In each eukaryotic cell, there is a """"""""cytoskeleton"""""""" of protein filaments that determine cell shape, morphology, movement and cytoplasmic organization. The most insoluble component of the cytoskeleton are intermediate filaments (IFs). Disruption of normal IF organization has been shown to occur in a number of neuropathies, including Alzheimer's disease. IFs are encoded by a complex, multigene family whose expression is closely linked to cellular differentiation. To study IF function, I will use the purified IF subunit protein vimentin adsorbed to nitrocellulose paper and purified vimentin filaments attached to chromatography resins to identify and isolate vimentin-binding proteins (VBPs). AntiVBP antibodies will be used to localize VBPs within the cell, and through intracellular injection studies, to study their function. Where antibodies against a VBP would not be useful for the study of function, we will subject vimentin to proteolytic or chemical treatment, in order to cleave it into its structural domains. I have used this approach to identify the phosphorylated portion of vimentin; antibodies directed against the """"""""phospho-domain"""""""" will be used to study the role of IF phosphorylation within the cell. The binding of VBPs to vimentin domains will be studies, and antibodies directed against these domains will be generated and tested for their ability to block the interaction between vimentin and VBPs. These antibodies will then be used to study the role of vimentin/VBP interaction in vivo. I have identified proteins present in cell residues that may be related to IF; using nitrocellulose-immobilized antigen presentation, we are now generating antibodies against these proteins, which we will use to study their cellular distribution and function. Together these studies examine the function of a major cytoskeletal system, they should provide new information which may prove useful in understanding cellular behavior and its aberrations.
