The carbamate herbicide isopropyl N-(3-chloropheny)carbamate(CIPC) inhibits the growth of animal as well as plant cells. The immediate effects of CIPC include development of multipolar mitotic spindles, delayed and incomplete mitotic spindle assembly and aborted or defective cytokinesis. These responses indicate that the microtubule organizing centers (MTOCs) of mitotic cells may be particular targets of CIPC action. Similar defects occur upon withdrawal of taxol from the taxol-dependant Chinese Hamster Ovary cell mutant, CHO-tax-18. We postulate that CIPC interacts with a taxol-like component of mitotic MTOCs that serves to nucleate or stabilize microtubule assembly. The experiments proposed here investigate the occurrence, function and CIPC-sensitivity of this proposed mitotic MTOC- associated protein. We will first generate a library of monoclonal antibodies to mitotic spindle components and other MTOCs(centrioles, kinetochores). We have already indentified one important antibody, designated mitosis-specific antibody(MSA), that associates with spindle poles from prophase through metaphase and with spindle midbodies during cytokinesis. Antibody distributions will be determined by immunofluorescence microscopy and their target antigens will be identifed by Western blotting. Fluorescence-labelled antibodies will be microinjected into living cells. Their association with MTOCs and their effects on mitosis and cytokinesis will be observed by high resolution video intensification microscopy. Antibodies that mimic effects of CIPC and antibodies whose distribution is abnormal in CIPC-treated and CHO-tax-18 cells will be selected for further study. In particular we will determine the effects of these antibodies and CIPC on the assembly of bovine brain tubulin in vitro from partially purified interphase and mitotic MTOCs. The protein composition of spindles isolated from control, CIPC-treated and tax-18 CHO cells will be compared. The abillty of taxol to normalize the mitotic cycle in CIPC-treated cultures will be determined. These studies are expected to identify one or more CIPC-sensitive, taxol-like, mitosis-specific proteins whose association with MTOCs is essential for the orderly progression of mitosis and cytokinesis. Once identified, this protein(s) can be isolated and its synthesis, molecular characteristics, location during interphase, and effects on microtubule assembly in vitro can be studied. These data will provide a firm base to assess the mechanism of cell growth inhibition by CIPC and the safety of its continued use in agriculture
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