Chromosomal passenger complex condensates generate parallel microtubule bundles in vitro

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• The mitotic spindle relies on microtubule (MT) bundles like midzones and kinetochore fibers.
• Mechanisms of MT bundle formation within the mitotic spindle remain largely unknown.

Purpose of the Study:

• To investigate the role of the chromosomal passenger complex (CPC) in the in vitro formation of microtubule bundles.
• To elucidate the self-assembly properties of CPC and its interaction with microtubules.

Main Methods:

• Purification of the chromosomal passenger complex (CPC) from Escherichia coli.
• In vitro reconstitution assays with purified CPC, tubulin, and GTP to observe MT bundle formation.
• Analysis of CPC mutants with altered liquid-liquid demixing or MT-binding properties.
• Microscopy and imaging techniques to visualize MT structures and CPC localization.

Main Results:

• Purified CPC undergoes liquid-liquid demixing in vitro, forming condensates.
• These CPC condensates generate parallel microtubule bundles from free tubulin and GTP.
• MT bundles emerge from CPC droplets with protruding minus ends, growing into tapered structures.
• CPC reorganizes within condensates to coat and bundle the growing MTs.
• CPC mutants deficient in demixing or MT binding failed to produce MT bundles in vitro and showed reduced MTs at spindle midzones in HeLa cells.

Conclusions:

• In vitro biochemical activity for MT bundle production emerges upon CPC concentration.
• CPC concentration and liquid-liquid demixing are critical for generating parallel MT bundles.
• The study provides models for cellular generation of parallel-bundled MT structures essential for mitotic spindle assembly.
• Data suggest the existence of MT-organizing centers that produce MT bundles with opposite polarity to centrosomes.