Interaction of spindle assembly factor TPX2 with importins-α/β inhibits protein phase separation

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-alpha/beta until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-alpha/beta with nanomolar affinity in a 1:1:1 mono dispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-alpha. In addition, we establish that TPX2 interacts with importin-beta via dispersed, weak interactions. We show that interactions of both importin-alpha and -beta with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.

Automated summary

The study revealed high-affinity interactions between TPX2 and importins α/β, as well as a new nuclear localization sequence in TPX2. Additionally, it was found that interactions of importin α and β with TPX2 inhibit its ability to undergo phase separation.