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Title page for ETD etd-03232017-152918

Type of Document Dissertation
Author Landino, Jennifer Elaine
Author's Email Address jennifer.landino@vanderbilt.edu
URN etd-03232017-152918
Title Actin and Microtubule Cross-talk during Cytokinesis
Degree PhD
Department Cell and Developmental Biology
Advisory Committee
Advisor Name Title
Ryoma Ohi Committee Chair
David Bader Committee Member
Irina Kaverina Committee Member
Kathy Gould Committee Member
Matt Lang Committee Member
  • actin
  • microtubule
  • cytokinesis
  • chromosomosal passenger complex
Date of Defense 2017-03-16
Availability unrestricted
The final steps of cell division are tightly coordinated in space and time but whether mechanisms exist to couple the actin and microtubule cytoskeletons during anaphase and cytokinesis (C phase) is largely unknown. We show here that spindle midzone microtubules are stabilized as cells initiate cleavage furrow ingression. This stabilization is dependent on actomyosin contraction, suggesting that there is active coordination between furrow ingression and microtubule dynamics during C phase. Midzone microtubule stabilization also depends on the kinase activity of Aurora B, the catalytic subunit of the Chromosomal Passenger Complex (CPC), uncovering a feedback mechanism that couples furrowing with microtubule dynamics. We further show that the CPC scaffolding protein INCENP binds actin. Interaction between actin and INCENP is important for cytokinesis, and for midzone microtubule stabilization following furrow ingression. Pharmacological stabilization of midzone microtubules rescues cytokinesis in INCENP actin-binding mutant expressing cells, demonstrating that the CPC is integral for coupling furrow ingression with midzone microtubule stabilization. We also found that actin binding is required to localize the CPC to the midzone and equatorial cortex during C phase. As the sub-cellular localization of the CPC is tied to its mitotic functions we investigated the role of actin-binding in recruiting the CPC to the equatorial cortex. The transport of the CPC from the centromeres to the cell middle is thought to depend on microtubule plus-end directed transport by the kinesin Mklp2 (Kif20a), however, we observed that in the absence of motor-based transport on MTs, the CPC can still target the cortex, in manner that depends on INCNEP-actin binding. This demonstrates that the mechanisms involving both the actin and MT cytoskeletons cooperate to precisely position CPC during C phase. We also observed that Mklp2 and the CPC remain associated throughout C phase and that the Mklp2-CPC complex diffuses once it reaches midzone MT plus ends, or the cell cortex, suggesting that motor activity does not define the bulk of the dynamic behavior we observe during anaphase. We find that cortical diffusion of Mklp2-CPC relies on F-actin, suggesting INCENP-actin binding promotes cortical recruitment and diffusion. Finally, cortically-localized CPC is sufficient to rescue furrow closure in Mklp2-depleted cells, indicating that this population is functional to promote cleavage furrow ingression. Collectively, our work demonstrates that the activities of the actin and microtubule cytoskeletons are coordinated during C phase, through both cytoskeletal cross-talk and cooperative CPC positioning, in order to ensure successful cell division.
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