Microtubule associated proteins (MAPs) from sea urchin eggs will be characterized. Preliminary studies have yielded a new procedure for isolating microtubules from this source which involves the use of taxol to promote assembly. Several MAPs were identified in the purified microtubules with molecular weight values of 80,000, 105,000, 140,000 and approximately 300,00 to 350,000 (HMW MAPs). Fine arms were observed on the surface of the microtubules, quite similar in appearance to arms observed on microtubules in the mitotic spindle. The arms isolated in vitro appeared to cross-link microtubules in to bundles similar to arrays of microtubules observed in the spindle. Monoclonal antibodies were raised against the MAPs. So far two cloned hybridoma cell lines producing antibody respectively to a component of the HMW MAPs and to a minor 40,000 molecular weight MAP have been isolated. In addition, two hybridoma lines producing antibody to proteins of about 55,000 molecular weight that appear to be distinct from tubulin have also been isolated. All of the antibodies stain the mitotic spindle of dividing sea urchin eggs. The antibodies to the 40,000 molecular weight and HMW MAPs also stain the spindle of mouse 3T3 cells. We plan to continue our isolation of hybridomas to obtain antibodies against other MAPs. We will determine by immunofluorescence microscopy and immunoelectron microscopy which classes of microtubules the individual MAPs are associated with during development and within the mitotic spindle. We will microinject the antibodies into living cells to determine their effect on mitosis and other cellular functions, such as nuclear migration and ciliary movement. We will analyse the MAPs by biochemical means for microtubule assembly promoting activity, microtubule cross-linking activity, and ATPase activity, and will determine which MAP species represent arms. Since the major function of sea urchin microtubules seems to be in mitosis, this study should yield information primarily regarding MAPs in the mitotic spindle. Preliminary results have so far borne out this supposition. It is hoped that by the approach proposed here we will be able to obtain information on how cells divide, something of potentially great value for controlling normal or abnormal cell division in humans.
