An acentrosomal aster with atypical microtubule polarity recruits cytokinesis signals to its center in <i>Xenopus</i> egg extracts

Xenopus egg extracts can self-organize into cell-like compartments without the classic microtubule organizer centrosome. Compartment formation requires microtubules, but the organization of microtubules throughout the process remains unclear. Here, we show that the earliest organized microtubule structures to emerge during cell-like compartment formation are centrosome-independent asters. In contrast to the microtubule orientation of a centrosome-nucleated aster, most microtubules in the centrosome-independent aster point their plus ends toward the center. Formation of these asters requires the microtubule motor MKLP2 (also known as KIF20A) and Aurora kinase B activity. The aster center accumulates microtubule plus end-binding protein EB1-GFP and the plus end-tracking motor kinesin-1-GFP, and also recruits cytokinesis-related proteins GFP-MKLP1 (KIF23), active RhoA, and F-actin. Together, our findings identify an early microtubule structure in cell-like compartment self-organization and link it to the cytokinesis pathway.