An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

Abstract

Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly

“…In other species, CENP-T is thought to form a distinct CDKdependent kinetochore assembly pathway that works in parallel to the CENP-C assembly pathway to recruit the Ndc80 and Mis12Cs (Akiyoshi et al, 2013;Kim and Yu, 2015;Rago et al, 2015;Lang et al, 2018). In Xenopus egg extracts, both CENP-T and CENP-C contribute to outer kinetochore assembly, which suggests a similar dichotomy (Krizaic et al, 2015).…”

Reconstitution of human kinetochore in mitotic cell extracts reveals permitted and restricted assembly steps

“…Inspired by previous kinetochore reconstruction studies in yeast and frog egg systems (Akiyoshi et al 2010, Guse et al 2011, Haase et al 2017, Lang et al 2018, Bonner et al 2019, Hamilton et al 2020, we applied analogous strategies to investigate the assembly and microtubule-binding activities of the cytoplasmic pool of kinetochore proteins in mitotically arrested human cells ( Figure 7A). Native GFP-fused kinetochore proteins, such as the Ndc80 and Mis12 complexes and the CENP-T and CENP-C scaffold, were present at concentrations of 10-100 nM in the soluble fraction of extracts prepared from HeLa cells stably expressing these proteins.…”

“…Work in other systems identified phosphorylation of Dsn1 by Aurora B kinase as a key step that partially or completely relieves Mis12 autoinhibition, promoting its binding to kinetochore-localized CENP-C, followed by recruitment of Ndc80 complex and other outer kinetochore proteins (Yang et al 2008, Kim et al 2015, Rago et al 2015, Lang et al 2018, Bonner et al 2019. Indeed, when we used mitotic cell extracts containing autoinhibition-deficient Mis12 complex (GFP-Dsn1-Δ91-113), which localizes constitutively to HeLa kinetochores, binding of Mis12 complexes to the CENP-A-coated beads was noticeably improved relative to wild-type Mis12.…”

“…However, the activity of the soluble Aurora B kinase pool toward the Mis12 complex may be insufficient for robust Mis12 activation (Bonner et al 2019). In Xenopus, Saccharomyces cerevisiae, Kluyveromyces lactis, chicken and human systems the requirement of Mis12 phosphorylation for CENP-C binding can be bypassed by introducing the truncations or phosphomimetic substitutions at the Dsn1 N-terminus, thereby generating an autoinhibition-deficient Mis12 complex (Akiyoshi et al 2013, Kim et al 2015, Rago et al 2015, Hara et al 2018, Lang et al 2018, Bonner et al 2019, Hamilton et al 2020).…”

“…Indeed, Mis12 complex containing phospho-mimetic substitutions in Dsn1 (S77E, S84E) allows robust assembly of functional kinetochores in Xenopus egg extracts even in the absence of Aurora B activity (Bonner et al 2019). In yeast cell extracts, kinetochores can be assembled de novo using a 125-bp centromeric DNA as a template (Lang et al 2018). In this system, recruitment of KMN components was increased by up to 7-fold using extracts from cells with phospho-mimetic mutations in Dsn1 (S240D, S250D).…”