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First published online 22 November 2005
doi: 10.1242/jcs.02694

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A role for the fission yeast Rqh1 helicase in chromosome segregation

¹ Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK
² Cancer Research UK Laboratories, University of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK

View larger version (39K): [in a new window]   Fig. 1. Rqh1 is not required for bulk DNA replication. Wild-type (rqh1+) and rqh1 cells were arrested in G1 by nitrogen starvation at 25°C for 20 hours and released into nitrogen-rich medium to restart the cell cycle at the temperature of 25°C. Cells harvested at hourly intervals were processed for flow cytometry (A), and mitotic index was assessed by scoring bi-nucleate cells (n=200) (B). (C) Micrographs of DAPI-stained wild-type and rqh1 cells (lower panels), 9 hours after release from nitrogen starvation at 25°C. Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 2. Mitotic behaviour of microtubules in rqh1 cells. (A) Cells expressing GFP–2-tubulin were synchronously released from the G2 block imposed by a temperature-sensitive cdc25-22 mutation. Samples were taken every 10 minutes and percentages of cells with different lengths of mitotic spindles in the indicated strains were determined. Representative examples of cells with metaphase (I) and anaphase (II) spindles in each strain are shown: Spindle (GFP-Atb2) in green and DNA (DAPI) in blue. Bar, 10 µm. (B) Visualisation of mitotic spindle behaviours in living rqh1 cells. Individual wild-type and rqh1 cells expressing GFP–2-tubulin were observed over a period of 20 minutes or 36 minutes as in (C), with images collected every 4 minutes.

View larger version (57K): [in a new window]   Fig. 3. rqh1 cells are defective in nucleolar segregation. (A) Merged images of fluorescence micrographs showing Gar2-GFP and DNA (Hoechst 33342) localisation in living wild-type and rqh1 cells. Bar, 10 µm. (B) Visualisation of lagging Gar2-GFP signal in rqh1 cells. Individual cells of indicated strains expressing Gar2-GFP were observed as in A, over a period of 20 minutes, with images collected every 4 minutes. (C) Percentages of cells displaying aberrant nucleolar structures in each strain are shown (n=200).

View larger version (52K): [in a new window]   Fig. 4. Lagging chromosome behaviour in rqh1 cells. Merged images of fluorescence micrographs showing DNA (Hoechst 33342) and the localisation of (A) Ndc80-GFP, (B) the cut3 or (C) sod2 locus marked by LacI-NLS-GFP in living wild-type (rqh1+) and rqh1 cells. The frequencies of anaphase cells with segregated GFP signals in the indicated strains are shown. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 5. The spindle checkpoint is activated in rqh1 mutants. (A) mad2 and mad2 rqh1 cells expressing GFP–2-tubulin were synchronously released from the G2 block imposed by a temperature-sensitive cdc25-22 mutation. Samples were taken every 10 minutes and percentages of cells with mitotic spindles in the indicated strains were determined. (B) Merged images of fluorescence micrographs showing Mad2-GFP and DNA (Hoechst 33342) localisation in living wild-type and rqh1 cells. (C) Visualisation of Cut2-GFP signals in methanol/acetone-fixed, DAPI-stained cells. The frequencies of cells with GFP signals in B and C are shown. Bar, 10 µm.

View larger version (26K): [in a new window]   Fig. 6. Genetic interactions between mad2, reb1 and rqh1. (A) Tenfold serial dilutions of the indicated strain spanning the range from 106 to 102 cells, as indicated, were spotted onto YE agar plates containing 3 mM HU, 15 µg/ml TBZ or neither drugs (control). Plates were photographed after incubation for 3 days at 30°C. (B) Pulsed-field gel electrophoresis analyses of chromosomes from rqh1 mutants. Equal numbers of cells were prepared in agarose gel plugs from exponentially growing cultures of the indicated strains. Pulsed-field gel electrophoresis was carried out as described in Materials and Methods. Arrowhead indicates the anomalous mobility shift of chromosome III band seen in these samples.

View larger version (12K): [in a new window]   Fig. 7. Chromosome segregation is impaired in the rqh1 mutants as a result of a defect in the resolution of rDNA repeats, which activates the spindle checkpoint. Inactivation of the spindle checkpoint by deletion of mad2 suppresses the lagging chromosome behaviour but not the aberrant structure in rDNA repeats that leads to the reduction of chromosome III length in rqh1 and rqh1 mad2 mutants.