Impact of liquid-liquid phase separation on microtubule organization by spindle associated protein, PRC1

The cytoskeleton is a network of protein filaments including microtubules, actin filaments, and intermediate filaments, that interacts with an array of associated proteins to assemble and regulate sub-cellular structures such as the mitotic spindle. New research has been focused on the connection between cytoskeletal assemblies and liquid-liquid phase separation (LLPS) as a mechanism for their regulation and organization. Microtubules have a rich assortment of associated proteins (MAPs) that are known to undergo LLPS in vitro and in vivo, though the link between LLPS and the cytoskeleton is not fully characterized. In this study, we examine the in vitro LLPS and microtubule organization capabilities of PRC1: an antiparallel microtubule crosslinker from the MAP65/Ase1 family that is associated with the midzone of the metaphase and anaphase mitotic spindle. MAP65 and PRC1 have recently been found to form droplets that can bind free tubulin to nucleate and grow microtubule tactoids and asters, depending on the size. Using a variety of PRC1 constructs containing different combinations of PRC1s structural domains, we seek to uncover the mechanism by which PRC1 undergoes LLPS and organizes microtubules. We find that the c-terminal intrinsically disordered domain aids in LLPS and droplet formation is needed for microtubule organization.