Insights from biochemical reconstitution into the architecture of human kinetochores

Abstract

Chromosomes are carriers of genetic material and their accurate transfer from a mother cell to its two daughters during cell division is of paramount importance for life. Kinetochores are crucial for this process, as they connect chromosomes with microtubules in the mitotic spindle. Kinetochores are multi-subunit complexes that assemble on specialized chromatin domains, the centromeres, that are able to enrich nucleosomes containing the histone H3 variant centromeric protein A (CENP-A). A group of several additional CENPs, collectively known as constitutive centromere associated network (CCAN), establish the inner kinetochore, whereas a ten-subunit assembly known as the KMN network creates a microtubule-binding site in the outer kinetochore. Interactions between CENP-A and two CCAN subunits, CENP-C and CENP-N, have been previously described, but a comprehensive understanding of CCAN organization and of how it contributes to the selective recognition of CENP-A has been missing. Here we use biochemical reconstitution to unveil fundamental principles of kinetochore organization and function. We show that cooperative interactions of a seven-subunit CCAN subcomplex, the CHIKMLN complex, determine binding selectivity for CENP-A over H3-nucleosomes. The CENP-A:CHIKMLN complex binds directly to the KMN network, resulting in a 21-subunit complex that forms a minimal high-affinity linkage between CENP-A nucleosomes and microtubules in vitro. This structural module is related to fungal point kinetochores, which bind a single microtubule. Its convolution with multiple CENP-A proteins may give rise to the regional kinetochores of higher eukaryotes, which bind multiple microtubules. Biochemical reconstitution paves the way for mechanistic and quantitative analyses of kinetochores.

How Kinetochore Architecture Shapes the Mechanisms of Its Function

“…This interface is assembled by two parallel pathways initiated by the proteins CENP-C Mif2 and CENP-T Cnn1 [16–20]. CENP-C Mif2 and CENP-T Cnn1 are assembled on a well-defined territory on each chromosome, known as the centromere, through their interactions with the centromere-specific histone H3 variant CENP-A Cse4 [9, 19, 21]. Although the CENP-C Mif2 and CENP-T Cnn1 pathways are conserved, their contribution to kinetochore function is species-specific: both pathways are required for the function of the human kinetochore, whereas only the CENP-C Mif2 pathway is required in budding yeast [22].…”

“…Therefore, the architecture-function relationships derived from budding yeast will provide insight into the operation of the highly complex human kinetochore. Indeed, a recent study proposed an elegant conceptualization of the human kinetochore as the two-dimensional convolution of multiple yeast kinetochore-like subunits over a disk-shaped surface [9]. Nevertheless, the human kinetochore is built for entirely different performance specifications: it must coordinate the activities of its multiple microtubule binding sites to move the chromosome over longer distances (~ 5 μm versus < 0.5 μm in budding yeast, ref.…”

Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

“…Coupling performance improved with the incorporation of additional microtubule-binding kinetochore elements [153,154], with the use of native kinetochore particles isolated from yeast [43], and with the use of flexible tethers for linking sub-complexes to beads [155]. Further improvements seem likely, especially as continued advancements in kinetochore biochemistry enable reconstitutions of ever more complete and stable kinetochore assemblies [156][157][158]. However, the performance achieved in laser trap tip-coupling assays already provides a reasonably good match to physiological conditions.…”

A time out for CENP-A

“…Increasing evidence points toward the existence of an interdependent protein interaction meshwork formed by the centromeric proteins. 8,9 This suggests that following CENP-A depletion, the kinetochore might also be anchored by adjacent H3 nucleosomes or, likely, by the CENP-T/W/S/X complex. The spatial arrangement of CENP-A and H3 nucleosomes at the centromeric region is unknown.…”

“…10 However, the existence of point centromeres in budding yeast and the discovery that the centromere complex remains intact shortly after CENP-A depletion 4 support the idea that in mitosis, a single CENP-A molecule is sufficient to generate a microtubule-capturing centromere-kinetochore complex in humans, as proposed following in vitro reconstitution. 9 …”