INTERMEDIATE FILAMENT-CELL SURFACE INTERACTIONS

本提案的总体目标是确定动态

The overall goals of this proposal are to determine the dynamic properties of the intermediate filament (IF) cytoskeletal networks in fibroblasts and epithelial cells as well as to determine the nature of the interaction between IF and the cell surface. IF dynamics will be studied in mitotic cells in which the IF system is depolymerized and in daughter cells in which IF are repolymerized. Specifically, studies of the molecular basis of the extensive remodeling of Type III IF networks containing vimentin or vimentin/desmin will be undertaken. The explosive depolymerization which typifies early-mid prometaphase and the rapid reassembly of IF which takes place during anaphase-telophase can be explained, for the most part, by the hyperphosphorylation of IF structural proteins. Two mitotic kinases have been found in BHK cells which are involved in the phosphorylation reaction. We are undertaking an extensive analysis of one of these kinases termed vimentin protein kinase (VPK). VPK is a complex of three proteins: a 65kD component, a 110kD component, and p34cdc2 which is the catalytic subunit of maturation promoting factor (MPF) and is known to play a major role in triggering mitosis. We plan to carry out experiments aimed at determining the functional significance of the specific site(s) phosphorylated by VPK and other kinases which act on Type HI IF proteins using a variety of cell physiological (e.g., microinjection), morphological (immunofluorescence, confocal, and electron microscopy), biochemical and molecular biological (the use of bacterially expressed proteins and transient transfection of cultured mammalian cells) techniques. We will also attempt to study the interphase dynamic properties of IF. For these studies we will utilize the microinjection of derivatized IF proteins such as biotinylated vimentin and keratin into live cells and to determine their fate in situ using confocal and electron microscopy. Similar approaches using x-rhodamine labelled IF proteins will be used to determine whether or not a steady state or dynamic equilibrium exists in interphase cells through the use of fluorescence recovery after photobleaching (FRAP) experiments. Studies are also described which are aimed at determining the biochemical basis of the binding of IF to cell surface associated desmosomes in epithelial cells. These studies involve the isolation and biochemical characterization of bovine tongue desmosome components and the determination of how keratin polypeptides bind to them. The information derived from our investigations should shed new light on the properties and functions of IF in mammalian cells. A better understanding of IF structure and function is basic to understanding the pathogenesis of a variety of diseases in which changes in IF occur, such as Alzheimer's disease, Parkinson's disease, various cancers, and alcoholic cirrhosis.