STRUCTURE AND DYNAMICS OF ORGANIZED MICROTUBULE ARRAYS

The work in this thesis was directed towards understanding some of the factors involved in the spatial organization and dynamics of the microtubule cytoskeleton. Initially, we isolated centrosomes from mammalian cells in order to study their microtubule nucleation properties. These organelles were purified five thousand fold, to give a stable preparation retaining both morphological and functional attributes. Studying the nucleation process by electron microscopy, we discovered that the centrosome can influence the structure of nucleated microtubules, constraining the lattice to the physiological number of protofilaments.

Further studies on microtubule nucleation by centrosomes in vitro uncovered some unexpected features of microtubule growth. These included the coexistence of shrinking and growing microtubules, and nucleation below the critical concentration for bulk assembly. These observations were extended to free microtubules resulting in a new model for microtubule assembly kinetics, which we have termed dynamic instability. The essence of this model is that microtubules require a cap of GTP liganded subunits in order to elongate, and thus are only transiently stable, and highly dynamic.

Next we turned to the other main mammalian microtubule organizing center, the kinetochore, which is responsible for attaching chromosomes to the mitotic spindle. We have demonstrated five distinct interactions between the kinetochores of isolated chromosomes and tubulin in vitro. These are microtubule nucleation, tubulin binding, microtubule capture, microtubule capping, and ATP dependent translocation. The capture reaction, combined with dynamic instability, suggested a new model for spindle morphogenesis. The role of the ATP dependent translocation reaction is not yet clear, since it seems to be directed towards microtubule plus ends, and thus away from the spindle pole in vivo. It may play a role in spindle morphogenesis and maintaining steady state dynamics during metaphase.