Online

Thompson, Alex Frances

Cellular, Molecular, and Biomedical Sciences Graduate Program

2022

Ph. D.

Mitosis requires the control of mechanical forces generated by kinesin motors to ensure equal segregation of chromosomes into two daughter cells. Two kinesins contributing to these forces are KIF11 (or Eg5), which crosslinks and slides anti-parallel microtubules, and KIF22 (or Kid), which binds to microtubules and chromosome arms. This work addresses how post-translational modifications and pathogenic mutations alter the structure and function of KIF11 and KIF22. The homotetrameric kinesin KIF11 is subject to acetylation at lysine 146, a residue in the a2 helix of the motor domain. The effect of this acetylation was assessed at the single molecule and cellular levels using an acetylation mimetic mutant, KIF11 K146Q. In single molecule optical trapping assays, KIF11 K146Q dimers are more likely than wild type (WT) dimers to stall rather than dissociate from the microtubule under load. Based on these results, acetylated KIF11 motors would be predicted to stall microtubule sliding during spindle formation, acting as a brake and slowing pole separation. To test this prediction, mCherry (mCh) tagged KIF11 WT and K146Q motors were expressed at low levels in HeLa cells. To compare the functional activity of WT and K146Q KIF11, cells were treated with the KIF11 inhibitor monastrol, resulting in mitotic arrest and the formation of monopolar spindles. Bipolar spindle formation following monastrol washout was then imaged and measured. While spindle lengths at the completion of pole separation were similar in cells expressing mCh-KIF11 WT or K146Q, pole separation occurred at a significantly slower velocity in cells expressing mCh-KIF11 K146Q than in cells expressing mCh-KIF11 WT. This velocity difference is consistent with KIF11 acetylated at K146 stalling rather than dissociating from the microtubule and acting as a brake during pole separation. Acetylation at a2 helix lysine 146 represents a mechanism by which the activity of KIF11 may be controlled in mitotic cells. Structural changes in the a2 helix also affect the activity of KIF22. Point mutations at P148 and R149 in this domain, as well as at V475 in the tail of the motor, dominantly cause a skeletal developmental disorder. The effect of these pathogenic mutations on the function of KIF22 in mitosis was investigated. KIF22 uses plus end-directed motility and direct binding to chromosome arms to generate polar ejection forces, which contribute to chromosome congression and alignment in metaphase. Mutant KIF22 generated forces to move chromosomes toward microtubule plus ends in prometaphase, indicating that mutant motors are active. As cells proceeded through mitosis, however, mutations disrupted anaphase chromosome segregation and caused chromosome recongression, which resulted in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase, resulting in continued generation of polar ejection forces and impaired anaphase chromosome segregation. Consistent with this model, a phosphomimetic mutation in the tail of KIF22 which constitutively activates the motor phenocopied the effect of pathogenic mutations. Mimicking the phosphorylation of a2 helix residue T158 also prevented the inactivation of KIF22 in anaphase, demonstrating the importance of this region of the motor domain in controlling KIF22 activity.