Microtubules are cytoskeletal polymers that play essential roles in intracellular membrane trafficking and in segregating chromosomes during mitosis. To achieve these vital functions, the microtubule cytoskeleton must be capable of rapid reorganization. The dramatic reconstruction of the microtubule array at the onset of mitosis to form the mitotic spindle is an example of how microtubules can be rapidly disassembled and then assembled into a new configuration. Microtubules in axons, on the other hand, are extremely stable and resist disassembly by microtubule depolymerizing drugs. Since changes in the stability of microtubules occur during the cell cycle, development and migration, deciphering the molecular regulation of microtubule dynamics is of great importance for understanding the division and differentiation of normal and tumorigenic cells. At present, the properties of microtubule assembly from pure tubulin are fairly well understood, but the inventory of proteins that modulate microtubule dynamics and stability in living cells is still incomplete. Our long term goals are to combine in vitro assays with biochemical fractionation protocols to identify new proteins that regulate the assembly of the microtubule cytoskeleton. Previous work by this laboratory has identified a novel, cell cycle regulated microtubule severing activity from frog (Xenopus laevis) eggs, which may be involved in disassembling interphase microtubules when cells enter mitosis. This microtubule severing protein has recently been purified to homogeneity from sea urchin eggs. In this grant, the molecular properties of this ATPdependent severing protein will be characterized by physical studies and by obtaining cDNA clones. In an effort to understand how this protein disrupts tubulin bonds within a microtubule, microtubule severing will be studied using video and electron microscopy, and the interactions of the severing protein with tubulin and nucleotide will be examined using binding assays. To determine how severing is controlled by the cell cycle, the phosphorylation state of the severing protein in vivo and in vitro will be examined and the effects of other microtubule-associated proteins on severing will be explored. To understand the biological functions of the severing protein, antibodies will be prepared and used to disrupt its function in cell extracts and in living cells. Homologous proteins in yeast or Dictyostelium will be identified and their genes disrupted, as another strategy to identify the physiological functions of this class of proteins. In addition to work on the severing protein, a novel capping protein that stabilizes microtubule minus-ends in cells will be purified and characterized. This protein may contribute to the nucleation, stability, and organization of microtubules in a variety of eukaryotic cells. Together, these studies will characterize two important protein modulators of the microtubule dynamics, which will constitute an important step in understanding the organization of the microtubule cytoskeleton at a molecular level.
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