MOLECULAR ANALYSIS OF MITOTIC SPINDLE COMPONENTS

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The purpose of this proposal is to identify and study the function of components of the mitotic spindle at the molecular level. Although tubulin is the most abundant component of the spindle, it serves primarily a structural role. The activity of microtubules is likely to be understood through the molecules that govern their assembly and interaction with poles, chromosomes and perhaps other spindle structures. This proposal will focus on specific molecules recently identified as components of the spindle and on identifying components hitherto unknown. The recently identified components are the microtuble-associated proteins (MAPs) of 210,000 and 125,000 molecule weight first isolated by us from the carcinoma cell line, HeLa. The as yet unknown components will be sought by a combination of new and old technologies. Microtubule affinity methods will be used to prepare MAPs from cultured cells. Cell fractionation will be used to prepared mitotic spindlese, centrosomes and mid-bodies from cultured cells. Monoclonal antibody techniques will be used to prepare antibodies to MAPs and to shotgun for other mitotic spindle components. The cloned hybridomas will be screened by a rapid indirect immunofluorescence method for secretion of spindle-positive antibodies. Likely spindle component antibodies will be characterized for specificity and cross-reactivity by a gel binding assay and by immunoprecipitation. Antigens will be localized in cells by a combination of light and electron microscopic immunocytochemistry. The function of MAPs and other identified spindle components will be investigated through the use of antibody inhibition of appropriate in vitro assays. For MAPs, the assays will test for inhibition of binding to microtubles to define reagents specific for functional domains of the molecules. For molecules of the pericentriolar cloud, the assays will test for inhibition of nucleation of microtuble formation from tubulin subunits. Function will also be tested by determining the effects of microinjecting selected antibodies into living cells. Selected living cells will be analyzed by light microscopy and subsequently the same cell will be analyzed by immunocytochemical and/or electron microcopy to define the action of the antibody. These basic studies on cell division may reveal control mechanisms important for understanding the biochemical defects which result in the uncontrolled division of cancer cells.