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Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells

School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK
¹ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston MA 02115, USA
^* Present address: Department of Biology, Imperial College of Science Technology and Medicine, London SW7 2AZ, UK

View larger version (88K): [in a new window]   Fig. 1. Characterisation of antibodies against Bub1 and BubR1. (A and B) Protein extracts from BHK cells ectopically expressing either GST-mBub1 (lanes 1, 5, 9 and 13) or GST-BubR1 (lanes 2, 6, 10 and 14), TA-HeLa cells (lanes 3, 7, 8, 11, 15 and 16) and mouse L929 cells (lane 4 and 12) analysed by western blotting with (A) the anti-Bub1 antibodies 4B12 (mouse monoclonal) and SB1.3 (sheep polyclonal) and (B) the anti-BubR1 antibodies 5F9 (mouse monoclonal) and SBR1.1 (sheep polyclonal). Where indicated, HeLa cells were either asynchronous (–) or treated with 0.2 µg/ml nocodazole for 16 hours (+). (C) Immunofluorescence images of DLD-1 cells stained with monoclonal (red) and polyclonal (green) antibodies against Bub1 (left column) and BubR1 (middle column). The cells were also stained with Hoechst to identify the chromosomes (blue, right column). (i, iii) Late prophase cells showing colocalisation of Bub1 and BubR1 at kinetochores. (ii, iv) Fields showing multiple mitotic cells. Note that in anaphase cells (arrowheads), Bub1 and BubR1 are virtually undetectable at kinetochores yet at prometaphase (ii) and metaphase (iv) cells (arrows) kinetochore-associated Bub1 and BubR1 is clearly detectable. Note also that in early prophase (double arrowhead) Bub1 localises to kinetochores before BubR1.

View larger version (73K): [in a new window]   Fig. 2. Bub1 and BubR1 localise to the same domain within the kinetochore. (A) Projection of a deconvoluted image stack showing DLD-1 prometaphase kinetochore pairs stained with 4B12 (anti-Bub1, red) and RCE1 (anti-Cenp-E, green), showing that localisation of Cenp-E extends distally beyond Bub1. (B) 3D model showing that Cenp-E also extends beyond Bub1 in the z axis. (C) Projection of a deconvolved image stack showing a nocodazole-treated DLD-1 cell stained with 4B12 (anti-Bub1, red) and SBR1.1 (anti-BubR1, green). Because Bub1 and BubR1 colocalise perfectly the kinetochores appear orange in this merged image. Scale bar represents 5 µm. (D) 3D model of one of the kinetochores from (C) showing perfect overlap of Bub1 and BubR1.

View larger version (74K): [in a new window]   Fig. 3. Sister kinetochores exhibit asymmetric Bub1/BubR1 staining. Projections of deconvoluted image stacks showing DLD-1 cells stained with 4B12 (anti-Bub1, red), SBR1.1 (anti-BubR1, green) and Hoechst (blue). Scale bars represent 5µm. (A) Merged red/green image of an untreated cell showing that one kinetochore within a pair often appears green while the other appears orange/yellow, indicative of asymmetric Bub1/BubR1 staining. The arrowheads identify four clear examples. (B) A taxol-treated cell, showing splayed-out chromosomes due to microtubule stabilisation, also exhibits asymmetric Bub1/BubR1 staining. (C) A nocodazole-treated cell, showing collapsed chromosomes owing to microtubule depolymerisation, exhibits reduced Bub1/BubR1 asymmetry: most of the kinetochores appear orange/yellow.

View larger version (121K): [in a new window]   Fig. 4. Monoclonal and polyclonal anti-Bub1 antibodies give identical patterns. A deconvolved image of a prometaphase DLD-1 cell stained with (A) 4B12 and (B) SB1.3 anti-Bub1 antibodies. (C) shows a red/green merged image and (D) shows the Hoechst-stained chromosomes. The scale bar represents 5 µm. Both 4B12 and SB1.3 give identical patterns.

View larger version (39K): [in a new window]   Fig. 5. Asymmetric Bub1 staining is microtubule dependent. (A) Enlarged images of kinetochore pairs taken from Fig. 3A showing Bub1 (red), BubR1 (green) and the merged image (right column). Although the BubR1 signal at sister kinetochores is roughly equivalent, the Bub1 signal is often asymmetric, giving rise to a green and yellow spots in the merged image. (B) 3D plots of a kinetochore pair confirming BubR1 symmetry and Bub1 asymmetry. (C) A bar graph plotting the fluorescence ratio (K1/K2) for Bub1 (red) and BubR1 (green) where K1 is the kinetochore within a pair exhibiting stronger Bub1 fluorescence. The cells were either untreated (U) or treated with nocodazole (N) or taxol (T) for one hour. (D) A histogram plotting the percentage of kinetochore pairs exhibiting a given K1/K2 Bub1 fluorescence ratio. (E) A bar graph plotting the average ratio of BubR1/Bub1 signal at kinetochores K1 (green) and K2 (red). Values represent the mean and the standard error of the mean (s.e.m.).

View larger version (63K): [in a new window]   Fig. 6. Kinetochores with weaker Bub1 staining are oriented towards the spindle pole. (A) A projection of a deconvolved image stack showing a prometaphase DLD-1 cell stained with 4B12 (anti-Bub1, red), RAA.1 (anti-aurora a, green) and Hoechst (blue). (B) An enlarged image of the region boxed in (A) showing three kinetochore pairs. The two pairs that exhibit clear Bub1 asymmetry are oriented such that the weaker staining kinetochore is closer to the pole. (C) shows a dot plot of the interkinetochore distance (K1-K2) against the fluorescence ratio (K1/K2), confirming that kinetochores exhibiting weaker Bub1 staining are generally closer to the nearest spindle pole. The scale bars represent 5 µm in (A) and 1 µm in (B).

View larger version (53K): [in a new window]   Fig. 7. Bub1 is phosphorylated in response to spindle damage. (A) HeLa cells synchronised by a double thymidine block analysed by flow cytometry (top panel) and western blotting using 4B12 (anti-Bub1, bottom left panel) and 5F9 (anti-BubR1, bottom right panel) at various times following release from G1/S, shown in hours. The anti-tubulin antibody TAT-1 (Woods et al., 1989) was used to monitor protein loading. Between 9 and 12 hours after release, the majority of the cells had completed mitosis and returned to G1. In the presence of nocodazole, both Bub1 and BubR1 exhibit slower migrating forms. (B) Proteins from mitotic (M) HeLa cells treated with phosphatase (+) or CIP (+C), as indicated, then blotted for Bub1 using SB1.3 or BubR1 using 5F9. Phosphatase treatment results in the disappearance of the slower migrating forms indicating that they are phosphorylated forms. (C) Mitotic L929 (M, upper panel) or mitotic HeLa cells (M, lower two panels) were treated with 0.2 µg/ml nocodazole (N) or 10 µM taxol (T) for the times indicated in minutes then blotted for Bub1 using 4B12 or BubR1 using 5F9. Phosphorylated Bub1 is only detectable in response to spindle damage but BubR1 is phosphorylated in the absence of spindle toxins. (D) Bub1 (lanes 1 and 4) and BubR1 (lanes 3 and 6) were immunoprecipitated from nocodazole-arrested TA-HeLa cells using the 4B12 and 5F9 antibodies, respectively. The immunoprecipitates were then analysed by western blotting using 4B12 (anti-Bub1, right panel) and 5F9 (anti-BubR1, left panel). The lower panel shows that when the two forms of BubR1 are well resolved, it becomes apparent that the phosphorylated form of BubR1 preferentially immunoprecipitates with Bub1.

View larger version (85K): [in a new window]   Fig. 8. Loss of tension results in Bub1 recruitment to kinetochores. Projections of deconvolved image stacks showing metaphase HeLa cells stained with Hoechst (blue, top panels), 4B12 (anti-Bub1) and anti-centromere antibodies (red and green respectively in the merged image shown in the middle and bottom panels). The bottom panels show enlarged views of the boxed areas shown in the middle panels. Scale bars represent 5 µm. (A) In the absence of taxol, the metaphase is broad, centromeres are stretched and Bub1 staining at kinetochores is weak. (B) Following 30 minutes of taxol treatment, the metaphase is tight, centromere stretching is reduced and Bub1 staining at kinetochores is increased.