This is a proposal to investigate the structure and function of the cytoskeleton of the mammalian red blood cell using techniques of surface chemistry. The elements of the cytoskeleton of the human red blood cell that contribute to its elasticity will be reassembled beneath a phospholipid monolayer at an air/water interface. The phospholipid monolayer will provide 1) a physiologically relevant surface for cytoskeletal reconstitution and 2) convenient access to the cytoskeleton for sensitive mechanical sensors able to detect small changes in surface viscoelasticity and for potential modifiers of the reconstituted cytoskeleton, such as enzymes, metabolites and regulatory molecules. The cytoskeleton will be reconstituted in the following stages and its 2-dimensional rigidity will be carefully determined using instrumentation designed for the purpose. First, the shear properties of acidic phospholipid films will be established. Next, the consequences of the interaction of spectrin with that film will be examined using both static and dynamic surface measurements. Then band 4.1 and actin will be added to reconstitute a cross-linked cytoskeletal array, which may exhibit hyperelastic behavior characteristic of the erythrocyte membrane. After the mechanical behavior of spectrin-band 4.1-actinphospholipid structure is understood, the effects of physiological modifiers and minor cytoskeletal proteins will be examined. At critical steps in reconstitution, the cytoskeleton will be analyzed for protein composition and examined in the electron microscope. Determination of the dependece of the shear modulus and shear viscosity on the composition (surface density of each component) of the reconstituted cytoskeleton coupled with electron microscopy of selected preparations will yield fundamental information about the structure of the cell surface: 1) characteristics of cytoskeletal architecture, such as the number of cross-linking units, their lengths and their capacity for and the dynamics of exchange, 2) inherent elasticity of the major components and potential for interaction with cellular phospholipids, 3) how the mechanical properties of membranes are under cellular control, and 4) what role minor cytoskeletal components may play. This study of the novel, monolayer system to be developed will facilitate similar investigations of the cytoskeletal function of membranes other than erythrocyte membranes, as well as future studies of the function of linkages between intrinsic membrane proteins and the cytoskeleton.
