Molecular basis of outer kinetochore assembly on CENP-T

Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.

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Molecular basis of outer kinetochore assembly on CENP-T

eLife ◽

10.7554/elife.21007 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 55

Author(s):

Pim J Huis in 't Veld ◽

Sadasivam Jeganathan ◽

Arsen Petrovic ◽

Priyanka Singh ◽

Juliane John ◽

...

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

Molecular Basis ◽

Cell Cycle Regulation ◽

Cyclin B ◽

Kinetochore Assembly ◽

Microtubule Attachment ◽

Centromere Proteins ◽

Ndc80 Complex ◽

Cycle Regulation

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Cell cycle regulation in Aspergillus by two protein kinases

Biochemical Journal ◽

10.1042/bj3170633 ◽

1996 ◽

Vol 317 (3) ◽

pp. 633-641 ◽

Cited By ~ 61

Author(s):

Stephen A. OSMANI ◽

Xiang S. YE

Keyword(s):

Cell Cycle ◽

Protein Kinases ◽

Cell Cycle Regulation ◽

Cell Cycle Progression ◽

Human Cells ◽

Dominant Negative ◽

Cyclin B ◽

Cyclin Dependent Kinases ◽

Cycle Regulation ◽

Mitotic Regulation

Great progress has recently been made in our understanding of the regulation of the eukaryotic cell cycle, and the central role of cyclin-dependent kinases is now clear. In Aspergillus nidulans it has been established that a second class of cell-cycle-regulated protein kinases, typified by NIMA (encoded by the nimA gene), is also required for cell cycle progression into mitosis. Indeed, both p34cdc2/cyclin B and NIMA have to be correctly activated before mitosis can be initiated in this species, and p34cdc2/cyclin B plays a role in the mitosis-specific activation of NIMA. In addition, both kinases have to be proteolytically destroyed before mitosis can be completed. NIMA-related kinases may also regulate the cell cycle in other eukaryotes, as expression of NIMA can promote mitotic events in yeast, frog or human cells. Moreover, dominant-negative versions of NIMA can adversely affect the progression of human cells into mitosis, as they do in A. nidulans. The ability of NIMA to influence mitotic regulation in human and frog cells strongly suggests the existence of a NIMA pathway of mitotic regulation in higher eukaryotes. A growing number of NIMA-related kinases have been isolated from organisms ranging from fungi to humans, and some of these kinases are also cell-cycle-regulated. How NIMA-related kinases and cyclin-dependent kinases act in concert to promote cell cycle transitions is just beginning to be understood. This understanding is the key to a full knowledge of cell cycle regulation.

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The structure of the Ctf19c/CCAN from budding yeast

eLife ◽

10.7554/elife.44239 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 36

Author(s):

Stephen M Hinshaw ◽

Stephen C Harrison

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

Budding Yeast ◽

Histone H3 ◽

Kinetochore Assembly ◽

Microtubule Attachment ◽

Cryo Electron Microscopy ◽

Histone H3 Variant

Eukaryotic kinetochores connect spindlemicrotubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

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