The overall goal of this research is to determine how cytoplasmic microtubules are regulated and how they in turn participate in regulation of cell proliferation and other cell functions. We have rencently shown that a unique monoclonal anti-tubulin antibody (Ab 1-6.1) binds to only a subset of microtubules within single fibroblastic cells. This discovery indicates that certain microtubule subsets are configured to differentially express certain antigenic sites or that isotubulin molecules are segregated into these microtubule subsets. The proposed studies will determine the physical and biochemical differences between antigenically dissimilar cytoplasmic microtubules. We will generate a """"""""library"""""""" of monoclonal anti-tubulin antibodies which we will use to immunologically dissect the cytoplasmic microtubule complex into antigenic subsets and then determine the nature of the antigenic differences and any role of these subsets in controlling various physiological states of the cells. Specific antigenic determinants on tubulin molecules will be characterized by immunohistochemical modification of antigenic sites, Western blot antibody binding to electrophoresed isotubulins, immunoprecipitation or affinity chromatography of isotubulins and tubulin fragments and peptide mapping and amino acid analysis of unique peptides. To determine the role of microtubule subsets we will microinject monoclonal antibodies into individual living cells and observe the effects of these antibodies on physiological properties including mitogenesis, induced cell migration and maintenance of cell morphology. We will also microinject isolated tubulin mRNA into cells which do not express these subsets to examine the processing and effects of subset tubulin. The effects of micro-injected antibodies or the appearance of new microtubule subsets will be examined by immunofluorescent staining of fixed cells or by directly observing the fate of fluorescently labeled antibodies in living cells. To establish any correlations between altered microtubule subsets and various cell functions, the immunofluorescent patterns of various microtubule subsets will be examined in fixed cells which have been treated with a variety of agents to induce chemotaxis, mitogenesis or transformation. Because microtubules appear to be involved in initiation of normal cell proliferation and many transformed cells have been reported to have altered microtubules, the proposed investigation of microtubule subsets may provide a key to understanding the regulation of normal cell function and the molecular lesions which lead to neoplasia.
