Branching microtubule nucleation is controlled by importin-mediated inhibition of TPX2 phase separation

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed by several microtubule nucleation pathways. In the spindle center, Targeting Protein for XKlp2 (TPX2) promotes branching microtubule nucleation, where new microtubules are nucleated from pre-existing ones. Until the onset of spindle assembly, TPX2 is sequestered by importins-/{beta}, yet the molecular nature of this regulation remains unclear, particularly since TPX2 was recently found to undergo a liquid-liquid phase separation to execute its function. Here we demonstrate that TPX2 interacts with importins-/{beta} with nanomolar affinity as a 1:1:1 mono-dispersed trimer. We identify a new nuclear localization sequence (NLS) on TPX2, which contributes to its high-affinity interaction with importin-. Interestingly, importin-{beta} alone can also associate with TPX2, and does so via dispersed, weak interactions. Interactions of both importin- and importin-{beta} with TPX2 each inhibit its propensity for phase separation, and consequently its ability to orchestrate branching microtubule nucleation. In sum, our study explains how TPX2 is regulated in order to facilitate spindle assembly, and provides novel insight into how a protein phase separation can be inhibited via weak biomolecular interactions. Significance StatementThe discovery that proteins can undergo phase separation is revolutionizing biology. Characterization of dozens of phase separating proteins in vitro over the past several years has mainly focused on how macromolecules undergo liquid-liquid phase separation (LLPS). The next, and possibly bigger challenge is to investigate how LLPS is regulated in the cell, namely how it is inhibited to spatiotemporally control a certain cellular function. Here, we addressed this challenge by identifying how the spindle assembly factor TPX2 is inhibited by importins from undergoing LLPS and thereby turning on spindle assembly.