Higher-order assembly is a regulatory switch that promotes outer kinetochore recruitment

To faithfully segregate chromosomes during vertebrate mitosis, kinetochore-microtubule interactions must be restricted to a single site on each chromosome. Prior work on pair-wise kinetochore protein interactions has been unable to identify the mechanisms that prevent kinetochore formation in regions with a low density of CENP-A nucleosomes. To investigate the impact of higher-order assembly on kinetochore formation, we generated defined oligomers of the inner kinetochore protein CENP-T using two distinct, genetically engineered systems in human cells. Although individual CENP-T molecules interact poorly with other kinetochore proteins, oligomers that mimic the centromeric density of CENP-T trigger the robust formation of functional, cytoplasmic kinetochore-like particles. Both in cells and in vitro, each molecule of oligomerized CENP-T recruits substantially higher levels of outer kinetochore components than monomeric CENP-T molecules. Thus, the density-dependence of CENP-T restricts outer kinetochore recruitment to centromeres, where densely packed CENP-A recruits a high local concentration of CENP-T.