Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 promote accurate chromosome segregation - PubMed (original) (raw)

Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 promote accurate chromosome segregation

Mathijs Vleugel et al. J Cell Biol. 2013.

Abstract

Fidelity of chromosome segregation relies on coordination of chromosome biorientation and the spindle checkpoint. Central to this is the kinetochore scaffold KNL1 that integrates the functions of various mitotic regulators including BUB1 and BUBR1. We show that KNL1 contains an extensive array of short linear sequence modules that encompass TxxΩ and MELT motifs and that can independently localize BUB1. Engineered KNL1 variants with few modules recruit low levels of BUB1 to kinetochores but support a robust checkpoint. Increasing numbers of modules concomitantly increase kinetochore BUB1 levels and progressively enhance efficiency of chromosome biorientation. Remarkably, normal KNL1 function is maintained by replacing all modules with a short array of naturally occurring or identical, artificially designed ones. A minimal array of generic BUB recruitment modules in KNL1 thus suffices for accurate chromosome segregation. Widespread divergence in the amount and sequence of these modules in KNL1 homologues may represent flexibility in adapting regulation of mitotic processes to altered requirements for chromosome segregation during evolution.

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Figures

Figure 1.

Figure 1.

The N-terminal MDLT-KI module in KNL1 independently recruits BUB proteins. (A) Immunolocalization of BUB1 (left panels, red) and BUBR1 (right panels, red) in LacI-LAP-KNL11–261–transfected, nocodazole-treated U2OS-LacO cells. LacI-LAP-KNL11–261 is shown in green and DNA (DAPI) is in blue. Insets show magnifications of the boxed regions. KNL1KI1 denotes LacI-LAP-KNL11–261 in which KIDTTSF is mutated to KIDATSA, KNL1KI2 is KIDFNDF mutated to KIDANDA, and KNL1MDLT mutated is MDLT to MDLA. Bars, 5 µm (insets, 0.5 µm). (B–D) Representative images (B and C) and quantification (D) of LAP-KNL1–expressing Flp-in HeLa cells transfected with siRNAs to luciferase (siLUC) or to KNL1 (siKNL1) and treated with nocodazole. LAP-KNL1 is shown in green, BUB proteins in red, centromeres (CREST) in blue, and DNA (DAPI) in white. Bars, 5 µm. Quantification in D shows total kinetochore signal intensity (+SD) of LAP-KNL1 and BUB proteins over CREST. Data are from >15 cells and representative of 3 experiments. Levels of kinetochore BUBs in control cells and of kinetochore LAP-KNL1 in KNL1-FL–expressing cells are set to 1.

Figure 2.

Figure 2.

The N-terminal MDLT-KI module in KNL1 is sufficient to support SAC activity but not chromosome biorientation. (A) Time-lapse analysis of Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with nocodazole and 250 nM reversine. Data (n = 40 representative of 3 independent experiments) indicate cumulative fraction of cells that exit from mitosis (as scored by cell morphology using DIC) at the indicated time after NEB. (B) As in A, but with transfection of the indicated siRNAs. (C) Immunostaining and quantification of centromeric H2A-Thr120 phosphorylation in Flp-in HeLa cells expressing LAP-KNL1 variants and transfected with siLUC or siKNL1. pH2A-Thr120 is shown in green, centromeres (CREST) in red, and DNA (DAPI) in blue. Bars, 5 µm. pH2A-Thr120 is quantified over CREST (n = 10 representative of 3 independent experiments). (D) Immunostaining and quantification of chromosome alignment in Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with MG132 for 45 min. Tubulin is shown in green, centromeres (CREST) in red, and DNA (DAPI) in blue. Bars, 5 µm. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 50. (E) Time-lapse analysis of Flp-in HeLa cells expressing LAP-KNL1 variants and transfected with siLUC or siKNL1. Data (n = 40 representative of 3 independent experiments) indicate cumulative fraction of cells that exit from mitosis at the indicated time after NEB (as scored by GFP-H2B).

Figure 3.

Figure 3.

KNL1 contains multiple independent BUB recruitment modules. (A) Schematic representation of KNL1 showing the microtubule- and PP1-binding domain in green and the kinetochore recruitment domain in orange. KI1 and KI2 motifs are shown as green bars, MELT-like sequences in red, and TΩ-like sequences in blue. Dashed lines indicate the generated LacI-LAP-KNL1 fragments used in B. (B) Immunolocalization of BUB1 (red) in nocodazole-treated U2OS-LacO cells transfected with LacI-LAP-KNL1 fragments. LacI-LAP-KNL1 fragments are shown in green, centromeres (CREST) in blue, and DNA (DAPI) in white. Insets show magnifications of the boxed regions. Bars, 5 µm (insets, 0.5 µm). Table indicates the ability (− or +) to recruit BUB1 and BUBR1 by the indicated KNL1 fragments (see also

Fig. S3, A and B

). (C) Alignment of identified TΩ-MELT modules showing conserved (green/purple/red/blue) and atypical (orange/yellow) amino acids. (D) Sequence logo of the 19 TΩ-MELT units. (E) As in B, but with LacI-LAP-KNL1818–1051 (M3) or mutant variants thereof. These variants are: M3-TAΩA (TxxΩ to AxxA), M3-MELTA (MELT to MELA), and A3 (TxxΩ-MELT to AxxA-AELA), as shown in

Fig. S3 C

.

Figure 4.

Figure 4.

Engineered KNL1 proteins reveal differential requirements for TΩ-MELT modules in the SAC and chromosome biorientation. (A) Schematic representation of synthetic LAP-KNL1 constructs, showing the microtubule- and PP1-binding domain in green and the kinetochore recruitment domain in orange. KI1 and KI2 motifs are shown as green bars, MELT-like sequences in blue, and TxxΩ-like sequences in red. See main text for details about constructs. (B and C) Representative images (B) and quantification (C) of LAP-KNL1-expressing Flp-in HeLa cells transfected with siRNAs to luciferase (siLUC) or to KNL1 (siKNL1) and treated with nocodazole. LAP-KNL1 is shown in green, BUB1 in red, centromeres (CREST) in blue, and DNA (DAPI) in white. Bars, 5 µm. Quantification in C shows total kinetochore signal intensity (+SD) of LAP-KNL1 and BUB proteins over CREST. Data are from >15 cells and representative of 3 experiments. Levels of kinetochore BUBs in control cells and of kinetochore LAP-KNL1 in KNL1-FL–expressing cells are set to 1. (D) Schematic as in A. See main text for details about constructs. (E) Time-lapse analysis of Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with nocodazole and 250 nM reversine. Data (n = 40 representative of 3 independent experiments) indicate cumulative fraction of cells that exit from mitosis (as scored by cell morphology using DIC) at the indicated time after NEB. (F) As in E, with the indicated constructs. (G) Time-lapse analysis of Flp-in HeLa cells expressing LAP-KNL1 variants and transfected with siLUC or siKNL1. Data (n = 40 representative of 3 independent experiments) indicate cumulative fraction of cells that exit from mitosis at the indicated time after NEB (as scored by GFP-H2B). (H) Quantification of chromosome alignment in Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with MG132 for 45 min. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 40.

Figure 5.

Figure 5.

TΩ-MELT modules in KNL1 are redundant and exchangeable. (A) Schematic representation of synthetic LAP-KNL1 constructs. For color codes, see Fig. 4 A. See main text for details about constructs. (B and C) Representative images (B) and quantification (C) of LAP-KNL1–expressing Flp-in HeLa cells transfected with siRNAs to luciferase (siLUC) or to KNL1 (siKNL1) and treated with nocodazole. LAP-KNL1 is shown in green, BUB1 in red, centromeres (CREST) in blue, and DNA (DAPI) in white. Bars, 5 µm. Quantification in C shows total kinetochore signal intensity (+SD) of LAP-KNL1 and BUB proteins over CREST. Data are from >15 cells and representative of 3 experiments. Levels of kinetochore BUBs in control cells and of kinetochore LAP-KNL1 in KNL1-FL–expressing cells are set to 1. (D) Quantification of chromosome alignment in Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with MG132 for 45 min. The data shown are from a single representative experiment out of three repeats. For the experiment shown, n = 40. (E) Time-lapse analysis of Flp-in HeLa cells expressing LAP-KNL1 variants, transfected with siLUC or siKNL1, and treated with nocodazole and 250 nM reversine. Data (n = 40 representative of 3 independent experiments) indicate cumulative fraction of cells that exit from mitosis (as scored by cell morphology using DIC) at the indicated time after NEB.

Figure 6.

Figure 6.

TΩ-MELT module evolution and model. (A) Schematic representation of eukaryotic tree of life showing KNL1 homologues from indicated species. Repeating units are shown in blue and red with the number of repeats in corresponding colors. Repeat sequences are shown as sequence logos. (B) Model for TΩ-MELT function in human KNL1. Conserved (dark blue) and degenerated (light blue) TΩ-MELT modules (essential amino acids in red) in KNL1 can independently recruit BUB protein complexes (BUBs) to promote H2A-Thr120 phosphorylation and SAC activity (few modules, low BUB levels) and chromosome biorientation (increasing fidelity with increasing modules and BUB levels).

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References

    1. Bailey T.L., Boden M., Buske F.A., Frith M., Grant C.E., Clementi L., Ren J., Li W.W., Noble W.S. 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37(Web Server issue):W202–W208 10.1093/nar/gkp335 - DOI - PMC - PubMed
    1. Bolanos-Garcia V.M., Blundell T.L. 2011. BUB1 and BUBR1: multifaceted kinases of the cell cycle. Trends Biochem. Sci. 36:141–150 10.1016/j.tibs.2010.08.004 - DOI - PMC - PubMed
    1. Bolanos-Garcia V.M., Lischetti T., Matak-Vinković D., Cota E., Simpson P.J., Chirgadze D.Y., Spring D.R., Robinson C.V., Nilsson J., Blundell T.L. 2011. Structure of a Blinkin-BUBR1 complex reveals an interaction crucial for kinetochore-mitotic checkpoint regulation via an unanticipated binding Site. Structure. 19:1691–1700 10.1016/j.str.2011.09.017 - DOI - PMC - PubMed
    1. Chao W.C., Kulkarni K., Zhang Z., Kong E.H., Barford D. 2012. Structure of the mitotic checkpoint complex. Nature. 484:208–213 10.1038/nature10896 - DOI - PubMed
    1. Cheeseman I.M., Desai A. 2008. Molecular architecture of the kinetochore-microtubule interface. Nat. Rev. Mol. Cell Biol. 9:33–46 10.1038/nrm2310 - DOI - PubMed

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