Assembly principles and stoichiometry of a complete human kinetochore module

Abstract

Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes, and permits further assembly of all other kinetochore subunits in vitro with relative ratios that closely match those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module, and may have important implications for trans-generational inheritance of centromeric chromatin.

The Aurora B gradient sustains kinetochore stability in anaphase

“…Indeed, Cyclin B1 destruction reaches completion in early anaphase 17,34 , and many Cdk1-generated phosphosites are removed rapidly in anaphase 35 , including in the N-terminal region of CENP-T 8 . Third, the affinity of Aurora B-phosphorylated Mis12C for CENP-C and Cdk1-phosphorylated CENP-T is similar, as reported 21 . The model can recapitulate key features of the results, such as the similar kinetics of Dsn1-WT and Dsn1-EE dissociation from kinetochores in early anaphase (likely largely due to loss of Cdk1-dependent CENP-T binding), and the failure of approximately 50% of Dsn1 to dissociate from kinetochores in telophase when the half-life of Aurora B-dependent Mis12C is increased (Figure S4a), as seen for the Dsn1-EE mutant (Figure 4c).…”

“…Two mechanisms each maintain up to 50% of Mis12C (containing Dsn1) at kinetochores prior to anaphase: binding to CENP-C or CENP-T 11,33 . The phosphorylation of Dsn1 at S100 and/or S109 by Aurora B is required for the stable association of Mis12C with both CENP-C and CENP-T 3,5-7,21 . Dsn1 S100/S109 phosphorylation relieves the inhibitory effect of a basic region of Dsn1 on the interaction with CENP-C and CENP-T 7,12,19-21 .…”

“…The phosphorylation of Dsn1 at S100 and/or S109 by Aurora B is required for the stable association of Mis12C with both CENP-C and CENP-T 3,5-7,21 . Dsn1 S100/S109 phosphorylation relieves the inhibitory effect of a basic region of Dsn1 on the interaction with CENP-C and CENP-T 7,12,19-21 . The interaction of Mis12C with CENP-T, but not with CENP-C, is additionally dependent on phosphorylation of CENP-T by Cyclin B-Cdk1 8-12,21 .…”

“…Dsn1 S100/S109 phosphorylation relieves the inhibitory effect of a basic region of Dsn1 on the interaction with CENP-C and CENP-T 7,12,19-21 . The interaction of Mis12C with CENP-T, but not with CENP-C, is additionally dependent on phosphorylation of CENP-T by Cyclin B-Cdk1 8-12,21 . To determine whether, like Dsn1 S109ph, total Dsn1 is also found in a gradient, we compared the distribution of phosphorylated and total Dsn1 in HeLa cells undergoing normal anaphase.…”

“…However, it is striking that key processes that control kinetochore assembly and function are driven by the activity of Cyclin B-Cdk1, which strongly declines at the metaphase to anaphase transition 16,17 , and Aurora B, which dissociates from centromeres and transfers to the central spindle in anaphase 18 . For example, the phosphorylation of the KMN (Knl1 complex, Mis12 complex, Ndc80 complex) protein Dsn1 at S100 and/or S109 by Aurora B is required for the Mis12 complex (Mis12C) to stably associate with the constitutive centromere-associated network (CCAN) proteins CENP-C and CENP-T 3,5-7,12,19-21 . However, it is widely believed that the function of Aurora B at kinetochores ceases in anaphase 15,22-24 .…”