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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.

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

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The CENP-C4A mutant abrogates rescue of CENP-HK 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-HK were measured and normalized to CREST. Graphs and bars indicate means ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-HK protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (C) GFP–CENP-C1–544 but not the GFP–CENP-C1–544-4A mutant coimmunoprecipitates CENP-HK and CENP-TW. α-GFP coimmunoprecipitation analysis was performed on protein extracts from cycling Flp-In T-REx HeLa cells expressing GFP, GFP–CENP-C, or GFP–CENP-C4A from an inducible promoter. Total protein extracts (Input) and immunoprecipitates (α-GFP co-IP) were separated by SDS-PAGE and blotted with the indicated antibodies. Vinculin served as loading control. IP, immunoprecipitation.
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fig3: The CENP-C4A mutant abrogates rescue of CENP-HK 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-HK were measured and normalized to CREST. Graphs and bars indicate means ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-HK protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (C) GFP–CENP-C1–544 but not the GFP–CENP-C1–544-4A mutant coimmunoprecipitates CENP-HK and CENP-TW. α-GFP coimmunoprecipitation analysis was performed on protein extracts from cycling Flp-In T-REx HeLa cells expressing GFP, GFP–CENP-C, or GFP–CENP-C4A from an inducible promoter. Total protein extracts (Input) and immunoprecipitates (α-GFP co-IP) were separated by SDS-PAGE and blotted with the indicated antibodies. Vinculin served as loading control. IP, immunoprecipitation.

Mentions: We asked whether the CENP-HIKM binding interface of CENP-C is important for kinetochore localization of CCAN subunits in HeLa cells. For this, we generated inducible stable cell lines that expressed full-length wild-type GFP–CENP-C, or the 3A, W317A, or 4A mutants (Fig. S3, F and G). After depletion of endogenous CENP-C by RNAi and induction of transgene expression, the levels of CENP-HK at kinetochores were quantified (Fig. 3 A). Expression of GFP–CENP-Cwt largely rescued the kinetochore levels of CENP-HK observed in control cells. GFP–CENP-C3A and GFP–CENP-CW317A, on the other hand, only produced a partial rescue of the kinetochore levels of CENP-HK (Fig. 3 A), in agreement with their partial retention of the interaction with CENP-HIKM observed in SEC experiments (Fig. S1, C–F). Conversely, GFP–CENP-C4A was unable to produce significant rescue of the CENP-HK levels (Fig. 3, A and B), in line with the biochemical experiments.


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)

The CENP-C4A mutant abrogates rescue of CENP-HK 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-HK were measured and normalized to CREST. Graphs and bars indicate means ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-HK protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (C) GFP–CENP-C1–544 but not the GFP–CENP-C1–544-4A mutant coimmunoprecipitates CENP-HK and CENP-TW. α-GFP coimmunoprecipitation analysis was performed on protein extracts from cycling Flp-In T-REx HeLa cells expressing GFP, GFP–CENP-C, or GFP–CENP-C4A from an inducible promoter. Total protein extracts (Input) and immunoprecipitates (α-GFP co-IP) were separated by SDS-PAGE and blotted with the indicated antibodies. Vinculin served as loading control. IP, immunoprecipitation.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The CENP-C4A mutant abrogates rescue of CENP-HK 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-HK were measured and normalized to CREST. Graphs and bars indicate means ± SEM of three independent experiments. (B) Representative images of the dataset quantified in A and documenting the levels of CENP-HK protein in cells expressing GFP–CENP-C or GFP–CENP-C4A. Bars, 10 µM. Magnification = 630×. Insets show a magnification of the white boxed areas that capture one or more kinetochores in each panel. (C) GFP–CENP-C1–544 but not the GFP–CENP-C1–544-4A mutant coimmunoprecipitates CENP-HK and CENP-TW. α-GFP coimmunoprecipitation analysis was performed on protein extracts from cycling Flp-In T-REx HeLa cells expressing GFP, GFP–CENP-C, or GFP–CENP-C4A from an inducible promoter. Total protein extracts (Input) and immunoprecipitates (α-GFP co-IP) were separated by SDS-PAGE and blotted with the indicated antibodies. Vinculin served as loading control. IP, immunoprecipitation.
Mentions: We asked whether the CENP-HIKM binding interface of CENP-C is important for kinetochore localization of CCAN subunits in HeLa cells. For this, we generated inducible stable cell lines that expressed full-length wild-type GFP–CENP-C, or the 3A, W317A, or 4A mutants (Fig. S3, F and G). After depletion of endogenous CENP-C by RNAi and induction of transgene expression, the levels of CENP-HK at kinetochores were quantified (Fig. 3 A). Expression of GFP–CENP-Cwt largely rescued the kinetochore levels of CENP-HK observed in control cells. GFP–CENP-C3A and GFP–CENP-CW317A, on the other hand, only produced a partial rescue of the kinetochore levels of CENP-HK (Fig. 3 A), in agreement with their partial retention of the interaction with CENP-HIKM observed in SEC experiments (Fig. S1, C–F). Conversely, GFP–CENP-C4A was unable to produce significant rescue of the CENP-HK levels (Fig. 3, A and B), in line with the biochemical experiments.

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