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.
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