Dynamics of the Vascular Smooth Muscle Cytoskeleton

描述（由申请人提供）：这是一个跨学科项目的新申请，由一个有凝聚力的研究小组领导。该项目的中心主题是了解分化血管平滑肌细胞（dVSMC）中肌动蛋白非收缩细胞骨架动力学的性质和调控。该工作组已经收集的初步数据表明，dVSMC的肌动蛋白细胞骨架确实是动态的。此外，对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。从本质上讲，该项目旨在确定dVSMC细胞骨架的综合蛋白质生物学，这一目标对于单个RO1来说过于宽泛，但非常适合PPG机制。项目1将聚焦于dVSM组织和细胞水平，使用包括蛋白敲除、诱饵肽、定量成像、细胞内FRET和交联在内的技术来研究肌动蛋白重塑的机制，验证非收缩性细胞骨架重塑调节VSM收缩性的假设。项目2将专注于物理蛋白质化学和细胞生物学方法，研究磷酸化诱导的肌动蛋白结合蛋白构象的改变，并验证构象变化解释协同蛋白-蛋白质相互作用的假设。项目3旨在通过螺旋重建、单粒子分析和激光陷阱等方法，了解肌动蛋白结合蛋白（actin binding proteins， ABP）对dVSMCs细胞骨架的结构和力学影响。项目4将测试串联WH2结构域在丝成核中起作用的假设，以及邻近富含脯氨酸的区域参与丝伸长的假设，并将利用x射线晶体学、生物物理方法、EM和细胞生物学来确定α -肌动蛋白和plectin与肌动蛋白相互作用的机制。核心B将通过生产定制肽和蛋白质工具以及提供使用复杂生物物理设备的专业知识和培训来支持所有项目。

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.