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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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CENP-C4A abrogates rescue of CENP-TW localization to kinetochores. (A) Quantification of immunofluorescence experiment in fixed Flp-In T-REx HeLa cells depleted of endogenous CENP-C (where indicated) and expressing the indicated GFP constructs. The kinetochore levels of CENP-TW were measured and normalized to CREST. Graphs and bars indicate mean ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-TW protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. (C) GST–CENP-C2–545 does not bind directly to CENP-TW in a GST pull-down assay. Bands were visualized with Coomassie brilliant blue staining. (left) Only when HIKM is included in the assay, CENP-TW is incorporated into the complex. Because CENP-I and CENP-T comigrate on the SDS-PAGE gel shown, 10% of the pull-down samples were separated on a new SDS-PAGE and subjected to Western blotting with either a CENP-T or CENP-I antibody. (D) Recasting of the kinetochore assembly scheme based on our new results. (E) The CENP-C sequence contains a linear array of motifs whose succession appears to correlate with the order of kinetochore assembly from the CENP-A nucleosome toward the outer kinetochore. N, N terminus; C, C terminus.
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fig4: CENP-C4A abrogates rescue of CENP-TW localization to kinetochores. (A) Quantification of immunofluorescence experiment in fixed Flp-In T-REx HeLa cells depleted of endogenous CENP-C (where indicated) and expressing the indicated GFP constructs. The kinetochore levels of CENP-TW were measured and normalized to CREST. Graphs and bars indicate mean ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-TW protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. (C) GST–CENP-C2–545 does not bind directly to CENP-TW in a GST pull-down assay. Bands were visualized with Coomassie brilliant blue staining. (left) Only when HIKM is included in the assay, CENP-TW is incorporated into the complex. Because CENP-I and CENP-T comigrate on the SDS-PAGE gel shown, 10% of the pull-down samples were separated on a new SDS-PAGE and subjected to Western blotting with either a CENP-T or CENP-I antibody. (D) Recasting of the kinetochore assembly scheme based on our new results. (E) The CENP-C sequence contains a linear array of motifs whose succession appears to correlate with the order of kinetochore assembly from the CENP-A nucleosome toward the outer kinetochore. N, N terminus; C, C terminus.

Mentions: In Fig. 1 (D and E), we demonstrated that kinetochore localization of CENP-TW depends on CENP-C. Kinetochore localization of CENP-TW had been previously shown to depend also on the CENP-HIKM complex (Hori et al., 2008; Basilico et al., 2014). Thus, we analyzed how CENP-C mutants impairing CENP-HIKM localization to kinetochores impacted kinetochore localization of CENP-TW. As for CENP-HK, wild-type CENP-C fully rescued kinetochore localization of CENP-TW, but there was a progressive reduction of CENP-TW kinetochore levels when CENP-C mutants were expressed (Fig. 4, A and B). In agreement with these observations, we readily identified CENP-TW in precipitates of GFP–CENP-C1–544-wt but not in precipitates of GFP–CENP-C1–544-4A (Fig. 3 C).


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)

CENP-C4A abrogates rescue of CENP-TW localization to kinetochores. (A) Quantification of immunofluorescence experiment in fixed Flp-In T-REx HeLa cells depleted of endogenous CENP-C (where indicated) and expressing the indicated GFP constructs. The kinetochore levels of CENP-TW were measured and normalized to CREST. Graphs and bars indicate mean ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-TW protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. (C) GST–CENP-C2–545 does not bind directly to CENP-TW in a GST pull-down assay. Bands were visualized with Coomassie brilliant blue staining. (left) Only when HIKM is included in the assay, CENP-TW is incorporated into the complex. Because CENP-I and CENP-T comigrate on the SDS-PAGE gel shown, 10% of the pull-down samples were separated on a new SDS-PAGE and subjected to Western blotting with either a CENP-T or CENP-I antibody. (D) Recasting of the kinetochore assembly scheme based on our new results. (E) The CENP-C sequence contains a linear array of motifs whose succession appears to correlate with the order of kinetochore assembly from the CENP-A nucleosome toward the outer kinetochore. N, N terminus; C, C terminus.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: CENP-C4A abrogates rescue of CENP-TW localization to kinetochores. (A) Quantification of immunofluorescence experiment in fixed Flp-In T-REx HeLa cells depleted of endogenous CENP-C (where indicated) and expressing the indicated GFP constructs. The kinetochore levels of CENP-TW were measured and normalized to CREST. Graphs and bars indicate mean ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-TW protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. (C) GST–CENP-C2–545 does not bind directly to CENP-TW in a GST pull-down assay. Bands were visualized with Coomassie brilliant blue staining. (left) Only when HIKM is included in the assay, CENP-TW is incorporated into the complex. Because CENP-I and CENP-T comigrate on the SDS-PAGE gel shown, 10% of the pull-down samples were separated on a new SDS-PAGE and subjected to Western blotting with either a CENP-T or CENP-I antibody. (D) Recasting of the kinetochore assembly scheme based on our new results. (E) The CENP-C sequence contains a linear array of motifs whose succession appears to correlate with the order of kinetochore assembly from the CENP-A nucleosome toward the outer kinetochore. N, N terminus; C, C terminus.
Mentions: In Fig. 1 (D and E), we demonstrated that kinetochore localization of CENP-TW depends on CENP-C. Kinetochore localization of CENP-TW had been previously shown to depend also on the CENP-HIKM complex (Hori et al., 2008; Basilico et al., 2014). Thus, we analyzed how CENP-C mutants impairing CENP-HIKM localization to kinetochores impacted kinetochore localization of CENP-TW. As for CENP-HK, wild-type CENP-C fully rescued kinetochore localization of CENP-TW, but there was a progressive reduction of CENP-TW kinetochore levels when CENP-C mutants were expressed (Fig. 4, A and B). In agreement with these observations, we readily identified CENP-TW in precipitates of GFP–CENP-C1–544-wt but not in precipitates of GFP–CENP-C1–544-4A (Fig. 3 C).

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