Functional and Structural Validation of a Novel Nuclear Localization Sequence within the Microtubule Effector TPX2

Abstract

Living organisms rely on mitosis for their growth and proliferation. During mitosis, a highly dynamic network of microtubules comes together to form the mitotic spindle, which drives chromosome segregation. Targeting Protein for Xklp2, or TPX2, contributes to the formation of the mitotic spindle by promoting the generation of new microtubules from preexisting microtubules, a process known as branching microtubule nucleation. Understanding the role and regulation of TPX2 during mitosis can guide our treatment of diseases that involve aberrant mitotic behavior, such as cancer. TPX2 is primarily regulated by the Importin-a/b heterodimer, a nuclear transport factor that sequesters TPX2 to the cell nucleus. Previously, Importin-ɑ was thought to bind to TPX2 via two nuclear localization signals, or NLSs, spanning amino acid residues 280-330 (NLS12) on the N-terminal region of TPX2. However, recent findings from the Petry Lab suggest that there is a third NLS spanning amino acid residues 120-130 (NLS3) on TPX2 that exhibits strong binding to Importin-ɑ. In this thesis, I assessed the ability of the novel NLS to bind to Importin-a. I designed three constructs of TPX2 containing different combinations of these three NLSs: TPX2-NLS3, TPX2-NLS12, and TPX2-NLS312. My results indicate that constructs TPX2-NLS12 and TPX2-NLS3 are able to form a co-complex with Importin-a, validating NLS3 as a new NLS. Additionally, I set forth a method for TPX2 and Importin-a purification that allows for stoichiometric manipulation of the protein mixture. This suggests that the crystallization of this co-complex is possible. The resulting co-complexes from these purifications were set up for crystallization trails using the hanging drop vapor diffusion method to gain structural insight into this new interaction central to the regulation of mitosis.