现的。这些复合体中的空间关系将通过距离测量和荧光猝灭来评估。除了ERK，还将检测其他已知的磷酸化CAD和Cortactin的激酶的作用。通过亲和分离结合蛋白质分解和质谱分析确定相互作用的位置，通过三维重建和弯曲刚性测量确定CAD/Cortactin结合的肌动蛋白细丝的结构和对其力学性能的影响。(3)确定CAD-Cortactin相互作用的生理作用及其对细胞骨架的影响。A7r5细胞或VSM组织中的内源性CAD或Cortactin将首先通过siRNA处理被击倒，然后重新表达突变蛋白，以测试磷酸化的调节效果。与相互作用界面相对应的合成肽将作为“诱饵”被引入siRNA处理的细胞中。将通过检查细胞骨架结构的形态、足体的生物发生以及迁移和收缩特性来分析这些修饰所产生的影响。所有这些信息有望使我们能够机械地剖析CAD和Cortactin在VSM细胞肌动蛋白细胞骨架重塑过程中的生理作用，这将有助于我们与动脉粥样硬化等心血管疾病作斗争。420字。公共卫生相关性：数百万美国人患有心血管疾病。一个主要的问题是动脉粥样硬化和再狭窄，这是由血管平滑肌细胞的病理性运动引起的。然而，由于平滑肌细胞的复杂性，其调控细胞迁移的分子机制尚不清楚。在这个项目中，我们将着手研究两个与平滑肌肌动蛋白动力学有关的关键蛋白，并验证异常细胞外信号可以改变这些蛋白之间的相互作用并诱导平滑肌细胞迁移的假设。获得的信息对抗击心血管疾病将是无价的，心血管疾病仍然是美国的头号杀手。

DESCRIPTION (provided by applicant): The actin cytoskeleton, which plays an important role in differentiated vascular smooth muscle (VSM) cells, requires close collaboration between actin and actin-binding proteins (ABPs) under physiological and pathological conditions. Our overall aim is to determine the mechanisms by which the actin architecture of VSM cells is regulated via interactions with the ABPs. The immediate goal is to define the physiological roles of caldesmon (CaD) and cortactin and their interaction in the control of smooth muscle cytoskeleton during contraction and migration. Specifically, we propose: (1) To test the hypothesis that CaD interacts with cortactin in VSM cells where the actin cytoskeleton undergoes dynamic reorganization, and that such interaction requires specific modifications on both proteins. We will carry out pull-down assays and affinity binding in A7r5 cells and intact mouse VSM tissues under agonist stimulations, and the in situ co-localization studies by immuno-microscopy. Potential post- translational modifications (e.g., phosphorylation) of these proteins will then be examined by mass spectrometry. (2) To define the structural basis of the CaD-cortactin interaction and phosphorylation on actin regulation. We will test how the phosphorylation effect on CaD is manifested through interaction with cortactin by studying the binding between cortactin and CaD in the absence and presence of actin using kinase treated or phospho-mimetic proteins. The spatial relationship in these complexes will be assessed by distance measurements and fluorescence quenching. In addition to ERK, the effect of other kinases that are known to phosphorylated CaD and cortactin will also be examined. The interaction sites will be mapped out by affinity separation coupled with proteolysis and mass spectrometric analysis, and the structure of CaD/cortactin-bound actin filaments and the effect on the mechanical properties of the filament will be determined by 3D reconstruction and flexural rigidity measurements. (3) To determine the physiological role and of the CaD-cortactin interaction and the effects on cytoskeleton. The endogenous CaD or cortactin in the A7r5 cells or VSM tissues will first be knocked down by siRNA treatment, followed by re-expression of mutant proteins to test the regulatory effect of phosphorylation. Synthetic peptides corresponding to the interaction interface will be introduced to the siRNA treated cells as "decoys". The effects resulting from these modifications will be analyzed by examining the morphology of the cytoskeleton structures, podosome biogenesis, and the migratory and contractile properties. All this information is expected to enable us to mechanistically dissect the physiological roles of CaD and cortactin in the process of the actin cytoskeleton remodeling in VSM cells, which will help us to battle cardiovascular diseases such as atherosclerosis. 420 words. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from cardiovascular diseases. A major problem is atherosclerosis and restenosis, which results from pathological movement of vascular smooth muscle cells. However, owing to the complexity of the smooth muscle cells, the molecular mechanism by which the smooth muscle cell migration is controlled remains elusive. In this project we are setting out to investigate two key proteins that are involved in the smooth muscle actin dynamics and test the hypothesis that abnormal extracellular cues can modify the interaction between these proteins and induce smooth muscle cells to migrate. Information obtained will be invaluable to battle cardiovascular diseases which remain to be number one killer in the U.S.