Dynamics of the Vascular Smooth Muscle Cytoskeleton

描述（由申请人提供）：这是一个由一个有凝聚力的研究小组领导的跨学科计划项目的新申请。该项目的中心主题是了解分化的血管平滑肌细胞（dVSMC）中肌动蛋白非收缩性细胞骨架动力学的性质和调节。这个工作组已经收集的初步数据表明，肌动蛋白细胞骨架的dVSMC，确实是动态的。此外，肌动蛋白结合蛋白在非肌肉细胞骨架中的功能的关键分子见解，部分由该小组的成员发表，已经定位了该领域，使我们在未来五年内对该系统的理解取得重大进展。该计划，在本质上，旨在确定dVSMC细胞骨架的整合蛋白生物学，一个目标过于广泛的个人RO 1，但非常适合PPG机制。项目1将集中在dVSM组织和细胞水平，使用包括蛋白质敲除，诱饵肽，定量成像，细胞内FRET和交联的技术来研究肌动蛋白重塑的机制，测试非收缩性细胞骨架重塑调节VSM收缩性的假设。项目2将集中于物理、蛋白质、化学和细胞生物学方法来研究磷酸化诱导的肌动蛋白结合蛋白构象的改变，并检验构象变化解释协同蛋白质-蛋白质相互作用的假设。项目3的目的是了解肌动蛋白结合蛋白（ABP）的结构和力学作用的dVSMCs的细胞骨架使用螺旋重建和单粒子分析和激光陷阱的方法。项目4将测试的假设，串联WH 2结构域功能的细丝成核，和相邻的脯氨酸丰富的地区参与细丝伸长，并将确定的机制，α-辅肌动蛋白和plectin与肌动蛋白相互作用，使用X射线晶体学，生物物理学方法，EM和细胞生物学。核心B将通过生产定制肽和蛋白质工具以及提供使用先进生物物理设备的专门知识和培训来支持所有项目。 敷设总结：该计划的跨学科，综合性方法将允许单个蛋白质序列与血管功能直接相关，因此，将为治疗心血管疾病的新疗法的可能开发确定全新的靶点。

DESCRIPTION (provided by applicant) :This is a new application for an interdisciplinary program project led by a cohesive group of investigators. The central theme of the Program Project is to understand the nature and regulation of the actin noncontractile cytoskeletal dynamics in the differentiated vascular smooth muscle cell (dVSMC). Preliminary data already collected by this working group demonstrate that the actin cytoskeleton of the dVSMC is, indeed, dynamic. Additionally, key molecular insights into the function of actin binding proteins in the nonmuscle cytoskeleton, published in part by members of this group, have positioned the field to allow major advances in our understanding of this system over the next five years. This program, in essence, aims to determine the integrative protein biology of the dVSMC cytoskeleton, a goal too broad for an individual RO1, but ideally suited for the PPG mechanism. Project 1 will focus at the level of dVSM tissue and cells, using techniques including protein knockdown, decoy peptides, quantitative imaging, in-cell FRET and crosslinking to investigate the mechanisms of actin remodeling, testing the hypothesis that remodeling of the noncontractile cytoskeleton modulates VSM contractility. Project 2 will focus on physical protein chemical and cell biology approaches to study phosphorylation-induced alterations of the conformation of actin binding proteins and test the hypothesis that conformational changes explain synergistic protein-protein interactions. Project 3 aims to understand the structural and mechanical effects of actin binding proteins (ABP) on the cytoskeleton of dVSMCs using helical reconstruction and single-particle analysis and laser trap methods. Project 4 will test the hypotheses that tandem WH2 domains function in filament nucleation, and that the adjacent proline-rich regions participate in filament elongation and will determine the mechanism by which alpha-actinin and plectin interact with actin using X-ray crystallography, biophysical methods, EM and cell biology. Core B will support all projects by producing custom peptide and protein tools and providing expertise and training in the use of sophisticated biophysical equipment. Lay Summary: The interdisciplinary, integrative, approach of the program will allow individual protein sequences to be directly related to vascular function and, thus, will identify entirely novel targets for the possible development of new therapeutics for the treatment of cardiovascular disease.