(742i) Inhibition of TPX2 Condensation By Importins

Spindle Assemble Factors (SAFs) play essential role in generating and regulating microtubules nucleation during cell division. SAFs are transported to the nucleus by a family of transporter proteins known as Importins. One important SAF for microtubules nucleation is TPX2, which binds via nuclear localization sequence (NLS) (residues 270-370) to importin-a mediated by importin-b. Recently our lab has discovered that the long disordered-N terminus region of TPX2 promotes liquid-liquid phase separation of GFP-TPX2 in vitro under physiological conditions. Interestingly, addition of importin-ab can prevent this condensation. In this study, we explore the mechanism, by which individual importins control TPX2 condensation. Our study identifies a second NLS sequence, rich in lysine residues located at 120-125 region of TPX2, which can bind to Importin-a alone (Kd~ 30 nM). Furthermore, we discover that without presence of Importin-a, TPX2 exhibits only non-specific weak binding to Importin-b (Kd~400 nM) and the C-terminus of TPX2 exhibits super weak binding to both importins (Kd > 2uM). With the knowledge of interaction strength of TPX2-importins, we investigated how individual importins regulate TPX2 condensation. Surprisingly, we found out that Importin-a with two strong NLS sequence can not inhibit TPX2 condensation, whereas weak non-specific interactions of Importin-b-TPX2 is sufficient to block TPX2 condensation. To gain a better understanding of molecular picture, we applied size exclusion chromatography in line with light scattering and discovered that TPX2 exists as an ensemble of metastable oligomers at lower temperatures. Addition of negatively charged Importin-a (pI ~ 5.4) facilitates charge neutralization and rapid phase separation of TPX2. By contrast, Importin-b can stabilize these oligomers and completely inhibit phase separation. Our work can potentially introduce new insights in preventing protein aggregation disease.