The major objectives of this project are: 1) to identify the molecular complexes which directly associate with mitotic apparatus microtubules in Strongylocentrotus purpuratus eggs: 2) to investigate whether similar organizations exist in the mitotic apparatus microtubular structures and in the axoneme of sperm flagellum; 3) to study the function and the assembly of substructures connected with the microtubules of the spindle fibers and of the astral rays. The study will be performed on a discrete number of proteins which are solubilized from mitotic apparatus preparations. Those components are extracted along with microtubular proteins under conditions which are specific for microtubule depolymerization. Constituents of fractions prepared by different physical or chemical treatments will be compared by one- and two-dimensional gel electrophoresis. Putative microtubule-associated polypeptides should be present in all the fractions with a constant molar ratio versus the tubulin. The comparative analysis will be extended to components of the whole axoneme or to polypeptides preferentially solubilized from different axonemal substructures. A large number of hybridomas secreting monoclonal antibodies directed against polypeptides of the mitotic apparatus and of the axoneme, will be selected by a sensitive solid phase radioimmunoassay. With the monoclonal antibodies the search for similarities between the two peptides which have similar antigenic determinants could be studied by peptide analysis and by structural analysis of the complexes formed with other molecules from the same source. Mitotic apparatus polypeptides which appear to be associated with microtubules will be localized in the cell during different phases of mitosis by the antibodies. Quantitative analysis of antigens which bind in vitro to microtubules will be performed by radioimmunoassay.
