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First published online 18 December 2002
doi: 10.1242/jcs.00273

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A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment

James G. Wakefield^,*, David J. Stephens and Jeremy M. Tavaré

Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
Present address: Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

View larger version (59K): [in a new window]   Fig. 1. GSK-3 interacts with spindle microtubules during mitosis. (A) A microtubule co-sedimentation assay using cycling or mitotic HeLa cell extracts. S, high speed supernatant; P, microtubule pellet; -/+ indicates the absence or presence of taxol in the assay; U, unsynchronised cells; M, Mitotic cells. Western blots were probed with antibodies against GSK-3ß. (B) HeLa cells permeabilised with 0.5% Triton before fixation with 4% paraformaldehyde and stained with affinity purified antibodies raised against GSK-3. Bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 2. GSK-3 is phosphorylated during mitosis and accumulates at centrosomes and spindle poles. (A) GSK-3 phosphorylation increases during mitosis. Total HeLa cell lysates were probed using antibodies against GSK-3ß and Phospho-GSK-3/ß (Ser21/9). S, stimulated HeLa cells (see Materials and Methods); U, unsynchronised, unstimulated HeLa cells; M, mitotic HeLa cells. (B) HeLa cells stained for Phospho-GSK-3/ß (Ser21/9) and DNA. (C) HeLa cell stained for Phospho-GSK-3/ß (Ser21/9), total GSK-3 and DNA. (D) Localisation of phospho-GSK-3 and -tubulin in HeLa cells treated with 1 µM nocodazole for 2 hours. Bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 3. PKB/Akt is phosphorylated in mitosis and associates with the centrosome. (A) HeLa whole cell lysates probed for total PKB and phospho-PKB (T308). S, serum-stimulated HeLa cells; U, unsynchronised HeLa cells; M, mitotic HeLa cells. (B) Immunofluorescence microscopy of HeLa cells. Cells were fixed and stained with anti-PKB antibody. DNA was counterstained with DAPI. (C) Localisation of Phospho-PKB (Thr308) in HeLa cells. Cells were fixed using methanol (-20°C). Bar, 10 µm.

View larger version (68K): [in a new window]   Fig. 4. Inhibition of GSK-3 during mitosis causes spindle defects and chromosome mis-alignment. HeLa cells were incubated with 10 µM or 30 µM of the GSK-3 inhibitors SB-216763 and SB-415286, respectively for 90 minutes. DNA, blue; tubulin, green. (A) Untreated metaphase cell; (B) SB-216763-treated metaphase cell; (C,D) SB-415286-treated metaphase cell; (E) Mitotic cell treated with 40 mM LiCl for 90 minutes; (F) Mitotic cell treated for 90 minutes with 1 nM taxol. Bar, 10 µm. (G) Effect of treating HeLa cells with SB-415286 for 16 hours. Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 5. Inhibition of GSK-3 does not affect centrosome separation. Actively cycling HeLa cells were treated with the GSK-3 inhibitor SB-415286 for 90 minutes before fixation with methanol (-20°C). Coverslips were stained with antibodies to -tubulin and with DAPI to visualise DNA. In cells possessing mono-oriented chromosomes -tubulin staining is seen as two discrete dots, one at either side of the metaphase plate. Identical results were obtained using SB-216763.

View larger version (58K): [in a new window]   Fig. 6. Inhibition of GSK-3 dramatically reduces chromosome dynamics in mitosis. HeLa cells stably expressing Histone 2B-GFP were imaged at 37°C at 3 second intervals for 30 minutes in the absence (A) or presence (B) of the GSK-3 inhibitor, SB-415286. (A) The arrow in the image at 300 seconds marks the position of a chromosome that undergoes significant movement. This movement is shown enlarged in panels 279-458s. (B) The arrow in the image at 300 seconds marks the position of a chromosome that does not undergo significant movement. This is shown enlarged in panels 312-492s. Bars, 10 µm. QuickTimeTM movies of time-lapse sequence shown in A and B are available online as Movies 1 and 2, respectively (http://jcs.biologists.org/supplemental).

View larger version (41K): [in a new window]   Fig. 7. Inhibition of GSK-3 results in inhibition of chromosome oscillation and congression. Manual particle tracking was used to quantitate the time-lapse data from Fig. 6. Tracks shown in green in Fig. 7 represent movements of the chromosomes highlighted in the zoomed images in Fig. 6C. Starting points of two dynamic chromosomes for control (A) or inhibitor treated cells (B) are shown as white spots with their subsequent trajectories during the time-lapse sequence shown as red or green overlays. (C,D) The total distance moved of each of these chromosomes is shown as a function of time. Bars, 10 µm. (E-F) Tracking record of mono-oriented chromosomes. (E) In an untreated cell, the chromosome performs several rapid, reversible movements before moving into the metaphase plate. (F) A cell treated with SB-415286. The unaligned chromosome fails to show similar oscillations. Even after a further 30 minutes this chromosome does not approach the metaphase plate (D.J.S., unpublished) (for the movies of these chromosomes, see http://jcs.biologists.org/supplemental).