Microtubules are used by eukaryotic cells to segregate chromosomes at mitosis and to transport organelles to specific locations in the cell during interphase. The accuracy of organelle transport requires that the microtubules be arranged in a specific polarized array in interphase, whereas accurate segregation of chromosomes during mitosis requires a bipolar array of microtubules, the mitotic spindle. Knowledge of the specific proteins responsible for the reorganization of the microtubule array during the cell cycle will be essential for a complete understanding of the mechanisms of cell division and therefore of oncogenesis. The long term goal of this project is to elucidate the roles that individual proteins play in regulating microtubule arrays in vivo. Previous work by the principal investigator has resulted in the purification of katanin, a microtubule-severing protein, from sea urchin eggs. Preliminary experiments indicate that katanin is concentrated at the centrosomes, the microtubule organizing centers, of dividing sea urchin cells.
The specific aim of this project is to determine the specific role of katanin-based microtubule severing in the dynamics of interphase and mitotic microtubule arrays by using antibody-based methods to inhibit katanin's activity in vivo and in in vitro extracts. Specifically, antibody FAb fragments that inhibit katanin's in vitro activity will be microinjected into sea urchin zygotes to examine the effects on the structure of microtubule arrays in vivo. The existence of purified, active katanin will allow complementation by coinjection of katanin with the inhibitory FAb fragments. The effects of injecting excess katanin will also be examined. In vitro, sperm centriole complexes are assembled into centrosomes in extracts made from sea urchin eggs. Immunodepletion of katanin from these extracts will allow assembly of centrosomes devoid of katanin whereas addition of purified katanin to these extracts may allow assembly of centrosomes containing excess katanin. Two particular aspects of microtubule dynamics will be examined in detail. The first is the release of microtubules from their attachment to the centrosome, a phenomenon observed in vivo and in cell free extracts. The second is tubulin flux, a process observed in vivo and in vitro in which microtubules are disassembled at there sites of attachment at the centrosome. If inactivation of katanin inhibits either of these processes, these experiments will, for the first time, provide a test of the importance of tubulin flux and of microtubule release from the centrosome in cell division. Isolation of cDNAs encoding the katanin subunits and bacterial expression of these cDNAs will facilitate production of the antibodies required for these experiments.
