This proposal continues our studies on one of the major structural elements of the cytoplasm--the 10-nm filament cytoskeleton. Previous work has shown 10-nm filaments occupy virtually every volume of the cytoplasm and associate with organelles such as mitochondria, lysosomes, the golgi, the nucleus, the mitotic spindle and microtubules. We now propose to define: (A) auxiliary proteins that crosslink 10-nm filaments to microtubules and to other organelles, and (B) what part of the microtubule backbone (alpha- or beta-subunits) directly interacts with 10-nm filaments. Intact 10-nm filament and microtubule cytoskeletons will be isolated using a new technique of monoclonal antibody-induced aggregation and crosslinking induced by bi-specific monoclonal antibodies. Isolated cytoskeletons will be analyzed by two-dimensional gels to determine the proteins that co-isolate with both cytoskeletons as candidates for crosslinkers. Monoclonal antibodies will be made against these crosslinking proteins to be used for immunolocalization in cells, microinjection, and immunoaffinity column chromatography to purify the protein for biochemical studies. Monoclonal antibodies or the proteins will be microinjected into cells to see if crossbridging is perturbed. Using a new technique, microinjected cells will be microlabeled with [35S]-methionine, [3H]-amino acids, or [32P]-phosphate to determine if perturbation results in change of synthesis or modification (phosphorylation) of (A) the crossbridge protein and/or (B) to detect other new functional groups of protein that might regulate or operate in concert with the crossbridge proteins. In the second part of the proposal we will determine the domains in tubulin that interact with 10-nm filaments. Two monoclonal anti-tubulins (that block filament-microtubule association) will be used to isolate these domains (peptides) by immunoaffinity chromatography. These peptides will be microinjected back into cells to determine if crosslinking is disturbed and will be sequenced to learn their location in the tubulin molecule. The last part of the proposal will seek to determine the antigenic site on desmin and vimentin found to be in common with tropomyosin. Our goal is to learn the function of this domain and see if it links 10-nm filaments to the microfilament system. Our long-term goal is to understand why the 10-nm filament cytoskeleton, through its interactions with elements of the cytoplasm, is an exquisitely sensitive indicator of cell injury and disease states of the cardiovascular system, the neurologic system, and cancer.
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