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Regulation of spindle stability by Cdk1 and the APC

  • Author(s): Woodbury, Erika Lynne
  • Advisor(s): Morgan, David O
  • et al.
Abstract

At the onset of anaphase, changes in microtubule behavior lead to the stabilization of the mitotic spindle. Changes in the phosphorylation of microtubule-associated proteins are thought to be important for anaphase spindle stability. Here we identify the budding yeast protein Fin1 as a microtubule-stabilizing protein whose activity is strictly limited to anaphase by changes in its phosphorylation state and rate of degradation. Phosphorylation of Fin1 from S phase to early mitosis, by the cyclin-dependent kinase Clb5-Cdk1, inhibits Fin1 association with the spindle. In anaphase, when Clb5-Cdk1 is inactivated, Cdc14-dependent dephosphorylation of Fin1 targets it to the poles and microtubules of the elongating spindle, where it contributes to spindle integrity. A nonphosphorylatable Fin1 mutant (Fin15A) localizes to the spindle before anaphase and impairs efficient chromosome segregation. As cells complete mitosis and disassemble the spindle, the ubiquitin ligase APCCdh1 targets Fin1 for destruction.

To identify regions of Fin1 that are important for its localization and function, we characterized a series of Fin1 truncations. Fin1 mutants containing the N-terminal Cdk1 consensus sites, but lacking the C-terminal predicted coiled-coil domains, localized to spindle pole bodies (SPBs), but not along spindle microtubules. These mutants failed to self-associate and showed reduced binding to microtubules in vitro, yet were functional in vivo and stabilized anaphase spindles. Deletion of the Fin1 C-terminus suppressed the lethal phenotypes of Fin15A. Our findings suggest that monomeric Fin1 at SPBs contributes to anaphase spindle stability, but that Fin1 multimerization and association with spindle microtubules are required for the lethal effects of Fin15A.

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