The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

Suzuki, Aussie; Badger, Benjamin L.; Wan, Xiaohu; DeLuca, Jennifer G.; Salmon, Edward D.

doi:10.1016/j.devcel.2014.08.003

Cited by 77 publications

(118 citation statements)

References 51 publications

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“…We monitored monomeric (m)EGFP fused to the C terminus of Hec1 (also known as Ndc80) and tdTomato fused to the N terminus of CENP-C, which labels the outer and inner KTs, respectively (Suzuki et al, 2014). As expected, the mEGFP and tdTomato signals were frequently separated by >80 nm during metaphase of MII, indicative of intra-KT stretching ( Figures S2A and S2B).…”

Section: Aurora B/c Is Located In Close Proximity To Kt-mt Attachmentmentioning

confidence: 86%

Inherent Instability of Correct Kinetochore-Microtubule Attachments during Meiosis I in Oocytes

Yoshida¹,

Kaido²,

Kitajima³

2015

Developmental Cell

128

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A model for mitosis suggests that correct kinetochore-microtubule (KT-MT) attachments are stabilized by spatial separation of the attachment sites from Aurora B kinase through sister KT stretching. However, the spatiotemporal regulation of attachment stability during meiosis I (MI) in oocytes remains unclear. Here, we found that in mouse oocytes, Aurora B and C (B/C) are located in close proximity to KT-MT attachment sites after bivalent stretching due to an intrinsic property of the MI chromosomes. The Aurora B/C activity destabilizes correct attachments while allowing a considerable amount of incorrect attachments to form. KT-MT attachments are eventually stabilized through KT dephosphorylation by PP2A-B56 phosphatase, which is progressively recruited to KTs depending on the BubR1 phosphorylation resulting from the timer Cdk1 and independent of bivalent stretching. Thus, oocytes lack a mechanism for coordinating bivalent stretching and KT phosphoregulation during MI, which may explain the high frequency of KT-MT attachment errors.

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Section: Aurora B/c Is Located In Close Proximity To Kt-mt Attachmentmentioning

confidence: 86%

Inherent Instability of Correct Kinetochore-Microtubule Attachments during Meiosis I in Oocytes

Yoshida¹,

Kaido²,

Kitajima³

2015

Developmental Cell

128

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“…In contrast, mammalian CENP-T actively recruits the Ndc80 complex to CENs (Gascoigne et al 2011) and the middle region of CENP-T appears to be flexible and assists in kinetochore stretching when it undergoes tension (Perpelescu and Fukagawa 2011;Suzuki et al 2011;Westhorpe and Straight 2013). Similarly, the preanaphase Cnn1 linkages may allow a proper intrakinetochore stretch required for chromosome biorientation, as shown for CENP-T (Suzuki et al 2014). Next to altering the interactions between the KMN complexes, the preanaphase Cnn1 linkages may be involved in tension sensing during sister-kinetochore attachment and biorientation, which are also regulated by Mps1 (Weiss and Winey 1996).…”

Section: Discussionmentioning

confidence: 99%

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

et al. 2015

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Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1 CENP-T , the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1 , should be extended with flanking residues, Cnn1 , to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74, based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase. KEYWORDS Cnn1; Mps1; kinetochore; centromere; CENP-T K INETOCHORES are large protein structures that assemble hierarchically on the centromeres (CEN) of replicated chromosomes (sister chromatids). They biorient each sister chromatid pair to the microtubules (MTs) of the mitotic spindle and orchestrate chromatid segregation into the daughter cells (Cheeseman 2014;Malvezzi and Westermann 2014). At the core of each kinetochore lies a protein network named KMN (KLN1/Spc105, MIS12/Mtw1, and NDC80/Ndc80 complexes) that bridges the CEN and MTs (Cheeseman et al. 2006;Westermann et al. 2007). The Ndc80 complex attaches kinetochores to the MTs via its outer Ndc80/Nuf2 dimer (Wei et al. 2007;Ciferri et al. 2008;Alushin et al. 2010), while its CEN-proximal Spc24/Spc25 dimer interacts with a putatively CEN-associated protein, known as Cnn1 in budding yeast (CENP-T in metazoans). The N-terminal domain of Cnn1 and CENP-T hook onto the interface of the Spc24/Spc25 dimer (Malvezzi et al. 2013;Nishino et al. 2013). In its C terminus, Cnn1 harbors a histonefold domain (HFD) (Schleiffer et al. 2012), which may associate with CEN DNA, as does CENP-T (Hori et al. 2008;Nishino et al. 2012).Cnn1 levels at kinetochores are low from G1 through metaphase but increase two-to threefold at anaphase entry and drop back to base level at anaphase exit. Cnn1 interacts with the...

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“…Immunofluorescence was conducted using methods described previously (27). Human mitotic chromosomes were prepared by cytospin as previously described (28).…”

Section: Methodsmentioning

confidence: 99%

FBW7 Loss Promotes Chromosomal Instability and Tumorigenesis via Cyclin E1/CDK2–Mediated Phosphorylation of CENP-A

et al. 2017

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The centromere regulates proper chromosome segregation and its dysfunction is implicated in chromosomal instability (CIN). However, relatively little is known about how centromere dysfunction occurs in cancer. Here we define the consequences of phosphorylation by Cyclin E1/CDK2 on a conserved Ser18 residue of centromere-associated protein CENP-A, an essential histone H3 variant that specifies centromere identity. Ser18 hyperphosphorylation in cells occurred upon loss of FBW7, a tumor suppressor whose inactivation leads to chromosomal instability (CIN). This event on CENP-A reduced its centromeric localization, increased CIN and promoted anchorage-independent growth and xenograft tumor formation. Overall, our results revealed a pathway that Cyclin E1/CDK2 activation coupled with FBW7 loss promotes CIN and tumor progression via CENP-A-mediated centromere dysfunction.

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The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

Cited by 77 publications

References 51 publications

Inherent Instability of Correct Kinetochore-Microtubule Attachments during Meiosis I in Oocytes

Inherent Instability of Correct Kinetochore-Microtubule Attachments during Meiosis I in Oocytes

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

FBW7 Loss Promotes Chromosomal Instability and Tumorigenesis via Cyclin E1/CDK2–Mediated Phosphorylation of CENP-A

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