The long range goals of the project are to identify in a comprehensive manner the microtubule-associated proteins (MAPs) of the mammalian mitotic spindle, and to determine the functions of these proteins in both the mitotic and interphase stages of the cell cycle. The principal immediate goals will be to identify new spindle MAPs and to evaluate their roles in mitosis. Cultured mammalian cells wil be the main experimental subjects for this work. Microtubules and MAPs will be purified with the aid of taxol from CHO cells synchronized in mitosis and PtK1 cultures enriched in mitotic cells. Monoclonal antibodies will then be made using putative MAPs as antigens. Initially, the antibodies will be used to establish whether the immunoreactive proteins are genuine MAPs by two complementary techniques, immunoblotting and immunofluorescence microscopy. The former technique will be used to follow the fate of immunoreactive proteins through the stages of the taxol-based purification procedure for microtubules and MAPs. Immunofluorescence microscopy will be used to determine the intracellular distributions of these proteins. Immunoreactive proteins that co-purify with microtubules and MAPs, and are shown by immunofluorescence microscopy to be localized on microtubules in cells will be classified as MAPs. Intracellular functions of these proteins, as well as others that may partially fulfill these criteria, will then be probed by antibody microinjection experiments. The object of these studies will be to interfere with specific aspects of mitosis by microinjection of dividing cells with antibodies to particular MAP species, in order to shed light on MAP functions during mitosis. If time permits, non-dividing cells will be microinjected with antibodies to spindle MAPs that are also found on interphase microtubules. This will permit interphase functions of these MAPs to be evaluated. The final set of experiments proposed here will be the screening of various rat tissues by immunoblotting and immunofluorescence microscopy to determine the cellular distributions of MAPs in vivo. These experiments will identify convenient tissue sources for the future purification of individual MAP species, so that biochemical properties of these proteins may be studied in greater detail in subsequent stages of this project. Because microtubules are essential for mitosis, the studies proposed here should further our understanding of how this process occurs during normal development and in malignant cells.
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