Investigating RSA-1-dependent PP2A functions in C. elegans mitotic spindle assembly

  • Author / Creator
    Lange, Karen I
  • Spindle assembly pathways are conserved in many organisms. Defects in spindle assembly can result in aneuploidy, a common cause of cell death and cancer. A better understanding of spindle assembly pathways is required to fully appreciate how defects in spindle assembly manifest as aneuploidy and cancer. Phosphorylation of spindle assembly proteins is a key mechanism to regulate this process. Phosphatases, such as protein phosphatase 2A (PP2A), dephosphorylate these proteins to precisely regulate their activity. The substrate specificity and subcellular localization of the PP2A complex is regulated by protein interactions with regulatory subunits. In C. elegans embryos, a PP2A regulatory subunit, RSA-1, is required for mitotic spindle assembly. rsa-1(lf) embryos exhibit a reduction in the number of microtubules at the centrosomes. Loss of rsa-1 causes a dramatic collapse of the mitotic spindle and, subsequently, a failure in chromosome segregation that results in embryonic lethality. Although it is expected that RSA-1 promotes dephosphorylation of specific proteins important for spindle assembly and/or function, bona fide substrates have not been verified. Potential targets include the microtubule depolymerase, KLP-7, and the microtubule stabilizer, TPXL-1. In rsa-1(lf) embryos, centrosomes exhibit increased levels of KLP-7 but decreased levels of TPXL-1, suggesting a potential molecular basis for the rsa-1 phenotypes. However, it is not clear how RSA-1 regulates KLP-7 and TPXL-1. To investigate the function of the RSA-1/PP2A complex, I performed a genetic screen for suppressors of a temperature-sensitive missense allele, rsa-1(or598). This screen recovered interaction suppressor mutations that altered amino acids within either the RSA-1 regulatory subunit itself or the PP2A structural subunit, PAA-1. These mutations highlight key residues that likely play a role in modulating the regulatory subunit preference of the PP2A holoenzyme. Through my characterization of RSA-1 function in embryos using microscopy-based experiments, I discovered a novel role for RSA-1 in localizing a major mitotic kinase, Aurora A, to microtubules. Furthermore, I propose a model whereby RSA-1 is required for an interaction between Aurora A and TPXL-1, which promotes microtubule localization of the kinase. I also observed that TPXL-1 is located on the chromatin in C. elegans embryos in an RSA-1, Aurora A, and RanGTP-dependent manner. I propose that TPXL-1 at the chromatin promotes spindle assembly in a manner similar to nuclear TPX2 in vertebrates.

  • Subjects / Keywords
  • Graduation date
    Spring 2015
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Specialization
    • Molecular Biology and Genetics
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Dave Pilgrim (Biological Sciences)
    • Frank Nargang (Biological Sciences)
    • Jean-Claude Labbe (Pathology and Cell Biology, University of Montreal)
    • Gordon Chan (Oncology)