Chromosome segregation is a complex imperative faced by all eukaryotes, as failure to accurately distribute genetic material to daughter cells in mitosis and meiosis causes potentially lethal aneuploidy (Cimini et al., 2001; Cimini, 2008; Santaguida and Amon, 2015). Eukaryotic cells rely on a network of protein complexes (Figure 1A and Table 1) to tether centromeres to the dynamic spindle microtubules that pull them to opposite poles of the dividing cell (Musacchio and Desai, 2017).

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This network, the kinetochore, mediates the vital process of chromosome segregation by coupling microtubule dynamics to chromosome movement. The inner kinetochore binds the centromere; the outer kinetochore binds microtubules. A massive molecular machine, the kinetochore must harness the force of depolymerizing microtubules, transmit this force to the centromere, and sense aberrant attachments that fail to generate force. The kinetochore also serves as a regulatory hub for detection of unattached kinetochores and destabilization of improper kinetochore-microtubule attachments (Musacchio, 2011). Tension is the signal of correct attachment (i.e., biorientation) (Nicklas and Ward, 1994), and it directly stabilizes kinetochore-microtubule connections (Akiyoshi et al., 2010). But which of the myriad kinetochore proteins actually take part in force transmission and how much tension the kinetochore is under are unresolved questions.

Estimates of mitotic forces experienced by kinetochores in vivo span orders of magnitude (Nicklas, 1988; Fisher et al., 2009; Ye et al., 2016). In the case of budding yeast, each kinetochore contacts a single microtubule and is estimated to experience forces between 0.2 and 16 pN during metaphase (Fisher et al., 2009; Chacón et al., 2014; Suzuki et al., 2016). Purified kinetochore particles isolated from budding yeast couple to microtubule plus ends with an average rupture strength in this range: 9.1 pN (Akiyoshi et al., 2010; Miller et al., 2016). Previous studies using purified proteins have concluded that (1) Ndc80c and Dam1c are both capable of coupling to microtubules independently, although Dam1c improves microtubule coupling by Ndc80c; (2) this process is regulated by Aurora B kinase (Ipl1 in budding yeast) (Cheeseman et al., 2002; Cheeseman et al., 2006; Caldas et al., 2013; Sarangapani et al., 2013; Umbreit et al., 2014; Kim et al., 2017); (3) Ndc80c transmits force to MIND (Kudalkar et al., 2015; Dimitrova et al., 2016), and (4) the kinetochore as a whole behaves as a tension-sensitive catch bond (Akiyoshi et al., 2010). Load-bearing tip-couplers can form in vitro by self-assembly of recombinant Ndc80c (Powers et al., 2009), MIND (Kudalkar et al., 2015), and Dam1c (Asbury et al., 2006), demonstrating that these components are sufficient to recapitulate a fundamental function of the outer kinetochore. Relatively little is known about how force is transmitted to the centromeric DNA. It is not even certain which inner kinetochore proteins are involved.

There are only two essential inner kinetochore protein complexes in budding yeast: Mif2 and OA (Okp1/Ame1) (Meeks-Wagner et al., 1986; Ortiz et al., 1999; Pot et al., 2005; Hara and Fukagawa, 2017; Table 1). Mif2, the budding yeast homolog of human CENP-C, is reported to interact with centromeric nucleosomes (Xiao et al., 2017), OA (Hornung et al., 2014), and MIND (Hornung et al., 2014; Dimitrova et al., 2016). Homologous to human CENP-QU, S. cerevisiae OA interacts with the centromeric histone Cse4 and MIND, the scaffold that bridges inner and outer kinetochore (Hornung et al., 2014; Dimitrova et al., 2016; Anedchenko et al., 2019; Fischböck-Halwachs et al., 2019). Because force transmission is a vital function of the kinetochore, we hypothesized that either Mif2 or OA must transmit force through the inner kinetochore.

Here, using recombinantly purified S. cerevisiae proteins, we reconstitute kinetochore assemblies capable of tethering centromeric nucleosomes to dynamic microtubule plus-ends. We show that the assemblies are load-bearing, in the sense that they sustain tensile forces in the mitotically relevant piconewton range. In doing so, we demonstrate that there are at least two possible paths of force transmission through the inner kinetochore: the Mif2 and OA protein complexes. Each of these components can independently form load-bearing interactions with both MIND and the centromeric nucleosome.

We have reconstituted kinetochore assemblies capable of transmitting piconewton-scale forces from dynamic microtubules to Cse4-containing nucleosomes (Figure 5). To our knowledge, these are the first kinetochore assemblies made from individually purified, recombinant S. cerevisiae components that reconstitute what is arguably the kinetochore’s most fundamental activity: coupling centromeric nucleosomes to dynamic microtubule tips under physiologically relevant loads.

The self-assembly of full-length kinetochore subcomplexes at nanomolar concentrations suggests that they might have greater affinities for one another than suggested by the micromolar dissociation constants obtained using short peptides (Hornung et al., 2014; Dimitrova et al., 2016; Petrovic et al., 2016), possibly because of additional points of contact, although the immobilization of one subcomplex on beads might also affect binding dynamics. Both OA and Mif2 are individually capable of forming load-bearing interactions with MIND, demonstrating that there are two distinct paths of force transmission through the inner kinetochore. However, these paths are not equivalent. They are differentially sensitive to regulation by the major mitotic kinase, Ipl1 (Aurora B in humans), with the Mif2 path depending on the presence of phosphomimetic mutations on the MIND complex, whereas the OA pathway is only slightly strengthened by them. Also, while both the Mif2/2D-MIND and OA/2D-MIND interfaces are load-bearing, the former is mechanically weak relative to the Ndc80c/microtubule interface, but the latter is strong (Figure 2 and Figure 3).

We also found that OA can strengthen Mif2/2D-MIND/Ndc80c linkers, whereas Mif2 slightly weakens OA/2D-MIND/Ndc80c linkers. Considering that ‘2D’ phosphomimetic mutations on MIND partially alleviate autoinhibition but do not abrogate it completely (Dimitrova et al., 2016), we speculate that OA binding to 2D-MIND might alleviate this residual autoinhibition, promoting MIND’s interaction with Mif2 and strengthening the Mif2-based chain. In contrast, for the OA-based linker, we hypothesize that OA disinhibition of 2D-MIND might permit Mif2 to outcompete OA for binding to 2D-MIND (Figure 4A,B). In other words, Mif2 might decrease the rupture force of the OA/2D-MIND/Ndc80c linkers by ‘stealing’ the disinhibited 2D-MIND from bead-bound OA, thus detaching MIND from the bead. Competition for binding to MIND is plausible, because previous work has shown that OA and Mif2 bind to adjacent sites on the same head of MIND (Dimitrova et al., 2016).

When NCPs are on the beads, adding both OA and Mif2 in solution strengthens their attachment to microtubules (Figure 5B,C). OA and Mif2 probably do not compete with each other for binding to a centromeric nucleosome, because OA binds the N-terminus of Cse4, whereas Mif2 likely binds the C-terminus of Cse4 (Carroll et al., 2010; Guse et al., 2011; Anedchenko et al., 2019; Fischböck-Halwachs et al., 2019). If OA and Mif2 each recruit one MIND complex to the nucleosome, then together they would increase the total number of Ndc80c complexes in the assembly. Both OA and Mif2 self-assemble exclusively on centromeric nucleosomes (Figure 5A), a kinetochore function essential for genome stability. Yet we consistently find that Cse4-NCP-based chains are weak compared to shorter chains without NCPs. We hypothesize that this relative weakness occurs because the Cse4-NCPs were wrapped in 601 DNA rather than centromeric DNA. Budding yeast Mif2 is reported to have a 40-fold greater affinity for Cse4-containing nucleosomes if they are wrapped in centromeric DNA (Xiao et al., 2017). We were unable to test the effect of this increased affinity on rupture force because wrapping nucleosomes in centromeric DNA proved intractable. Nonetheless, the rupture strengths of our reconstituted assemblies fall within the range of mitotic forces estimated for budding yeast kinetochores in vivo.

In addition to the absence of centromeric DNA, there are other possible reasons why the strongest chains studied here still had a median force below the 9.1 pN strength measured previously for native purified kinetochore particles (Akiyoshi et al., 2010). First, studies of native kinetochore particles have not tested force transmission through the inner kinetochore; force has been exerted only through outer kinetochore components via MIND-based bead linkages (Akiyoshi et al., 2010). Thus, the inner kinetochore connections might also be weaker in the native particles. Second, post-translational modifications retained on the native proteins but absent from our recombinant proteins might strengthen interactions within the inner kinetochore. Given that methylation of Cse4 is known to weaken the OA-Cse4 interface (Anedchenko et al., 2019), it seems plausible that additional modifications that strengthen the inner kinetochore might also be found.

Although both OA and Mif2 can transmit force from Cse4-NCPs to MIND, they are neither interchangeable nor redundant. There are several reasons that having multiple paths of force transmission might be advantageous. First, each path can be differentially regulated, which our data suggest is the case. Second, OA and Mif2 together increase the total number of outer kinetochore components that can be recruited. Estimates of the number of Ndc80 complexes present in each kinetochore vary from six to thirty copies (Joglekar et al., 2006; Lawrimore et al., 2011; Dhatchinamoorthy et al., 2017), and biophysical experiments demonstrate that multiple Ndc80 complexes are necessary for effective microtubule coupling (Powers et al., 2009; Volkov et al., 2018). But MIND and Ndc80c interact in a 1:1 ratio (Dimitrova et al., 2016), implying that the inner kinetochore must serve as an oligomerization platform, assembling on a single centromeric nucleosome and recruiting multiple copies of MIND and Ndc80c. If one OA complex and one Mif2 dimer assembled onto each of the two faces of a centromeric nucleosome, they could collectively recruit up to 6 MIND/Ndc80c, with additional copies of Ndc80c potentially being recruited by the Cnn1 pathway (Lang et al., 2018).

Our findings also raise intriguing questions about how these multiple possible paths of force transmission and Ndc80c recruitment may be differentially utilized at different stages of the cell cycle or by different organisms. For instance, the human homolog of OA binds microtubules (Pesenti et al., 2018), but budding yeast OA clearly does not (Figure 2A). For some organisms, OA is essential (Pot et al., 2005; Kagawa et al., 2014); other species appear to lack OA homologs (Liu et al., 2016; van Hooff et al., 2017). A smaller subset of organisms has no known homolog of Mif2 (Drinnenberg et al., 2014; van Hooff et al., 2017). Evidently, the relative importance of the OA and Mif2 pathways varies between organisms. In budding yeast, the extreme N-terminus of Ame1 is essential, while the N-terminus of Mif2 is not (Hornung et al., 2014), suggesting the primacy of the OA-based path in this organism. There is also strong evidence for a third pathway of Ndc80c recruitment, through Cnn1 (Bock et al., 2012; Huis In 't Veld et al., 2016; Lang et al., 2018). Although Cnn1 is not essential in yeast (Giaever et al., 2002; De Wulf et al., 2003), an additive growth defect is observed when deletion of Cnn1 is combined with deletion of the N-terminus of Mif2 (Hornung et al., 2014). Homologs of Cnn1 also play important roles in several other organisms (Gascoigne et al., 2011; Nishino et al., 2013; Huis In 't Veld et al., 2016; Cortes-Silva et al., 2020). Thus, while the branched architecture of the inner kinetochore is a feature conserved across many species, the relative importance of particular branches apparently varies between species.

Taken together, our work represents a major advance towards the goal of reconstituting a complete, functional kinetochore from purely recombinant proteins. Going forward, the reconstituted ‘skeleton’ kinetochore presented here can be used as a scaffold to study the contributions of other kinetochore proteins to force transmission. Of particular interest are Cnn1, which could increase rupture force by recruiting additional copies of Ndc80c (Schleiffer et al., 2012; Lang et al., 2018), and Stu2, which is known to contribute significantly to kinetochore-microtubule coupling (Miller et al., 2016).

All data analyses during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5 and their supplements.

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Author details

1. Grace E Hamilton

   Department of Biochemistry, University of Washington, Seattle, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0522-0702

2. Luke A Helgeson

   Department of Biochemistry, University of Washington, Seattle, United States

   Contribution

   Resources, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5112-2751

3. Cameron L Noland

   Department of Structural Biology, Genentech Inc, South San Francisco, United States

   Contribution

   Resources, Histone purification and nucleosome core particle wrapping

   Competing interests

   is affiliated with Genentech Inc. The author has no financial interests to declare

   "This ORCID iD identifies the author of this article:" 0000-0001-6364-3167

4. Charles L Asbury

   Department of Physiology and Biophysics, University of Washington, Seattle, United States

   Contribution

   Conceptualization, Resources, Software, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - review and editing

   For correspondence

   casbury@uw.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0143-5394

5. Yoana N Dimitrova

   Department of Structural Biology, Genentech Inc, South San Francisco, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Investigation, Project administration, Writing - review and editing

   For correspondence

   dimitry4@gene.com

   Competing interests

   is affiliated with Genentech Inc. The author has no financial interests to declare

   "This ORCID iD identifies the author of this article:" 0000-0003-1547-5781

6. Trisha N Davis

   Department of Biochemistry, University of Washington, Seattle, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Project administration, Writing - review and editing

   For correspondence

   tdavis@uw.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4797-3152

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