Chromosomal passenger complex condensates generate parallel microtubule bundles in vitro

The mitotic spindle contains many bundles of microtubules (MT) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the Chromosomal Passenger Complex (CPC) purified from E. coli undergoes liquid-liquid demixing (LLD) in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, we found the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLD or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce microtubule bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain microtubule organizing centers that generate microtubule bundles that emerge with the opposite polarity from centrosomes.