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).

Figure 1 with 1 supplement see all

Download asset Open asset

Figure 1—source data 1

Plasmids used in this study.

https://cdn.elifesciences.org/articles/56582/elife-56582-fig1-data1-v2.docx

Download elife-56582-fig1-data1-v2.docx

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.

1. 1. Akiyoshi B
   2. Sarangapani KK
   3. Powers AF
   4. Nelson CR
   5. Reichow SL
   6. Arellano-Santoyo H
   7. Gonen T
   8. Ranish JA
   9. Asbury CL
   10. Biggins S

   (2010) Tension directly stabilizes reconstituted kinetochore-microtubule attachments

   Nature 468:576–579.

   https://doi.org/10.1038/nature09594

   • PubMed
   • Google Scholar
2. 1. Anedchenko EA
   2. Samel-Pommerencke A
   3. Tran Nguyen TM
   4. Shahnejat-Bushehri S
   5. Pöpsel J
   6. Lauster D
   7. Herrmann A
   8. Rappsilber J
   9. Cuomo A
   10. Bonaldi T
   11. Ehrenhofer-Murray AE

   (2019) The kinetochore module Okp1 CENP-Q /Ame1 CENP-U is a reader for N-terminal modifications on the centromeric histone Cse4 CENP-A

   EMBO Journal  38:e98991.

   https://doi.org/10.15252/embj.201898991

   • Google Scholar
3. 1. Asbury CL
   2. Gestaut DR
   3. Powers AF
   4. Franck AD
   5. Davis TN

   (2006) The Dam1 kinetochore complex harnesses microtubule dynamics to produce force and movement

   PNAS 103:9873–9878.

   https://doi.org/10.1073/pnas.0602249103

   • PubMed
   • Google Scholar
4. 1. Bock LJ
   2. Pagliuca C
   3. Kobayashi N
   4. Grove RA
   5. Oku Y
   6. Shrestha K
   7. Alfieri C
   8. Golfieri C
   9. Oldani A
   10. Dal Maschio M
   11. Bermejo R
   12. Hazbun TR
   13. Tanaka TU
   14. De Wulf P

   (2012) Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore

   Nature Cell Biology 14:614–624.

   https://doi.org/10.1038/ncb2495

   • PubMed
   • Google Scholar
5. 1. Bustamante C
   2. Chemla YR
   3. Forde NR
   4. Izhaky D

   (2004) Mechanical processes in biochemistry

   Annual Review of Biochemistry 73:705–748.

   https://doi.org/10.1146/annurev.biochem.72.121801.161542

   • Google Scholar
6. 1. Caldas GV
   2. DeLuca KF
   3. DeLuca JG

   (2013) KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting aurora B kinase activity

   The Journal of Cell Biology 203:957–969.

   https://doi.org/10.1083/jcb.201306054

   • PubMed
   • Google Scholar
7. 1. Carroll CW
   2. Milks KJ
   3. Straight AF

   (2010) Dual recognition of CENP-A nucleosomes is required for centromere assembly

   The Journal of Cell Biology 189:1143–1155.

   https://doi.org/10.1083/jcb.201001013

   • PubMed
   • Google Scholar
8. 1. Castoldi M
   2. Popov AV

   (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer

   Protein Expression and Purification 32:83–88.

   https://doi.org/10.1016/S1046-5928(03)00218-3

   • PubMed
   • Google Scholar
9. 1. Chacón JM
   2. Mukherjee S
   3. Schuster BM
   4. Clarke DJ
   5. Gardner MK

   (2014) Pericentromere tension is self-regulated by spindle structure in metaphase

   The Journal of Cell Biology 205:313–324.

   https://doi.org/10.1083/jcb.201312024

   • PubMed
   • Google Scholar
10. 1. Cheeseman IM
    2. Anderson S
    3. Jwa M
    4. Green EM
    5. Kang J
    6. Yates JR
    7. Chan CS
    8. Drubin DG
    9. Barnes G

    (2002) Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p

    Cell 111:163–172.

    https://doi.org/10.1016/S0092-8674(02)00973-X

    • PubMed
    • Google Scholar
11. 1. Cheeseman IM
    2. Chappie JS
    3. Wilson-Kubalek EM
    4. Desai A

    (2006) The conserved KMN network constitutes the core microtubule-binding site of the kinetochore

    Cell 127:983–997.

    https://doi.org/10.1016/j.cell.2006.09.039

    • PubMed
    • Google Scholar
12. 1. Cimini D
    2. Howell B
    3. Maddox P
    4. Khodjakov A
    5. Degrassi F
    6. Salmon ED

    (2001) Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

    The Journal of Cell Biology 153:517–528.

    https://doi.org/10.1083/jcb.153.3.517

    • PubMed
    • Google Scholar
13. 1. Cimini D

    (2008) Merotelic kinetochore orientation, aneuploidy, and Cancer

    Biochimica Et Biophysica Acta (BBA) - Reviews on Cancer 1786:32–40.

    https://doi.org/10.1016/j.bbcan.2008.05.003

    • Google Scholar
14. 1. Cortes-Silva N
    2. Ulmer J
    3. Kiuchi T
    4. Hsieh E
    5. Cornilleau G
    6. Ladid I
    7. Dingli F
    8. Loew D
    9. Katsuma S
    10. Drinnenberg IA

    (2020) CenH3-Independent kinetochore assembly in lepidoptera requires CCAN, including CENP-T

    Current Biology 30:561–572.

    https://doi.org/10.1016/j.cub.2019.12.014

    • PubMed
    • Google Scholar
15. 1. De Wulf P
    2. McAinsh AD
    3. Sorger PK

    (2003) Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes

    Genes & Development 17:2902–2921.

    https://doi.org/10.1101/gad.1144403

    • PubMed
    • Google Scholar
16. 1. Dhatchinamoorthy K
    2. Shivaraju M
    3. Lange JJ
    4. Rubinstein B
    5. Unruh JR
    6. Slaughter BD
    7. Gerton JL

    (2017) Structural plasticity of the living kinetochore

    Journal of Cell Biology 216:3551–3570.

    https://doi.org/10.1083/jcb.201703152

    • PubMed
    • Google Scholar
17. 1. Dimitrova YN
    2. Jenni S
    3. Valverde R
    4. Khin Y
    5. Harrison SC

    (2016) Structure of the MIND complex defines a regulatory focus for yeast kinetochore assembly

    Cell 167:1014–1027.

    https://doi.org/10.1016/j.cell.2016.10.011

    • Google Scholar
18. 1. Drinnenberg IA
    2. deYoung D
    3. Henikoff S
    4. Malik HS

    (2014) Recurrent loss of CenH3 is associated with independent transitions to holocentricity in insects

    eLife 3:e03676.

    https://doi.org/10.7554/eLife.03676

    • Google Scholar
19. 1. Evans E
    2. Ritchie K

    (1997) Dynamic strength of molecular adhesion bonds

    Biophysical Journal 72:1541–1555.

    https://doi.org/10.1016/S0006-3495(97)78802-7

    • PubMed
    • Google Scholar
20. 1. Fischböck-Halwachs J
    2. Singh S
    3. Potocnjak M
    4. Hagemann G
    5. Solis-Mezarino V
    6. Woike S
    7. Ghodgaonkar-Steger M
    8. Weissmann F
    9. Gallego LD
    10. Rojas J
    11. Andreani J
    12. Köhler A
    13. Herzog F

    (2019) The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore

    eLife 8:e42879.

    https://doi.org/10.7554/eLife.42879

    • PubMed
    • Google Scholar
21. 1. Fisher JK
    2. Ballenger M
    3. O'Brien ET
    4. Haase J
    5. Superfine R
    6. Bloom K

    (2009) DNA relaxation dynamics as a probe for the intracellular environment

    PNAS 106:9250–9255.

    https://doi.org/10.1073/pnas.0812723106

    • PubMed
    • Google Scholar
22. 1. Franck AD
    2. Powers AF
    3. Gestaut DR
    4. Davis TN
    5. Asbury CL

    (2010) Direct physical study of kinetochore-microtubule interactions by reconstitution and interrogation with an optical force clamp

    Methods 51:242–250.

    https://doi.org/10.1016/j.ymeth.2010.01.020

    • PubMed
    • Google Scholar
23. 1. Gascoigne KE
    2. Takeuchi K
    3. Suzuki A
    4. Hori T
    5. Fukagawa T
    6. Cheeseman IM

    (2011) Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes

    Cell 145:410–422.

    https://doi.org/10.1016/j.cell.2011.03.031

    • PubMed
    • Google Scholar
24. 1. Gestaut DR
    2. Graczyk B
    3. Cooper J
    4. Widlund PO
    5. Zelter A
    6. Wordeman L
    7. Asbury CL
    8. Davis TN

    (2008) Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation

    Nature Cell Biology 10:407–414.

    https://doi.org/10.1038/ncb1702

    • PubMed
    • Google Scholar
25. 1. Gestaut DR
    2. Cooper J
    3. Asbury CL
    4. Davis TN
    5. Wordeman L

    (2010) Reconstitution and functional analysis of kinetochore subcomplexes

    Methods in Cell Biology 95:641–656.

    https://doi.org/10.1016/S0091-679X(10)95032-2

    • PubMed
    • Google Scholar
26. 1. Giaever G
    2. Chu AM
    3. Ni L
    4. Connelly C
    5. Riles L
    6. Véronneau S
    7. Dow S
    8. Lucau-Danila A
    9. Anderson K
    10. André B
    11. Arkin AP
    12. Astromoff A
    13. El-Bakkoury M
    14. Bangham R
    15. Benito R
    16. Brachat S
    17. Campanaro S
    18. Curtiss M
    19. Davis K
    20. Deutschbauer A
    21. Entian KD
    22. Flaherty P
    23. Foury F
    24. Garfinkel DJ
    25. Gerstein M
    26. Gotte D
    27. Güldener U
    28. Hegemann JH
    29. Hempel S
    30. Herman Z
    31. Jaramillo DF
    32. Kelly DE
    33. Kelly SL
    34. Kötter P
    35. LaBonte D
    36. Lamb DC
    37. Lan N
    38. Liang H
    39. Liao H
    40. Liu L
    41. Luo C
    42. Lussier M
    43. Mao R
    44. Menard P
    45. Ooi SL
    46. Revuelta JL
    47. Roberts CJ
    48. Rose M
    49. Ross-Macdonald P
    50. Scherens B
    51. Schimmack G
    52. Shafer B
    53. Shoemaker DD
    54. Sookhai-Mahadeo S
    55. Storms RK
    56. Strathern JN
    57. Valle G
    58. Voet M
    59. Volckaert G
    60. Wang CY
    61. Ward TR
    62. Wilhelmy J
    63. Winzeler EA
    64. Yang Y
    65. Yen G
    66. Youngman E
    67. Yu K
    68. Bussey H
    69. Boeke JD
    70. Snyder M
    71. Philippsen P
    72. Davis RW
    73. Johnston M

    (2002) Functional profiling of the Saccharomyces cerevisiae genome

    Nature 418:387–391.

    https://doi.org/10.1038/nature00935

    • PubMed
    • Google Scholar
27. 1. Guse A
    2. Carroll CW
    3. Moree B
    4. Fuller CJ
    5. Straight AF

    (2011) In vitro centromere and kinetochore assembly on defined chromatin templates

    Nature 477:354–358.

    https://doi.org/10.1038/nature10379

    • PubMed
    • Google Scholar
28. 1. Hara M
    2. Fukagawa T

    (2017) Critical foundation of the kinetochore: the constitutive Centromere-Associated network (CCAN)

    Progress in Molecular and Subcellular Biology 56:29–57.

    https://doi.org/10.1007/978-3-319-58592-5_2

    • PubMed
    • Google Scholar
29. 1. Helgeson LA
    2. Zelter A
    3. Riffle M
    4. MacCoss MJ
    5. Asbury CL
    6. Davis TN

    (2018) Human ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments

    PNAS 115:2740–2745.

    https://doi.org/10.1073/pnas.1718553115

    • PubMed
    • Google Scholar
30. 1. Hinshaw SM
    2. Harrison SC

    (2013) An Iml3-Chl4 heterodimer links the core centromere to factors required for accurate chromosome segregation

    Cell Reports 5:29–36.

    https://doi.org/10.1016/j.celrep.2013.08.036

    • PubMed
    • Google Scholar
31. 1. Hornung P
    2. Troc P
    3. Malvezzi F
    4. Maier M
    5. Demianova Z
    6. Zimniak T
    7. Litos G
    8. Lampert F
    9. Schleiffer A
    10. Brunner M
    11. Mechtler K
    12. Herzog F
    13. Marlovits TC
    14. Westermann S

    (2014) A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

    Journal of Cell Biology 206:509–524.

    https://doi.org/10.1083/jcb.201403081

    • Google Scholar
32. 1. Huis In 't Veld PJ
    2. Jeganathan S
    3. Petrovic A
    4. Singh P
    5. John J
    6. Krenn V
    7. Weissmann F
    8. Bange T
    9. Musacchio A

    (2016) Molecular basis of outer kinetochore assembly on CENP-T

    eLife 5:e21007.

    https://doi.org/10.7554/eLife.21007

    • PubMed
    • Google Scholar
33. 1. Joglekar AP
    2. Bouck DC
    3. Molk JN
    4. Bloom KS
    5. Salmon ED

    (2006) Molecular architecture of a kinetochore-microtubule attachment site

    Nature Cell Biology 8:581–585.

    https://doi.org/10.1038/ncb1414

    • PubMed
    • Google Scholar
34. 1. Kagawa N
    2. Hori T
    3. Hoki Y
    4. Hosoya O
    5. Tsutsui K
    6. Saga Y
    7. Sado T
    8. Fukagawa T

    (2014) The CENP-O complex requirement varies among different cell types

    Chromosome Research 22:293–303.

    https://doi.org/10.1007/s10577-014-9404-1

    • PubMed
    • Google Scholar
35. Software

    1. Kassambra A
    2. Kosiniski M

    (2018)

    survminer: Drawing Survival Curves using 'ggplot2'

    survminer: Drawing Survival Curves using 'ggplot2'.
36. 1. Kato H
    2. Jiang J
    3. Zhou BR
    4. Rozendaal M
    5. Feng H
    6. Ghirlando R
    7. Xiao TS
    8. Straight AF
    9. Bai Y

    (2013) A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C

    Science 340:1110–1113.

    https://doi.org/10.1126/science.1235532

    • PubMed
    • Google Scholar
37. 1. Kim JO
    2. Zelter A
    3. Umbreit NT
    4. Bollozos A
    5. Riffle M
    6. Johnson R
    7. MacCoss MJ
    8. Asbury CL
    9. Davis TN

    (2017) The Ndc80 complex bridges two Dam1 complex rings

    eLife 6:e21069.

    https://doi.org/10.7554/eLife.21069

    • PubMed
    • Google Scholar
38. 1. Kudalkar EM
    2. Scarborough EA
    3. Umbreit NT
    4. Zelter A
    5. Gestaut DR
    6. Riffle M
    7. Johnson RS
    8. MacCoss MJ
    9. Asbury CL
    10. Davis TN

    (2015) Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

    PNAS 112:E5583–E5589.

    https://doi.org/10.1073/pnas.1513882112

    • PubMed
    • Google Scholar
39. 1. Lang J
    2. Barber A
    3. Biggins S

    (2018) An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

    eLife 7:e37819.

    https://doi.org/10.7554/eLife.37819

    • PubMed
    • Google Scholar
40. 1. Lawrimore J
    2. Bloom KS
    3. Salmon ED

    (2011) Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome

    The Journal of Cell Biology 195:573–582.

    https://doi.org/10.1083/jcb.201106036

    • PubMed
    • Google Scholar
41. 1. Liu Y
    2. Petrovic A
    3. Rombaut P
    4. Mosalaganti S
    5. Keller J
    6. Raunser S
    7. Herzog F
    8. Musacchio A

    (2016) Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

    Open Biology 6:150236.

    https://doi.org/10.1098/rsob.150236

    • PubMed
    • Google Scholar
42. 1. Lowary PT
    2. Widom J

    (1998) New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning

    Journal of Molecular Biology 276:19–42.

    https://doi.org/10.1006/jmbi.1997.1494

    • PubMed
    • Google Scholar
43. 1. Meeks-Wagner D
    2. Wood JS
    3. Garvik B
    4. Hartwell LH

    (1986) Isolation of two genes that affect mitotic chromosome transmission in S. cerevisiae

    Cell 44:53–63.

    https://doi.org/10.1016/0092-8674(86)90484-8

    • PubMed
    • Google Scholar
44. 1. Merkel R
    2. Nassoy P
    3. Leung A
    4. Ritchie K
    5. Evans E

    (1999) Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy

    Nature 397:50–53.

    https://doi.org/10.1038/16219

    • Google Scholar
45. 1. Migl D
    2. Kschonsak M
    3. Arthur CP
    4. Khin Y
    5. Harrison SC
    6. Ciferri C
    7. Dimitrova YN

    (2020) Cryoelectron microscopy structure of a yeast centromeric nucleosome at 2.7 Å Resolution

    Structure 28:363–370.

    https://doi.org/10.1016/j.str.2019.12.002

    • PubMed
    • Google Scholar
46. 1. Miller MP
    2. Asbury CL
    3. Biggins S

    (2016) A TOG protein confers tension sensitivity to Kinetochore-Microtubule attachments

    Cell 165:1428–1439.

    https://doi.org/10.1016/j.cell.2016.04.030

    • Google Scholar
47. 1. Musacchio A

    (2011) Spindle assembly checkpoint: the third decade

    Philosophical Transactions of the Royal Society B: Biological Sciences 366:3595–3604.

    https://doi.org/10.1098/rstb.2011.0072

    • Google Scholar
48. 1. Musacchio A
    2. Desai A

    (2017) A molecular view of kinetochore assembly and function

    Biology 6:E5.

    https://doi.org/10.3390/biology6010005

    • PubMed
    • Google Scholar
49. 1. Nicklas RB

    (1988) The forces that move chromosomes in mitosis

    Annual Review of Biophysics and Biophysical Chemistry 17:431–449.

    https://doi.org/10.1146/annurev.bb.17.060188.002243

    • PubMed
    • Google Scholar
50. 1. Nicklas RB
    2. Ward SC

    (1994) Elements of error correction in mitosis: microtubule capture, release, and tension

    The Journal of Cell Biology 126:1241–1253.

    https://doi.org/10.1083/jcb.126.5.1241

    • PubMed
    • Google Scholar
51. 1. Nishino T
    2. Rago F
    3. Hori T
    4. Tomii K
    5. Cheeseman IM
    6. Fukagawa T

    (2013) CENP-T provides a structural platform for outer kinetochore assembly

    The EMBO Journal 32:424–436.

    https://doi.org/10.1038/emboj.2012.348

    • PubMed
    • Google Scholar
52. 1. Ortiz J
    2. Stemmann O
    3. Rank S
    4. Lechner J

    (1999) A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore

    Genes & Development 13:1140–1155.

    https://doi.org/10.1101/gad.13.9.1140

    • PubMed
    • Google Scholar
53. 1. Palmer DK
    2. O'Day K
    3. Trong HL
    4. Charbonneau H
    5. Margolis RL

    (1991) Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone

    PNAS 88:3734–3738.

    https://doi.org/10.1073/pnas.88.9.3734

    • PubMed
    • Google Scholar
54. 1. Pesenti ME
    2. Prumbaum D
    3. Auckland P
    4. Smith CM
    5. Faesen AC
    6. Petrovic A
    7. Erent M
    8. Maffini S
    9. Pentakota S
    10. Weir JR
    11. Lin YC
    12. Raunser S
    13. McAinsh AD
    14. Musacchio A

    (2018) Reconstitution of a 26-Subunit human kinetochore reveals cooperative microtubule binding by CENP-OPQUR and NDC80

    Molecular Cell 71:923–939.

    https://doi.org/10.1016/j.molcel.2018.07.038

    • PubMed
    • Google Scholar
55. 1. Petrovic A
    2. Keller J
    3. Liu Y
    4. Overlack K
    5. John J
    6. Dimitrova YN
    7. Jenni S
    8. van Gerwen S
    9. Stege P
    10. Wohlgemuth S
    11. Rombaut P
    12. Herzog F
    13. Harrison SC
    14. Vetter IR
    15. Musacchio A

    (2016) Structure of the MIS12 complex and molecular basis of its interaction with CENP-C at human kinetochores

    Cell 167:1028–1040.

    https://doi.org/10.1016/j.cell.2016.10.005

    • PubMed
    • Google Scholar
56. 1. Pot I
    2. Knockleby J
    3. Aneliunas V
    4. Nguyen T
    5. Ah-Kye S
    6. Liszt G
    7. Snyder M
    8. Hieter P
    9. Vogel J

    (2005) Spindle checkpoint maintenance requires Ame1 and Okp1

    Cell Cycle 4:1448–1456.

    https://doi.org/10.4161/cc.4.10.2106

    • PubMed
    • Google Scholar
57. 1. Powers AF
    2. Franck AD
    3. Gestaut DR
    4. Cooper J
    5. Gracyzk B
    6. Wei RR
    7. Wordeman L
    8. Davis TN
    9. Asbury CL

    (2009) The Ndc80 kinetochore complex forms load-bearing attachments to dynamic microtubule tips via biased diffusion

    Cell 136:865–875.

    https://doi.org/10.1016/j.cell.2008.12.045

    • PubMed
    • Google Scholar
58. Software

    1. R Development Core Team

    (2013) R: A Language and Environment for Statistical Computing

    R Foundation for Statistical Computing, Vienna, Austria.

    http://www.r-project.org
59. 1. Santaguida S
    2. Amon A

    (2015) Short- and long-term effects of chromosome mis-segregation and aneuploidy

    Nature Reviews Molecular Cell Biology 16:473–485.

    https://doi.org/10.1038/nrm4025

    • PubMed
    • Google Scholar
60. 1. Sarangapani KK
    2. Akiyoshi B
    3. Duggan NM
    4. Biggins S
    5. Asbury CL

    (2013) Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

    PNAS 110:7282–7287.

    https://doi.org/10.1073/pnas.1220700110

    • PubMed
    • Google Scholar
61. 1. Scarborough EA
    2. Davis TN
    3. Asbury CL

    (2019) Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules

    eLife 8:e44489.

    https://doi.org/10.7554/eLife.44489

    • PubMed
    • Google Scholar
62. 1. Schleiffer A
    2. Maier M
    3. Litos G
    4. Lampert F
    5. Hornung P
    6. Mechtler K
    7. Westermann S

    (2012) CENP-T proteins are conserved centromere receptors of the Ndc80 complex

    Nature Cell Biology 14:604–613.

    https://doi.org/10.1038/ncb2493

    • PubMed
    • Google Scholar
63. 1. Screpanti E
    2. De Antoni A
    3. Alushin GM
    4. Petrovic A
    5. Melis T
    6. Nogales E
    7. Musacchio A

    (2011) Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore

    Current Biology 21:391–398.

    https://doi.org/10.1016/j.cub.2010.12.039

    • PubMed
    • Google Scholar
64. 1. Stoler S
    2. Keith KC
    3. Curnick KE
    4. Fitzgerald-Hayes M

    (1995) A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis

    Genes & Development 9:573–586.

    https://doi.org/10.1101/gad.9.5.573

    • PubMed
    • Google Scholar
65. 1. Suzuki A
    2. Badger BL
    3. Haase J
    4. Ohashi T
    5. Erickson HP
    6. Salmon ED
    7. Bloom K

    (2016) How the kinetochore couples microtubule force and centromere stretch to move chromosomes

    Nature Cell Biology 18:382–392.

    https://doi.org/10.1038/ncb3323

    • PubMed
    • Google Scholar
66. 1. Tien JF
    2. Umbreit NT
    3. Gestaut DR
    4. Franck AD
    5. Cooper J
    6. Wordeman L
    7. Gonen T
    8. Asbury CL
    9. Davis TN

    (2010) Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B

    Journal of Cell Biology 189:713–723.

    https://doi.org/10.1083/jcb.200910142

    • PubMed
    • Google Scholar
67. 1. Umbreit NT
    2. Miller MP
    3. Tien JF
    4. Cattin Ortolá J
    5. Gui L
    6. Lee KK
    7. Biggins S
    8. Asbury CL
    9. Davis TN

    (2014) Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation

    Nature Communications 5:4951.

    https://doi.org/10.1038/ncomms5951

    • Google Scholar
68. 1. van Hooff JJ
    2. Tromer E
    3. van Wijk LM
    4. Snel B
    5. Kops GJ

    (2017) Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

    EMBO Reports 18:1559–1571.

    https://doi.org/10.15252/embr.201744102

    • PubMed
    • Google Scholar
69. 1. Volkov VA
    2. Huis in 't Veld PJ
    3. Dogterom M
    4. Musacchio A

    (2018) Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces

    eLife 7:e36764.

    https://doi.org/10.7554/eLife.36764

    • Google Scholar
70. 1. Wei RR
    2. Sorger PK
    3. Harrison SC

    (2005) Molecular organization of the Ndc80 complex, an essential kinetochore component

    PNAS 102:5363–5367.

    https://doi.org/10.1073/pnas.0501168102

    • PubMed
    • Google Scholar
71. 1. Weir JR
    2. Faesen AC
    3. Klare K
    4. Petrovic A
    5. Basilico F
    6. Fischböck J
    7. Pentakota S
    8. Keller J
    9. Pesenti ME
    10. Pan D
    11. Vogt D
    12. Wohlgemuth S
    13. Herzog F
    14. Musacchio A

    (2016) Insights from biochemical reconstitution into the architecture of human kinetochores

    Nature 537:249–253.

    https://doi.org/10.1038/nature19333

    • PubMed
    • Google Scholar
72. Book

    1. Wickham H

    (2009) Ggplot2: Elegant Graphics for Data Analysis

    Springer-Verlag.

    https://doi.org/10.1007/978-0-387-98141-3

    • Google Scholar
73. 1. Xiao H
    2. Wang F
    3. Wisniewski J
    4. Shaytan AK
    5. Ghirlando R
    6. FitzGerald PC
    7. Huang Y
    8. Wei D
    9. Li S
    10. Landsman D
    11. Panchenko AR
    12. Wu C

    (2017) Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres

    Genes & Development 31:1958–1972.

    https://doi.org/10.1101/gad.304782.117

    • PubMed
    • Google Scholar
74. 1. Ye AA
    2. Cane S
    3. Maresca TJ

    (2016) Chromosome biorientation produces hundreds of piconewtons at a metazoan kinetochore

    Nature Communications 7:13221.

    https://doi.org/10.1038/ncomms13221

    • PubMed
    • Google Scholar

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

• 1,656

  views

• 308

  downloads

• 13

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.