The actin-cytoskeleton is a major determinant of the shape of vascular smooth muscle cells and is proposedto remodel during cell contraction and distension. Cytoskeletal remodeling requires the coordinated action ofactin binding proteins (ABPs) that stabilize, crosslink, cap and/or sever actin filaments. Under normalphysiological conditions the vascular system experiences a wide range of mechanical forces, and it is theinteractions of the cytoskeleton that enable vascular smooth muscle cells to sense and respond tomechanical distension and compression (and any disease related alterations in these forces). The goal ofthis proposal is to determine structural and mechanical effects of ABPs on the cytoskeleton of vascularsmooth muscle cells in order to understand the properties of this essential organelle. To elucidate the effectsof force on ABP binding as well as the effects of ABPs on cytoskeletal mechanics, information about themolecular structure and mechanics of the single cytoskeletal microfilaments is required. Our approach willtake advantage of helical and single-particle analysis to define the location of ABP binding sites on actin,thus revealing the potential for synergy or alternatively 'parking problems' between ABPs on actin filaments.In addition, we will fit atomic resolution structures into our reconstructions, thus pinpointing criticalintermolecular interactions between ABPs and actin. Since ABPs may alter both filament structure andmechanics, the effects of ABP binding on actin flexural and torsional rigidity will be determined. Likewise, themechanical effect of force applied to F-actin on the binding of ABP to filaments will be assessed. Thesestructural and physical studies will characterize the molecular domains of ABPs that regulate cytoskeletaldynamics and mechanical behavior. This information allows us to 1) define residues that are necessary forcytoskeletal filaments to transmit and perceive forces and 2) design decoy peptides to be used by PPGmembers to investigate physiological function in vascular cell preparations. Lay summary: Contraction andthe maintenance offeree by vascular smooth muscle cells that line blood vessels are key factors responsiblefor controlling blood pressure and blood flow to the organs.of the human body. We will determine thestructure and mechanics of an intracellular skeleton composed of microscopic filaments, which control theshape of vascular smooth muscle cells and hence help to regulate blood pressure and blood flow.
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