Recent evidence has shown that contractile simulation initiates the polymerization of actin that is required for tension development in airway smooth muscle tissues; however the function of the newly polymerized actin and the mechanisms for the regulation of actin filament assembly in response to a contractile stimulus are not known. The Arp2/3 complex and that the WASP family protein N-WASp (neuronal Wiskott-Aldrich Syndrome protein), have been recently identified as the first known activators of actin filament assembly in response to external signals in eukaryotic cells. The objectives of the proposed experiments are to evaluate the hypothesis that the contractile stimulation of airway smooth muscle activates N-WASp, which then activates the rp2/3 complex to initiate the formation of new actin filaments necessary for the development of contractile tension in airway smooth muscle. Furthermore, the actin-binding proteins profilin and cofilin are hypothesized to be essential participants in the regulation of actin dynamics during the contraction-relaxation cycle. The effects of modulating the amount of actin polymerization on the contractile mechanics of airway smooth muscle tissues will be determined. These objectives will be addressed in a series of experiments that take advantage of our ability to manipulate the expression of endogenous and recombinant proteins in isolated airway smooth muscle tissues in vitro.
The Specific Aims of this proposal are: 1) Evaluate the roles of N-WASp and the Arp2/3 complex in the regulation of actin polymerization and contraction in airway smooth muscle. 2.) Evaluate the signaling mechanisms that regulate the activation of N-WASp and actin polymerization in airway smooth muscle. 3). Evaluate the mechanisms for regulating the balance of monomeric globular (G) actin and filamentous (F) actin in airway smooth muscle, and determine how the modulation of actin dynamics affects the mechanical properties of the tissue during contractile stimulation. These experiments will provide new information regarding the mechanisms and function of actin polymerization and their role in regulating the contractile properties of smooth muscle tissues. This information may provide a foundation for the development of new therapeutic targets for the regulation of airway smooth muscle contraction in pathophysiologic conditions such as asthma.
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