Reconstitution of phospho-regulated mitotic chromatid assembly and disassembly

Exactly how cell cycle regulators control a sequential series of mitotic events is not fully understood. Here we report reconstitution assays that recapitulate the assembly and disassembly of mitotic chromatids in vitro using a minimal set of recombinant proteins, including condensin I. By incorporating cyclin B-Cdk1 and PP2A-B55, this system enables us to dissect the phospho-regulation of condensin I in these processes. We provide evidence that the terminal intrinsically disordered regions (tIDRs) of the non-SMC subunits suppress condensin I activity, and that this self-suppression is relieved by Cdk1 phosphorylation. Importantly, full activation of condensin I requires the phosphorylation of a conserved residue located in the central region of the kleisin subunit CAP-H. Conversely, PP2A-B55 induces dissociation of condensin I from reconstituted chromatids, leading to their disassembly. Complementary analyses using Xenopus egg extracts reveal that the tIDRs and the kleisin central region are phosphorylated and dephosphorylated with distinct kinetics during mitotic entry and exit. Together, these findings define an intricate regulatory network that coordinates chromatid assembly and disassembly with mitotic progression.