Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres

Joglekar, Ajit P.; Bouck, David C.; Finley, Ken; Liu, Xingkun; Wan, Yakun; Berman, Judith; He, Xiangwei; Salmon, Edward D.; Bloom, Kerry

doi:10.1083/jcb.200803027

Cited by 147 publications

(216 citation statements)

References 40 publications

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“…Structural analysis in vitro has shown that a Dam1 ring contains at least 16 copies of the complex (5,6). To test whether SpDam1 forms rings around MTs, we measured the copy number of Dam1 in individual spots on cytoplasmic MTs, using an established quantitative fluorescence microscopy approach (18,25,27). We examined strains expressing tri or di-GFP-tagged Dam1 complexes, and deduced the copy number of Dam1 ( Fig S2 and Materials and Methods).…”

Section: Identification Of Discrete Dam1 Assemblies On Cytoplasmic Mtmentioning

confidence: 99%

“…Similar to ScDam1, SpDam1 is localized at kinetochores as well as on the spindle and the intranuclear MT (20)(21)(22), and functions in coupling kinetochores to MTs (22)(23)(24). Using quantitative fluorescence microscopy, it was determined that on average only seven to eight copies of SpDam1 per kinetochore were found (25). If Dam1 is associated with each kMT (2-4 kMTs/kinetochore) (26) equally well, each kMT would have only two to three copies of SpDam1, arguing against the formation of a ring encircling the kMT.…”

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confidence: 99%

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A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

Gao

Courthéoux

Gachet

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The Dam1 complex is a kinetochore component that couples chromosomes to the dynamic ends of kinetochore microtubules (kMTs). Work in the budding yeast Saccharomyces cerevisiae has shown that the Dam1 complex forms a 16-unit ring encircling and tracking the tip of a MT in vitro, consistent with its cellular function as a coupler. Dam1 also forms smaller, nonring patches in vitro that track the dynamic ends of MTs. However, the identity of Dam1’s functional form in vivo remains unknown. Here we report a comprehensive in vivo characterization of Dam1 in the fission yeast Schizosaccharomyces pombe . In addition to their dense localizations on kinetochores and spindle MTs during mitosis, we identify that Dam1 is also localized onto cytoplasmic MTs as discrete spots in interphase, providing the unique opportunity to analyze Dam1 oligomers at the single-particle resolution in live cells. Such analysis shows that each oligomer contains one to five copies of Dam1, and is able to “switch-rail” while moving along MTs, precluding the possibility of a 16-unit encircling structure. Dam1 patches track the plus ends of the shortening, but not the elongating, MTs and retard MT depolymerization. Together with Mal3, the EB1-like MT-interacting protein, cytoplasmic Dam1 plays an important role in maintaining proper cell shape. In mitosis, kinetochore-associated Dam1 appears to facilitate kMT depolymerization. Together, our findings suggest that patches, instead of rings, are the physiologically functional forms of Dam1 in pombe. Our findings help establish the benchmark parameters of the Dam1 coupler and elucidate the mechanism of its functions.

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Section: Identification Of Discrete Dam1 Assemblies On Cytoplasmic Mtmentioning

confidence: 99%

mentioning

confidence: 99%

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

Gao

Courthéoux

Gachet

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…In C. albicans the nuclear spindle is visible for most of its cell cycle, the nuclear envelope remains intact during mitosis (17), and KT-MT interaction is established early during the cell cycle (42,44). Similar to S. cerevisiae, only one MT binds to a KT in C. albicans (25). C. albicans has 3-to 5-kb-long CENP-A-rich centromere sequences that are unique and different from each other on various chromosomes (3,43).…”

mentioning

confidence: 99%

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

Thakur

Sanyal

2011

Eukaryot Cell

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A fungus-specific outer kinetochore complex, the Dam1 complex, is essential in Saccharomyces cerevisiae, nonessential in fission yeast, and absent from metazoans. The reason for the reductive evolution of the functionality of this complex remains unknown. Both Candida albicans and Schizosaccharomyces pombe have regional centromeres as opposed to the short-point centromeres of S. cerevisiae. The interaction of one microtubule per kinetochore is established both in S. cerevisiae and C. albicans early during the cell cycle, which is in contrast to the multiple microtubules that bind to a kinetochore only during mitosis in S. pombe. Moreover, the Dam1 complex is associated with the kinetochore throughout the cell cycle in S. cerevisiae and C. albicans but only during mitosis in S. pombe. Here, we show that the Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation in C. albicans. The kinetochore localization of the Dam1 complex is independent of the kinetochoremicrotubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Strikingly, the Dam1 complex is required to prevent precocious spindle elongation in premitotic phases. Thus, constitutive kinetochore localization associated with a one-microtubule-one kinetochore type of interaction, but not the length of a centromere, is correlated with the essentiality of the Dam1 complex.The separation of sister chromatids during mitosis is driven by a dynamic interaction between two macromolecular structures: (i) the kinetochore (KT), a complex proteinaceous structure built on the centromere DNA (10, 47), and (ii) the mitotic spindle formed by microtubules (MTs). The process of chromosome segregation has been studied extensively in a wide range of organisms, from simple unicellular yeasts to humans. While the major sequence of events required for chromosome segregation remains the same, a few species-specific differences exist. The microtubule organizing centers (MTOCs) in budding yeasts, called spindle pole bodies (SPBs), are embedded in the nuclear envelope and assemble an intranuclear mitotic spindle (5). Thus, the interaction of spindle MTs with KTs does not require the breakdown of the nuclear envelope during closed mitosis in budding yeasts, and the KT-MT interaction is established early in the cell cycle (12, 23). The fission yeast Saccharomyces pombe also undergoes closed mitosis (23), but SPBs remain outside the nuclear envelope during interphase (27) and spindle MTs are nucleated and interact with KTs only when the duplicated SPBs enter the nuclear membrane following mitotic initiation (14). Metazoan cells, in contrast, undergo open mitosis (22,45) in which the MTs, originating from MTOCs, gain access to KTs only when the nuclear envelope breaks down during mitosis. Finally, the number of MTs that bind to a single chromosome differs in these organisms: in Saccharomyces cerevisiae only one MT binds per KT (52), in S. pombe two or three MTs bind per KT (13), and in metazoans multipl...

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“…At least one reason for the low estimation of Cnp1 copy number in our earlier study may be due to unexpected complication in the GFP-tagging construction (34,35). The previous yeast strain carrying Cnp1-GFP (S65T) is at the cnp1 ϩ endogenous locus (strain XL556) (39). In this strain, expression of an additional cryptic cnp1 ORF may produce untagged Cnp1p (missing the N-terminal 6 amino acids in the full-length Cnp1), resulting in reduced incorporation of Cnp1-GFP.…”

Section: Mapping Cnp1mentioning

confidence: 84%

“…2C). To minimize the impact of temperature on the measurement, all of the GFP signal measurement for the purpose of quantifying Cnp1-GFP (S65T) copy number was conducted at 25°C, and the Ndc80-GFP (S65T) was used as the reference (39).…”

Section: Mapping Cnp1mentioning

confidence: 99%

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

Yao¹,

Liu²,

Sakuno

et al. 2013

Journal of Biological Chemistry

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Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres

Cited by 147 publications

References 40 publications

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

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