Mitotic microtubule depolymerization and XMAP215

The work in this thesis was directed toward understanding the regulation of microtubule dynamics in the mitotic spindle, specifically focusing on microtubule depolymerization and the microtubule dynamics regulator XMAP215. Chapter 1 examines the role of microtubule depolymerization in a complex spindle microtubule behavior called poleward microtubule flux, defined as the poleward translocation of spindle microtubules coupled to net polymerization at microtubule plus ends and net depolymerization at minus ends. We present evidence here that net minus end depolymerization can be uncoupled from poleward microtubule translocation, using a combination of reagents that caused spindles made in Xenopus egg extract to elongate at the rate of microtubule translocation. Though the energy released from microtubule depolymerization can be converted to mechanical force, this result suggests that minus end depolymerization does not provide the driving force for poleward flux.; During the course of experiments described above, it became clear that Xenopus egg extract contained microtubule destabilizing activity independent of the known destabilizing factors. As detailed in Chapter 2, we purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a functional assay and identified it as XMAP215, previously identified as a prominent microtubule growth promoting protein in Xenopus extracts. The apparently contradictory behavior of XMAP215, able to increase both polymerization and depolymerization rates, led us to propose that XMAP215 might act as an anti-pause factor, destabilizing a semi-stable third state predicted to exist between growth and shrinkage. In Chapter 3, we describe our progress in exploring the molecular mechanism of XMAP215 and present evidence that XMAP215 stimulates nucleotide exchange in tubulin subunits of the microtubule lattice.