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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.01006

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Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression

School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK

View larger version (47K): [in a new window]   Fig. 2. Design of siRNA duplexes and repression of Bub1, Cenp-F and Survivin. (A) siRNA duplexes used in this study showing target sequence (AA-N19) including a scrambled siRNA duplex control. The control siRNA duplex was designed by scrambling the Bub1 target sequence and checking specificity in a BLAST search. (B) Immunoblots of asynchronous HeLa cell lysates 48 hours after transfection showing repression of Bub1, Cenp-F and Survivin. Tubulin was used as a loading control.

View larger version (47K): [in a new window]   Fig. 1. Mad2 is recruited to kinetochores after Bub1, BubR1 and Cenp-E. (A) A time line for the association of checkpoint proteins to the kinetochore during mitosis. (B) Protein extracts from DLD-1 cells expressing Myc or Myc-Mad2 were analysed by western blotting with anti-Mad2 antibody (SM2.2) or anti-Myc antibody (9E10). (C,D) Immunofluorescence images of DLD-1 Myc-Mad2 cells in different stages of mitosis stained to detect Myc-tagged Mad2 (9E10) and either Bub1 (SB1.3) (C) or BubR1 (SBR1.1). Cells were also stained to detect Aurora A (RAA.1) in order to define the spindle axis. (E) Quantitation of Cenp-E and Mad2 levels at kinetochores, normalized to the ACA signal, showing that Myc-Mad2 accumulates at kinetochores in late prometaphase, after Cenp-E.

View larger version (46K): [in a new window]   Fig. 3. Bub1 is required for kinetochore localization of BubR1, Cenp-F, Cenp-E and Mad2. DLD-1 or DLD-1 Myc-Mad2 cells were transfected with control or Bub1 siRNA duplexes, treated with nocodazole and then fixed and stained to detect BubR1 (blue), centromeres/kinetochores (ACA, green), DNA and BubR1, Myc-Mad2, Cenp-E or Cenp-F (red). The cells were then analysed by deconvolution microscopy and image stacks projected onto a single plane. (A) Representative projected image stacks showing that repression of Bub1 reduces kinetochore localization of BubR1, Mad2, Cenp-E and Cenp-F. Scale bar, 5 µm. (B) Normalized pixel intensities in control (black bar) and Bub1-repressed (white bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells.

View larger version (43K): [in a new window]   Fig. 4. BubR1 is required for kinetochore localization of Cenp-E. DLD-1 or DLD-1 Myc-Mad2 cells were transfected with control or BubR1 siRNA duplexes, treated with nocodazole and then fixed and stained to detect BubR1 (blue), centromeres/kinetochores (ACA, green), DNA and Bub1, Myc-Mad2, Cenp-E or Cenp-F (red). (A) Representative projected image stacks showing that repression of BubR1 reduces kinetochore localization of Cenp-E but does not affect Bub1, Cenp-F or Mad2. Scale bar, 5 µm. (B) Normalized pixel intensities in control (black bar) and BubR1-repressed (white bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells.

View larger version (44K): [in a new window]   Fig. 5. Cenp-E is required for efficient localization of Mad2 and Cenp-F. DLD-1 or DLD-1 Myc-Mad2 cells were transfected with control or Cenp-E siRNA duplexes, treated with nocodazole and then fixed and stained to detect Cenp-E (blue), centromeres/kinetochores (ACA, green), DNA and Bub1, BubR1, Myc-Mad2 or Cenp-F (red). (A) Representative projected image stacks showing that repression of Cenp-E reduces kinetochore localization of Mad2 and Cenp-F but does not affect Bub1 or BubR1. Scale bar, 5 µm. (B) Normalized pixel intensities in control (black bar) and Cenp-E-repressed (white bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells.

View larger version (33K): [in a new window]   Fig. 6. Cenp-F is not required for kinetochore localization of Bub1, BubR1 or Mad2. DLD-1 or DLD-1 Myc-Mad2 cells were transfected with control or Cenp-F siRNA duplexes, treated with nocodazole and then fixed and stained to detect Cenp-F (blue), centromeres/kinetochores (ACA, green), DNA and Bub1, BubR1, Myc-Mad2 or Cenp-E (red). (A) Representative projected image stacks showing that repression of Cenp-F reduces kinetochore localization of Cenp-E. Scale bar, 5 µm. (B) Normalized pixel intensities in control (black bar) and Cenp-F-repressed (white bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells.

View larger version (62K): [in a new window]   Fig. 7. Aurora B is required for localization of Bub1. DLD-1 cells were transfected with control or Aurora B siRNA duplexes, treated with nocodazole and then fixed and stained to detect Aurora B (blue), centromeres/kinetochores (ACA, green) DNA and Bub1, BubR1 or Cenp-E (red). (A) Projection of a deconvolved image stack showing repressed and unrepressed prometaphase DLD-1 cells transfected with Aurora B siRNA duplexes in the same field of view. (B) Normalized pixel intensities in nocodazole-treated control (black bar) and Aurora-B-repressed (white bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells. (C) Repression of Aurora B inhibits the localization of Bub1 during prophase. Projections of deconvolved image stacks showing prophase DLD-1 cells transfected with control (scramble) and Aurora B siRNA duplexes, fixed and stained, as described above. (D) Standard immunofluorescence of prometaphase DLD-1 cells that were transfected with control, Bub1, BubR1 or Cenp-E siRNA duplexes, fixed and stained to detect DNA, Aurora B (red) and Bub1, BubR1 and Cenp-E (Green). Representative examples showing that repression of either Bub1, BubR1 or Cenp-E has no apparent effect on Aurora B localization.

View larger version (8K): [in a new window]   Fig. 10. Model showing the temporal order and level of dependency of checkpoint proteins at the kinetochore. Bub1 acts as a master regulator for the assembly of checkpoint proteins at the kinetochore. The thick lines represent protein-protein interactions at the kinetochore but our data do not support the notion that the assembly pathway is a simple linear process. Therefore, other factors in addition to the linear pathway must also contribute to kinetochore targeting. One such factor might be Aurora B, which, as depicted by the thin lines, might cause priming of either the kinetochore and/or kinetochore proteins.

View larger version (40K): [in a new window]   Fig. 8. Repression of Bub1, BubR1, Cenp-E, Aurora B and Survivin result in distinct alignment defects. (A) Immunofluorescence images showing examples of alignment defects after repression of Bub1, Cenp-E, BubR1, Aurora B and Survivin in DLD-1 cells that were fixed and stained to detect DNA (blue) and Aurora A (red). (B) Percentages of normal and abnormal metaphases with incomplete congression at the metaphase plate (as shown in A) after repression of Bub1 or Cenp-E. (C) Proportion of mitotic cells exhibiting prophase, prometaphase, metaphase and anaphase chromosome alignments after a 1-3 hour exposure to MG132. Values represent the mean and s.e.m. derived from three independent experiments in which at least 100 mitotic cells were counted. (D) Prometaphase/metaphase ratios from the 1 hour time point (C).

View larger version (63K): [in a new window]   Fig. 9. Bub1-deficient cells have a robust spindle checkpoint. (A) DLD-1 cells were transfected with control, Bub1 and BubR1 siRNA duplexes. 48 hours after transfection, the cells were fixed, stained with Hoechst and the number of cells in each phase of mitosis determined. (B) 48 hours after transfection, cells were incubated with nocodazole and the number of mitotic cells scored by fluorescence microscopy after 3 hours and 6 hours. At least 1000 cells were counted for each time point and values represent the mean and s.e.m. from at least three independent experiments. (C) HeLa cells were transfected with control, Bub1 and BubR1 siRNA duplexes and, 40 hours after transfection, cells were treated with nocodazole for 18 hours, at which point untreated cell lysates were analysed by western blotting to determine levels of protein repression. (D) The percentage of mitotic cells in the population was determined by MPM2 staining followed by FACS analysis and plotted as a bar graph. Values represent the mean plus s.d. of two independent experiments. (E) Phase images of asynchronous control, Bub1 and BubR1 repressed cells after the 18 hour nocodazole block (top). Cells were spun onto slides to detect DNA (red) and phospho-histone H3 (green) (bottom).