The cytoskeleton is a dynamic network of interacting filamentous proteins which appears to play a critical role in a diverse group of cell functions including cell shape changes, organelle movement, exocytosis, and other secretory events. The cytoskeleton is composed of at least two discrete regions: the membrane cytoskeleton and the cytoplasmic cytoskeleton. The membrane cytoskeleton underlies the plasma membrane and serves to support the lipid bilayer. It is composed of proteins spectrin and 4.1 and is linked to the membrane by the protein, ankyrin. The other major region is composed of a matrix of proteins (including intermediate filaments) within the cytoplasm itself which serves to give shape to the cell and is involved in organelle movement. Our understanding of how these two regions are linked together in the cell remains quite unclear. Recent work indicates that ankyrin may be the link between the cytoplasmic cytoskeleton and the membrane.
The aim of this proposal is to define the nature of the linkage between the cytoskeleton and the plasma membrane. This will be pursued by exploring the interaction of the cytoskeletal protein ankyrin and intermediate filament proteins. The binding of intermediate filament protein vimentin to membrane vesicles isolated from human erythrocytes will be measured. The specificity of the binding will be tested with affinity purified antibodies. Following confirmation of the 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 studies should contribute to a molecular understanding of many cytoskeletal functions.
