Differential Targeting of Cytoplasmic Dynein During Mitosis by Site Specific Phosphorylation

Master's Thesis
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Abstract

The motor protein cytoplasmic dynein has been implicated in mitotic functions that require dynein localization to specific cellular locations. Recent studies in our lab have focused on the hypothesis that phosphorylation events determine the targeting of dynein to kinetochores and spindle poles. We previously identified a novel phosphorylation site at threonine 89 (T89) of the dynein intermediate chain that targets dynein to the kinetochore during mitosis (Whyte et al., 2008). In this study, we analyzed a second novel dynein phosphorylation site at tyrosine 130 (Y130) that results in dynein localization to spindle poles. We also examined the contributions of the spindle assembly checkpoint (SAC) protein Spindly to regulation of dynein kinetochore transitions. Preliminary experiments defined pY130-dynein as separate from the kinetochore dynein that functions as part of the spindle assembly checkpoint. To define the requirements for pY130-dynein targeting to spindle poles, we disrupted candidate dynein binding proteins and assessed localization of pY130-dynein. Dominant negative FAK, FRNK, ASAP, NudEL and CenpF constructs induced multipolar and fragmented spindle poles, but pY130-dynein targeting to spindle poles was unaffected. These results suggest a model in which specific proteins at spindle poles are necessary for dynein function, whereas other spindle pole proteins are required for dynein recruitment. Mass spectrometry analysis of isolated centrosomes confirmed the presence of pY130-dynein at centrosomes and suggests that pY130-dynein associates with the inner centrosome. Altogether, these results provide insight into the potential interactions of pY130-dynein at spindle poles. Spindly is a novel spindle assembly checkpoint protein and that has been recently implicated recruitment of dynein to kinetochores; however, the specific contributions of Spindly to dynein regulation and silencing of the spindle assembly checkpoint (SAC) are unclear. In this study, the effects of Spindly depletion on different populations of kinetochore dynein, and dynein poleward motility were measured. Together with previous studies demonstrating the ability of dynein to translocate to spindle poles in the absence of dynactin, these results of this work implicate Spindly in a novel dynactin-independent hand-off mechanism at kinetochores in which Spindly is required for dynein poleward motility. Overall, these findings provide a more thorough understanding of the role of phosphorylation in dynein differential targeting and function during mitosis.

Attributes

Attribute NameValues
URN
  • etd-03092011-173823

Author Colleen Therese Cole
Advisor Kevin T. Vaughan
Contributor Kevin T. Vaughan, Committee Chair
Contributor Zachary T. Schafer, Committee Member
Contributor Robert A. Schulz, Committee Member
Degree Level Master's Thesis
Degree Discipline Biological Sciences
Degree Name MS
Defense Date
  • 2011-03-09

Submission Date 2011-03-09
Country
  • United States of America

Subject
  • mitosis

  • phosphorylation

  • dynein

Publisher
  • University of Notre Dame

Language
  • English

Record Visibility and Access Public
Content License
  • All rights reserved

Departments and Units

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