Optimizing TACC3 protein purification and characterizing TACC3 regulation by importin-α and importin-β

Abstract

TACC3 (Transforming Acidic Coiled-Coil protein 3) is a conserved mitotic protein that plays a key role in microtubule nucleation and spindle organization during cell division. It belongs to a class of proteins known as spindle assembly factors (SAFs), which promote microtubule formation and ensure proper spindle assembly during mitosis. SAFs are tightly regulated by nuclear import receptors to prevent premature activation and maintain mitotic timing and fidelity. In this thesis, I investigated the regulation of Xenopus laevis TACC3 by the nuclear import receptors importin-α and importin-β. To do this, I generated a GFP-tagged TACC3 fusion construct, expressed and purified the protein using affinity and size exclusion chromatography, and performed pulldown assays to test for direct interactions with importin-α, importin-β, and a truncated ∆IBB-importin-α variant under various salt and concentration conditions. My results show that TACC3 specifically interacts with importin-β, but not with importin-α or ∆IBB-importin-α. The interaction is salt-sensitive and concentration-dependent, indicating it is electrostatic in nature and relatively weak. A GFP-only negative control confirmed that importin-β binding is specific to the TACC3 portion of the fusion protein. These findings challenge the traditional model in which SAFs are regulated via direct interaction with importin-α and instead suggest that TACC3 may be controlled directly by importin-β. This mechanism resembles importin-β–mediated regulation of other SAFs such as TPX2, and may reflect a broader regulatory pathway for spindle assembly. Further work is needed to map the binding interface, assess the role of regulatory factors like Ran–GTP, and ascertain any post-translational modifications. Overall, this study reveals a novel regulatory interaction between TACC3 and importin-β that advances our understanding of SAF control during mitosis.