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CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores.

Klare K, Weir JR, Basilico F, Zimniak T, Massimiliano L, Ludwigs N, Herzog F, Musacchio A - J. Cell Biol. (2015)

Bottom Line: A 16-subunit complex named the constitutive centromere-associated network (CCAN) creates the centromere-kinetochore interface.We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex.When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.

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Depletion of CENP-C abolishes localization of CCAN to kinetochores. (A) Schematic kinetochore organization. CCAN subunits are shown in hot pink. Light pink lines indicate presumed contacts with centromeric chromatin. Black arrows indicate direct and well-established interactions. Gray arrows represent interactions that are still uncharacterized and that were characterized in this study. For instance, whether localization of CENP-STWX to inner kinetochores requires CENP-C is controversial. Direct interaction of CENP-STWX to CENP-HIKM has been demonstrated recently and reflects in codependency for kinetochore localization. Both CENP-STWX and CENP-C contribute to recruitment of outer kinetochore components. (B) Schematic representation of human CENP-C depicting some of the protein’s crucial domains. N, N terminus; C, C terminus. (C) Whole cell protein extracts from Flp-In T-REx HeLa cells treated with control or CENP-C siRNAs were run on SDS-PAGE and immunoblotted for the indicated kinetochore proteins. Vinculin served as a loading control. MWM, molecular weight marker. (D) Representative images showing kinetochore levels of CENP-C, CENP-TW (with an antibody raised against the CENP-TW complex), and CENP-HK (with an antibody raised against the CENP-HK complex) in Flp-In T-REx HeLa cells upon treatment with control and CENP-C siRNA in interphase. Kinetochores were visualized with CREST sera. Upon CENP-C siRNA, CENP-C, CENP-HK, and CENP-TW were displaced from kinetochores. Bars, 10 µm. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (E) Quantifications are expressed as normalized protein/CREST fluorescence intensity ratios from the experiment in D. Graphs and bars indicate means ± SEM of three independent experiments for CENP-C, CENP-HK, and CENP-TW. (F) SEC elution profile shows CENP-C2–545 (∼62 kD) elutes earlier than predicted based on its molecular mass, consistent with an unfolded structure. The modest height of the CENP-C peak is caused by low abundance of aromatic amino acids. CENP-C2–545 and CENP-HIKM, both at 10 µM, form a stable, apparently stoichiometric complex on an analytical SEC. Shown is the typical outcome of at least five experiments. (G) SEC elution profile of CENP-C189–400 (∼24 kD) and of its combination with CENP-HIKM, both at 5 µM. Also CENP-C189–400 and CENP-HIKM form a stoichiometric complex and coelute in analytical SEC. Shown is the typical outcome of at least three experiments. The gray dotted lines show the elution profile of globular markers of known molecular masses, as indicated.
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fig1: Depletion of CENP-C abolishes localization of CCAN to kinetochores. (A) Schematic kinetochore organization. CCAN subunits are shown in hot pink. Light pink lines indicate presumed contacts with centromeric chromatin. Black arrows indicate direct and well-established interactions. Gray arrows represent interactions that are still uncharacterized and that were characterized in this study. For instance, whether localization of CENP-STWX to inner kinetochores requires CENP-C is controversial. Direct interaction of CENP-STWX to CENP-HIKM has been demonstrated recently and reflects in codependency for kinetochore localization. Both CENP-STWX and CENP-C contribute to recruitment of outer kinetochore components. (B) Schematic representation of human CENP-C depicting some of the protein’s crucial domains. N, N terminus; C, C terminus. (C) Whole cell protein extracts from Flp-In T-REx HeLa cells treated with control or CENP-C siRNAs were run on SDS-PAGE and immunoblotted for the indicated kinetochore proteins. Vinculin served as a loading control. MWM, molecular weight marker. (D) Representative images showing kinetochore levels of CENP-C, CENP-TW (with an antibody raised against the CENP-TW complex), and CENP-HK (with an antibody raised against the CENP-HK complex) in Flp-In T-REx HeLa cells upon treatment with control and CENP-C siRNA in interphase. Kinetochores were visualized with CREST sera. Upon CENP-C siRNA, CENP-C, CENP-HK, and CENP-TW were displaced from kinetochores. Bars, 10 µm. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (E) Quantifications are expressed as normalized protein/CREST fluorescence intensity ratios from the experiment in D. Graphs and bars indicate means ± SEM of three independent experiments for CENP-C, CENP-HK, and CENP-TW. (F) SEC elution profile shows CENP-C2–545 (∼62 kD) elutes earlier than predicted based on its molecular mass, consistent with an unfolded structure. The modest height of the CENP-C peak is caused by low abundance of aromatic amino acids. CENP-C2–545 and CENP-HIKM, both at 10 µM, form a stable, apparently stoichiometric complex on an analytical SEC. Shown is the typical outcome of at least five experiments. (G) SEC elution profile of CENP-C189–400 (∼24 kD) and of its combination with CENP-HIKM, both at 5 µM. Also CENP-C189–400 and CENP-HIKM form a stoichiometric complex and coelute in analytical SEC. Shown is the typical outcome of at least three experiments. The gray dotted lines show the elution profile of globular markers of known molecular masses, as indicated.

Mentions: Kinetochores mediate the attachment of sister chromatids to spindle microtubules. These large structures, consisting of several copies of ∼30 core subunits, become established on a segment of specialized chromatin named the centromere, whose main hallmark is the presence of the histone H3 variant CENP-A (or CenH3; Fig. 1 A; McAinsh and Meraldi, 2011; Fukagawa and Earnshaw, 2014). At low resolution, kinetochores appear as laminar structures, with an outer plate receiving the ends of spindle microtubules, and an inner plate adjacent to dense centromeric chromatin (Santaguida and Musacchio, 2009; Cheeseman, 2014). The outer kinetochore plate hosts the KMN (Knl1 complex, Mis12 complex, Ndc80 complex) network, a 10-subunit assembly that plays a crucial role as a receptor for microtubules (Cheeseman et al., 2006; DeLuca et al., 2006). The inner kinetochore hosts the constitutive centromere–associated network (CCAN), a complex of at least 16 different CENPs (centromeric proteins), several of which were originally identified in the CENP-A interactome of vertebrates (McCleland et al., 2004; Obuse et al., 2004; Foltz et al., 2006; Izuta et al., 2006; Okada et al., 2006; Hori et al., 2008).


CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores.

Klare K, Weir JR, Basilico F, Zimniak T, Massimiliano L, Ludwigs N, Herzog F, Musacchio A - J. Cell Biol. (2015)

Depletion of CENP-C abolishes localization of CCAN to kinetochores. (A) Schematic kinetochore organization. CCAN subunits are shown in hot pink. Light pink lines indicate presumed contacts with centromeric chromatin. Black arrows indicate direct and well-established interactions. Gray arrows represent interactions that are still uncharacterized and that were characterized in this study. For instance, whether localization of CENP-STWX to inner kinetochores requires CENP-C is controversial. Direct interaction of CENP-STWX to CENP-HIKM has been demonstrated recently and reflects in codependency for kinetochore localization. Both CENP-STWX and CENP-C contribute to recruitment of outer kinetochore components. (B) Schematic representation of human CENP-C depicting some of the protein’s crucial domains. N, N terminus; C, C terminus. (C) Whole cell protein extracts from Flp-In T-REx HeLa cells treated with control or CENP-C siRNAs were run on SDS-PAGE and immunoblotted for the indicated kinetochore proteins. Vinculin served as a loading control. MWM, molecular weight marker. (D) Representative images showing kinetochore levels of CENP-C, CENP-TW (with an antibody raised against the CENP-TW complex), and CENP-HK (with an antibody raised against the CENP-HK complex) in Flp-In T-REx HeLa cells upon treatment with control and CENP-C siRNA in interphase. Kinetochores were visualized with CREST sera. Upon CENP-C siRNA, CENP-C, CENP-HK, and CENP-TW were displaced from kinetochores. Bars, 10 µm. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (E) Quantifications are expressed as normalized protein/CREST fluorescence intensity ratios from the experiment in D. Graphs and bars indicate means ± SEM of three independent experiments for CENP-C, CENP-HK, and CENP-TW. (F) SEC elution profile shows CENP-C2–545 (∼62 kD) elutes earlier than predicted based on its molecular mass, consistent with an unfolded structure. The modest height of the CENP-C peak is caused by low abundance of aromatic amino acids. CENP-C2–545 and CENP-HIKM, both at 10 µM, form a stable, apparently stoichiometric complex on an analytical SEC. Shown is the typical outcome of at least five experiments. (G) SEC elution profile of CENP-C189–400 (∼24 kD) and of its combination with CENP-HIKM, both at 5 µM. Also CENP-C189–400 and CENP-HIKM form a stoichiometric complex and coelute in analytical SEC. Shown is the typical outcome of at least three experiments. The gray dotted lines show the elution profile of globular markers of known molecular masses, as indicated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494010&req=5

fig1: Depletion of CENP-C abolishes localization of CCAN to kinetochores. (A) Schematic kinetochore organization. CCAN subunits are shown in hot pink. Light pink lines indicate presumed contacts with centromeric chromatin. Black arrows indicate direct and well-established interactions. Gray arrows represent interactions that are still uncharacterized and that were characterized in this study. For instance, whether localization of CENP-STWX to inner kinetochores requires CENP-C is controversial. Direct interaction of CENP-STWX to CENP-HIKM has been demonstrated recently and reflects in codependency for kinetochore localization. Both CENP-STWX and CENP-C contribute to recruitment of outer kinetochore components. (B) Schematic representation of human CENP-C depicting some of the protein’s crucial domains. N, N terminus; C, C terminus. (C) Whole cell protein extracts from Flp-In T-REx HeLa cells treated with control or CENP-C siRNAs were run on SDS-PAGE and immunoblotted for the indicated kinetochore proteins. Vinculin served as a loading control. MWM, molecular weight marker. (D) Representative images showing kinetochore levels of CENP-C, CENP-TW (with an antibody raised against the CENP-TW complex), and CENP-HK (with an antibody raised against the CENP-HK complex) in Flp-In T-REx HeLa cells upon treatment with control and CENP-C siRNA in interphase. Kinetochores were visualized with CREST sera. Upon CENP-C siRNA, CENP-C, CENP-HK, and CENP-TW were displaced from kinetochores. Bars, 10 µm. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (E) Quantifications are expressed as normalized protein/CREST fluorescence intensity ratios from the experiment in D. Graphs and bars indicate means ± SEM of three independent experiments for CENP-C, CENP-HK, and CENP-TW. (F) SEC elution profile shows CENP-C2–545 (∼62 kD) elutes earlier than predicted based on its molecular mass, consistent with an unfolded structure. The modest height of the CENP-C peak is caused by low abundance of aromatic amino acids. CENP-C2–545 and CENP-HIKM, both at 10 µM, form a stable, apparently stoichiometric complex on an analytical SEC. Shown is the typical outcome of at least five experiments. (G) SEC elution profile of CENP-C189–400 (∼24 kD) and of its combination with CENP-HIKM, both at 5 µM. Also CENP-C189–400 and CENP-HIKM form a stoichiometric complex and coelute in analytical SEC. Shown is the typical outcome of at least three experiments. The gray dotted lines show the elution profile of globular markers of known molecular masses, as indicated.
Mentions: Kinetochores mediate the attachment of sister chromatids to spindle microtubules. These large structures, consisting of several copies of ∼30 core subunits, become established on a segment of specialized chromatin named the centromere, whose main hallmark is the presence of the histone H3 variant CENP-A (or CenH3; Fig. 1 A; McAinsh and Meraldi, 2011; Fukagawa and Earnshaw, 2014). At low resolution, kinetochores appear as laminar structures, with an outer plate receiving the ends of spindle microtubules, and an inner plate adjacent to dense centromeric chromatin (Santaguida and Musacchio, 2009; Cheeseman, 2014). The outer kinetochore plate hosts the KMN (Knl1 complex, Mis12 complex, Ndc80 complex) network, a 10-subunit assembly that plays a crucial role as a receptor for microtubules (Cheeseman et al., 2006; DeLuca et al., 2006). The inner kinetochore hosts the constitutive centromere–associated network (CCAN), a complex of at least 16 different CENPs (centromeric proteins), several of which were originally identified in the CENP-A interactome of vertebrates (McCleland et al., 2004; Obuse et al., 2004; Foltz et al., 2006; Izuta et al., 2006; Okada et al., 2006; Hori et al., 2008).

Bottom Line: A 16-subunit complex named the constitutive centromere-associated network (CCAN) creates the centromere-kinetochore interface.We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex.When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.

Show MeSH
Related in: MedlinePlus