Regulation of Actin Dynamics in Airway Smooth Muscle

描述（由申请人提供）：肌动蛋白聚合和肌动蛋白细胞骨架重塑是气道平滑肌（ASM）张力产生过程中的关键步骤。在肌动蛋白中，肌动蛋白细胞骨架重塑过程不同于肌动球蛋白过桥循环系统，并且可能独特地调节气道平滑肌的特殊生理特性，如机械适应，这对于呼吸时气道张力的调节至关重要。该项目的长期目标是确定ASM中调节肌动蛋白细胞骨架重塑的分子过程，并描述动态细胞骨架事件与受体激动剂和外部刺激引起的肌肉收缩激活相耦合的机制。研究提出了以下假设，即血管蛋白和cofilin是决定气道平滑肌机械和收缩特性的细胞骨架过程调节的关键效应器，并且这些蛋白质的激活状态通过激活Rho GTPases在ASM中受到收缩和放松刺激的调节。Vinculin被认为是调节细胞骨架肌动蛋白丝和膜粘附蛋白复合物之间连接的分子开关。这种连接的形成可能是调节张力从收缩器传递到平滑肌细胞外基质的关键。Cofilin被认为对严重的肌动蛋白丝起作用，并产生用于肌动蛋白重塑和聚合的G（单体）肌动蛋白池。该项目的具体目的是：1)确定血管蛋白对调节气道平滑肌收缩性的激动剂的反应，在调节肌动蛋白丝和膜粘附复合物之间的连接中的作用。2）. 确定cofilin在通过刺激调节气道收缩的肌动蛋白丝重塑中的作用，并评估肌动蛋白重塑的调节对ASM静态和动态力学性能的影响。3)。评估小Rho gtpase偶联激动剂刺激对ASM细胞骨架动力学调节的机制。这些目标将在一系列实验中得到解决，这些实验利用新技术，使内源性和重组蛋白的表达能够在完整的气道平滑肌组织中被操纵，并确定对其生理、细胞和生化特性的影响。这些关键的细胞骨架效应蛋白的激活可能直接将关键的细胞骨架过程与气道平滑肌的激动剂刺激偶联，因此它们可能成为治疗哮喘等气道张力和反应性调节异常的治疗干预的新靶点。项目描述：拟议的实验与公众健康直接相关。这些研究将描述正常呼吸条件下气道平滑肌收缩性和反应性调节的新基本机制，并确定气道平滑肌收缩的生理因子如何激活这些过程。这些研究将通过这些新机制确定调节气道平滑肌反应的关键分子的功能和调控。这些信息对于设计和开发适当的治疗药物来治疗哮喘和气道高反应性是必要的。

DESCRIPTION (provided by applicant): Actin polymerization and remodeling of the actin cytoskeleton are critical steps in the process of tension generation in airway smooth muscle (ASM). In ASM, the actin cytoskeletal remodeling process is distinct from the actomyosin crossbridge cycling system, and may uniquely regulate specialized physiological properties of airway smooth muscle such as mechanical adaptation, which is critical for the regulation of airway tone when they are stretched during breathing. The long term goals of this project are to determine the molecular processes that regulate actin cytoskeletal remodeling in ASM, and to describe the mechanisms by which dynamic cytoskeletal events are coupled to contractile activation of the muscle by receptor agonists and external stimuli. Studies are proposed to address the hypothesis that vinculin and cofilin are key effectors in the regulation of cytoskeletal processes that determine the mechanical and contractile properties of airway smooth muscle, and that the activation states of these proteins are regulated by contractile and relaxing stimuli in ASM through the activation of Rho GTPases. Vinculin is proposed to act as a molecular switch for the regulation of connections between cytoskeletal actin filaments and membrane adhesion protein complexes. The formation of such connections may be critical for the regulation of tension transmission from the contractile apparatus to the extracellular matrix in smooth muscle. Cofilin is proposed to function to severe actin filaments and to generate the pool of G (monomeric) actin used for actin remodeling and polymerization. The specific aims of the project are: 1) Determine the role of vinculin in regulating connections between actin filaments and membrane adhesion complexes in ASM in response to agonists that regulate airway smooth muscle contractility. 2). Determine the role of cofilin in the remodeling of actin filaments by stimuli that regulate airway contractility, and evaluate the effects of the modulation of the actin remodeling on the static and dynamic mechanical properties of ASM. 3). Evaluate the mechanisms by which small Rho GTPases couple agonist stimulation to the regulation of cytoskeletal dynamics in ASM. These aims will be addressed in a series of experiments that take advantage of novel technology that enables the expression of endogenous and recombinant proteins to be manipulated in intact airway smooth muscle tissues and the effects on their physiologic, cellular and biochemical properties determined. The activation of these key cytoskeletal effector proteins may directly couple critical cytoskeletal processes to agonist stimulation in airway smooth muscle, thus they may constitute novel targets for therapeutic intervention in the treatment of abnormalities in the regulation of airway tone and responsiveness such as asthma. PROJECT NARRATIVE: The proposed experiments are directly relevant to public health. These studies will characterize novel fundamental mechanisms by which airway smooth muscle contractility and responsiveness is regulated under the normal conditions of breathing, and determine how physiologic agents that contract airway smooth muscle activate these processes. The studies will determine the function and regulation of key molecules that act to regulate airway smooth muscle responsiveness through theses novel mechanisms. This information is necessary for the design and development of appropriate therapeutic agents to treat asthma and airway hyperreactivity.