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Open Access Dissertation
Doctor of Philosophy (PhD)
Molecular and Cellular Biology
Year Degree Awarded
Month Degree Awarded
Biochemistry, Biophysics, and Structural Biology | Cell Biology
Cell division is the fundamental process by which the replicated genetic material is faithfully segregated to form two identical daughter cells. The mitotic spindle is the macromolecular cytoskeletal structure that is built during every round of cell division to successfully separate the duplicated genome equally into the daughter cells. Errors in spindle formation can thus causegenetic aberrations and can potentially lead to cancer. Understanding the mechanisms that govern proper spindle assembly and function is thus important. Eg5 and Kif15 are two important kinesins which play a major role in establishing and maintaining bipolarity of the mitotic spindle. Both Eg5 and Kif15 have been shown to be regulated by the spindle assembly factor Targeting Protein for Xklp2, or TPX2 the mechanistic details of which remains less clear. The studies presented in this dissertation are aimed at understanding how TPX2 regulates Eg5 and Kif15 using a combination of in vitro reconstitution experiments and live cell imaging.
The microtubule co-sedimentation experiments show that removal of the Eg5 interaction domain located on the C-terminus of TPX2 does not abolish the microtubule binding ability of TPX2. My data show that the microtubule binding of TPX2 is vii electrostatic but does not involve the negatively charged tubulin E-hook region. In in vitro reconstitution Total Internal Reflection Fluorescence (TIRF) experiments, the Eg5-EGFP molecules derived from mammalian cells extracts display biophysical properties similar to the purified Eg5-EGFP molecules. In single molecule TIRF assays, full length TPX2 inhibited Eg5 motion on microtubules and removal of the Eg5 interaction domain from the C-terminus of TPX2 (TPX2-710) significantly reduced the inhibitory effect of TPX2 on Eg5. Data from microtubule surface gliding assays using monomeric and dimeric Eg5 molecules show that dimerization of Eg5 or the residues located in the neck and stalk region of Eg5 are important for the interaction of TPX2 with Eg5. These results suggest that both microtubule binding and ability of TPX2 to interact with Eg5 contribute to the regulation of Eg5 by TPX2.
My data show that the presence of C-terminus of TPX2 enhances Kif15 recruitment of Kif15 onto spindle microtubules and is also required for Eg5 independent bipolar spindle assembly. Characterization of Kif15-GFP molecules from cell extracts suggest that the motor molecules exist as tetramers. In single molecule TIRF experiments, only full length TPX2 suppresses Kif15 motor walking but not the C-terminally truncated TPX2-710. In live cells, fluorescent Kif15-GFP puncta stream towards microtubule plus-ends at rates consistent with microtubule growth rates. Treatment with Paclitaxel suppresses the motility of Kif15 puncta suggesting that dynamic microtubules contribute to the Kif15 behavior in cells. These results offer some mechanistic insights into how TPX2 regulates both the motors Eg5 and Kif15 through its C-terminus.
Balchand, Sai Keshavan, "MECHANISM OF REGULATION OF KINESINS EG5 AND KIF15 BY TPX2" (2018). Doctoral Dissertations. 1152.
Available for download on Wednesday, August 01, 2018