Investigating the Role of Importin Binding in Regulation of TPX2 Phase Transition

Abstract

The rapid and accurate assembly of microtubules that form the mitotic spindle is necessary to ensure proper chromosome segregation during cell division. This occurs through the coordination of many spindle assembly factors (SAFs). One important SAF is TPX2, a protein that promotes microtubule nucleation throughout the spindle. TPX2 can undergo phase transition to form liquid-like droplets. This droplet formation is thought to enhance TPX2’s ability to promote microtubule nucleation by increasing the local concentrations of TPX2 and tubulin, which are otherwise diffuse in the cytoplasm. Importins inhibit TPX2 phase transition. This study aims to explore how TPX2 is regulated by probing the interactions between TPX2 and importins. Importins are known to interact with TPX2 via two previously-identified four-amino-acid motifs known as nuclear localization sequences (NLSs). However, bioinformatic sequence analysis suggested that TPX2 may contain more NLSs. To explore the role of NLS interactions in regulation of TPX2 by importins, we created a construct of TPX2 with mutations at these two previously-identified NLSs (TPX2 DM). We show that TPX2 DM interacts with importins as well as unmutated TPX2 and that our mutations have no effect on TPX2’s ability to undergo a phase transition. This demonstrates that importins likely interact with TPX2 via non-annotated NLSs or non-NLS importin-binding sequences, allowing importins to coat TPX2 molecules and prevent phase separation. This research may help inform future studies exploring how importins interact with other SAFs to regulate their activity by preventing phase transition.