Spindle orientation is important in multiple developmental processes as it determines cell fate and function. The orientation of the spindle depends on the assembly of a proper astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates astral microtubules. TPX2 in the spindle pole area is activated by GM130 on Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by competing for importin α binding. Mitotic phosphorylation of importin α at Ser62 by CDK1 switches its substrate preference from TPX2 to GM130, thereby enabling competition-based activation. Importin α S62A impedes local TPX2 activation and compromises astral microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130-importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for TPX2 in the organization of astral microtubules. Furthermore, we show that the substrate preference of the important mitotic modulator importin α is regulated by CDK1-mediated phosphorylation.

Spindle orientation is important in multiple developmental processes as it determines cell fate and function. The correct orientation of the mitotic spindle depends on the assembly of a proper astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates the astral microtubule network. TPX2 in the spindle pole area is activated by GM130 on mitotic Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by competing for importin α binding. During mitosis, phosphorylation of importin α at Serine 62 by Cdk1 switches its substrate preference from TPX2 to GM130, thereby enabling competitionbased activation. Importin α S62A impedes local TPX2 activation and compromises astral microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for TPX2 in the organization of astral microtubules. Furthermore, we show that the substrate preference of the important mitotic modulator importin α is regulated by Cdk1 phosphorylation.