Nanoscale structural organization and stoichiometry of the budding yeast kinetochore

Abstract

Proper chromosome segregation is crucial for cell division. In eukaryotes, this is achieved by the kinetochore, an evolutionarily conserved multi-protein complex that physically links the DNA to spindle microtubules, and takes an active role in monitoring and correcting erroneous spindle-chromosome attachments. Our mechanistic understanding of these functions, and how they ensure an error-free outcome of mitosis, is still limited, partly because we lack a comprehensive understanding of the kinetochore structure in the cell. In this study, we use single molecule localization microscopy to visualize individual kinetochore complexes in situ in budding yeast. For all major kinetochore proteins, we measured abundance and position within the metaphase kinetochore. Based on this comprehensive dataset, we propose a quantitative model of the budding yeast kinetochore. While confirming many aspects of previous reports based on bulk imaging of kinetochores, our results present a somewhat different but unifying model of the inner kinetochore. We find that the centromere-specialized histone Cse4 is present in more than two copies per kinetochore along with its binding partner Mif2.

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

“…The SMLM study of the S. cerevisiae kinetochore by Cieslinski et al (Cieslinski et al, 2021), which was carried out in parallel to our work, is a perfect complement to our work. Together, the data consistently shows that kinetochores possess similar architecture when comparing the phylogenetically quite distant yeasts S. pombe and S. cerevisiae.…”

“…This is inverted for S. cerevisiae where mif2 (cnp3 CENP-C ) is essential but cnn1 (cnp20 CENP-T ) is not (Bock et al, 2012; De Wulf et al, 2003; Giaever et al, 2002; Kim et al, 2010; Meeks-Wagner et al, 1986; Schleiffer et al, 2012). This is also reflected in the protein copy numbers as mif2 (cnp3 CENP-C ) is more abundant than cnn1 (cnp20 CENP-T ) in S. c erevisiae , chicken and humans (Cieslinski et al, 2021; Johnston et al, 2010; Suzuki et al, 2015). For the COMAc, fta7 (Ame1) and mis17 (Okp1) are essential for both organisms (Hayles et al, 2013; Kim et al, 2010) and deletions of fta2 and mal2 are non-viable in S. pombe.…”

Kinetochore Architecture Employs Diverse Linker Strategies Across Evolution

“…Recent comparative analysis of the CENP-C Mif2 and CENP-Q Okp1 -U Ame1 linker modules in budding yeast revealed both as being able to produce similar forces, although the CENP-Q Okp1 -U Ame1 linkage more readily bound microtubules ( Hamilton et al, 2020 ). Linker pathways act as an oligomerization platform for the Ndc80C, where copy numbers of the Ndc80C are greater than their linker counterparts ( Joglekar et al, 2008 ; Suzuki et al, 2015 ; Dhatchinamoorthy et al, 2017 ; Cieslinski et al, 2021 ; Virant et al, 2021 ). Indeed, it is known that multivalency of the Ndc80C is required for effective tracking and force coupling ( Volkov et al, 2018 ; Takenoshita et al, 2022 ), although the critical density sufficient for kinetochore functions is unknown.…”

Functional Plasticity in Chromosome-Microtubule Coupling on the Evolutionary Time Scale

“…In an organism like S. cerevisiae , a single kinetochore module, assembled on 1-2 CENPA nucleosomes, serves as a MT receptor on each chromosome (Joglekar et al 2006; Furuyama and Biggins 2007; Cieslinski et al 2021). Hence, the single critical point of contact between the receptor and a single MT solely determines sister chromatid biorientation and their subsequent segregation.…”

“…In this study, we uncover another potential advantage associated with regional centromeres, more specifically surplus CENPA nucleosomes, in tolerating conditions sub-optimal for chromosome segregation. Although the presence of peripheral CENPA as a cloud is conserved across the point and regional centromere structures (Burrack et al 2011; Coffman et al 2011b; Lawrimore et al 2011; Scott and Bloom 2014; Wisniewski et al 2014; Cieslinski et al 2021), the opportunity to nucleate a mature kinetochore with the ability to bind to a MT, is only possible in regional centromeres that can accommodate additional CENPA nucleosomes. This adaptive edge provided by regional centromeres in enabling compensatory mechanisms may explain their recurring presence across Eukaryota (Figure S4B, Table S1).…”