RECONSTRUCTION OF RBC CYTOSKELETAL ELEMENTS IN VITRO

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We intend to investigate the role of cytoskeletal proteins of human red blood cell (RBC) in formation of structural elements capable of lending elastic properties to the intact RBC membrane. Our approach is to reconstitute in vitro possible elements of the cytoskeleton using highly purified cytoskeletal proteins. We will use, as a first step, the elastic modulus measured by an oscillating rheometer as a measure of elasticity in vitro. We have shown that spectrin and actin assemble spontaneously to form gels possessing respectable elastic modulus. We will investigate the role of other proteins such as band 4.1 as well as the role of phosphorylation and specific ion binding to spectrin, actin and other cytoskeletal components. We will determine the temperature dependence of the elastic modulus of our multiprotein gels. As the next step we propose to develop a 2-D model for reconstitution utilizing monolayers of phospholipids and a Wilhelmy balance. We hope to insert purified band 3 into such monolayer and attach reconstituted elements of spectrin-actin-band 4.1 to the solution side of the monolayer. Although the absolute elasticity and viscosity of such a monolayer is difficult to measure due to the presence of solvent, the apparent elastic modulus determined by oscillating Pt ring provides a good way to follow the effects of changing protein composition and phosphorylation. Our goal is to determine quantitatively the elasticity of such reconstructed protein complexes and determine by comparison to appropriate data for whole red cells whether the structures are likely to represent the cytoskeletal architecture in vivo.