The actin-cytoskeleton is a major determinant of the shape of vascular smooth muscle cells and is proposed to remodel during cell contraction and distension. Cytoskeletal remodeling requires the coordinated action of actin binding proteins (ABPs) that stabilize, crosslink, cap and/or sever actin filaments. Under normal physiological conditions the vascular system experiences a wide range of mechanical forces, and it is the interactions of the cytoskeleton that enable vascular smooth muscle cells to sense and respond to mechanical distension and compression (and any disease related alterations in these forces). The goal of this proposal is to determine structural and mechanical effects of ABPs on the cytoskeleton of vascular smooth muscle cells in order to understand the properties of this essential organelle. To elucidate the effects of force on ABP binding as well as the effects of ABPs on cytoskeletal mechanics, information about the molecular structure and mechanics of the single cytoskeletal microfilaments is required. Our approach will take 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 critical intermolecular interactions between ABPs and actin. Since ABPs may alter both filament structure and mechanics, the effects of ABP binding on actin flexural and torsional rigidity will be determined. Likewise, the mechanical effect of force applied to F-actin on the binding of ABP to filaments will be assessed. These structural and physical studies will characterize the molecular domains of ABPs that regulate cytoskeletal dynamics and mechanical behavior. This information allows us to 1) define residues that are necessary for cytoskeletal filaments to transmit and perceive forces and 2) design decoy peptides to be used by PPG members to investigate physiological function in vascular cell preparations. Lay summary: Contraction and the maintenance offeree by vascular smooth muscle cells that line blood vessels are key factors responsible for controlling blood pressure and blood flow to the organs.of the human body. We will determine the structure and mechanics of an intracellular skeleton composed of microscopic filaments, which control the shape of vascular smooth muscle cells and hence help to regulate blood pressure and blood flow.
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