The plasma membrance is supported an linked to the interior of the cell by the cytoskeleton. The cytoskeleton is a diverse network of interacting proteins that plays a critical role in maintaining cell shape. Interposed between the cytoskeleton and the membrane is the protein ankyrin which serves to anchor the cytoskeleton and perhaps to regulate cell shape. Ankyrin is a large (200,000 daltons) globular protein with multiple binding domains for cytoskeletal proteins including spectrin, tubulin and intermediate filaments.
The aim of this proposal is to define how ankyrin links the cytoskeleton to the membrane normal and diseased states by building on a structural and functional model of ankyrin. The first part of this proposal explores the binding of the intermediate filament protein vimentin to membrane vesicles isolated from human erythrocytes. The specificity of the binding will be tested with affinity purified antibodies. Following confirmation of binding, the vimentin binding site on ankyrin will be isolated using affinity columns prepared from intermediate filament proteins. To confirm and extend the binding specificity obtained from biochemical analysis, morphological examination of the binding will also be employed. The freeze-fracture/mica technique will be used to directly visualize the binding of intermediate filament proteins to membrane vesicles. The affinity purified binding domains and antibodies will offer a very important additional confirmation of the specificity and location of the binding sites. These results should yield a detailed structural and functional model of the binding sites on the cytoskeletal linker, ankyrin. The second part of this proposal is to extend this analysis by examining the role of ankyrin in the defective cytoskeleton found in the erythrocytes of patients with sickle cell anemia. This will be approached by measuring the binding of cytoskeletal proteins in solution and to erythrocyte membranes. With a detailed structural model of ankyrin available, the exploration of specific binding defects in sickled erythrocytes will be possible with the use of affinity-purified binding domains. This proposal should aid in our understanding of the cytoskeleton and how it interacts and communicates with plasma membrane. Such an understanding could contribute to modifications of such interactions in sickle cell anemia.
