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The S. pombe aurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation

¹ School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK
² Universite de Rennes I, CNRS UMR 6061, Avenue Pr Léon Bernard, 35043 Rennes Cédex, France

View larger version (78K): [in a new window]   Fig. 1. Spindle formation and chromosome segregation defects upon overproduction of Xenopus Aurora A in S. pombe. Panels A and B show immunofluorescence/DAPI staining. The first panel in each series bears the label and shows microtubules, the next spindle poles, while the last one shows a combined DAPI and DIC image. Expression of Eg2 induces defects in chromosome segregation (A) and spindle formation (B). Wild-type cells bearing a pREP3XEg2 plasmid were grown at 30°C so that 20 hours after induction they would be in mid-log phase and samples were fixed every 2 hours from 14-22 hours later. The three mitotic cells in A are clearly unable to segregate their chromosomes. The microtubules in B emanating from a single focus of Sad1 staining indicates that spindle formation is defective. (C) Quantitation of the phenotypes arising from expression of Xenopus aurora A. The graph shows the frequency with which each particular mitotic defect is seen in an entire population of cells. Open squares, monopolar spindles; open circles, chromatin stretched along an anaphase spindle; triangles, ‘cut’ phenotype; green diamonds cells with bi-polar spindles and condensed chromatin; filled squares, diploid cells with large amorphous nuclei.

View larger version (116K): [in a new window]   Fig. 2. Cells lacking Ark1 function have defects in spindle formation, chromosome segregation, chromosome resolution during anaphase and cell cycle progression. Spores from diploid strain IH1877 were germinated in minimal medium supplemented with all amino acids except leucine in order to select for those spores containing the ark1.1 allele. 24 hours after germination cells were stained to reveal microtubules and chromatin. (A-C) The first panel in each series bears the label and shows chromatin, the next microtubules, the third a combined chromatin and DIC image. (A) Chromosome resolution is defective in ark1.1 cells. Chromatin is stretched out along the spindle. (B) ark1.1 cells have abnormal mitotic figures in which condensed chromosomes associate with a normal spindle. In each case the chromosomes have not split into two sister chromatids. In the left and middle cells the chromosomes are clustered at one end of the spindle. In the cell on the right, one chromosome is at either end and one is in the middle of the spindle. As the spindle on the right is elongating the chromosomes should already have split into two sister chromatids and moved to the spindle poles. The fact that a chromosome is lagging on this spindle and that one is apparently unattached to the spindle (arrow) indicates that kinetochore function is compromised when Ark1 function is compromised. (C) Ark1 is required for spindle formation. Instead of forming a bipolar spindle, microtubules extend from a single focus around which the chromosomes cluster. These figures became prominent at later time points when the germination was at 30°C, suggesting that spindle formation does not require as much Ark1 activity as chromosome resolution in anaphase B, the first defect to be seen. (D) ark1. 1 cells exhibit a novel cdc– phenotype. From left to right; DAPI, microtubules, DAPI/DIC. An extensive microtubule network extends between the cell tips while a small compact nucleus is present in the middle of the cell. Note that this panel is printed to 80% of the scale of the others in the figure.

View larger version (129K): [in a new window]   Fig. 3. Antibodies against phospho-serine 10 of histone H3 stain S. pombe nuclei through the cell cycle, but staining is radically reduced in G1 cells and highest on mitotic centromeres. In each case, the top panel shows H3SP staining and the bottom DAPI/DIC. All panels use the same concentration of primary antibodies. For (A-C) Cy3 anti-rabbit antibodies were used at a dilution of 1:2000, for (D-H) FITC anti-rabbit antibodies were used at a dilution of 1:100. (A) Asynchronous wild-type cells. Cell 1 is in G2, cells 2 and 3 are in M, and cell 4 is in G1/S phase. Note the strong H3SP chromatin fluorescence in all cells except for 4. (B) H3SP staining is strong in cells arrested in early S phase. Cells were grown to early log phase in rich medium at 30°C before the addition of hydroxyurea. The chromatin of these early S phase cells had bright H3SP staining. (C) A mixed culture of cdc10.v50 and cdc7.A20 cells were grown to early log phase in rich medium at 25°C before the temperature was changed to 36°C for a further 6 hours. The H3SP staining of the single nuclei in the three cdc10.v50 arrested cells is much lower than that of the multi-nucleate, actively cycling cdc7.A20 mutant cell on the left. (D-E) Asynchronous wild-type cells stained by a less sensitive procedure to visualise H3SP than that used in A-C. Nuclear staining is only seen in the mitotic cells. (F) H3SP and Nuf2.GFP localisation. In each case the first (top) image is a merge of the Nuf2.GFP fluorescence (second panel) and H3SP staining (third panel). The fourth panel shows DAPI staining, while the fifth is stained with DAPI/DIC. In each case the pole proximal spot of H3SP associates with Nuf2.GFP kinetochore fluorescence. The two signals are not entirely overlapping as revealed by the presence of red, yellow and green sectors in many such pole associated spots - for example, in the middle cell.

View larger version (68K): [in a new window]   Fig. 4. H3SP staining of germinating spores reveals a deficiency of H3SP reactivity in ark1. cells. (A) Spores arising from sporulation of wild-type strain IH534 were inoculated into full supplemented minimal medium and processed for immunofluorescence using the procedures for Fig. 3D-F 24 hours later. Left panels, H3SP staining; middle, DAPI; right; DAPI/DIC. No staining was seen in interphase spores. Upon mitotic commitment a lumpy pattern of general chromatin staining was seen alongside some much brighter spots of H3SP staining. The number of spots of strong H3SP staining decreased with mitotic progression. During anaphase B a spot of strong staining was always seen at the extreme ends of the nuclei indicative of close proximity to the spindle pole. Towards the end of anaphase B staining generally diminished to two chromatin-associated spots. (B) Spores arising from sporulation of IH1877 were inoculated into full supplemented minimal medium. Left, H3SP; middle, DAPI; right, DAPI/DIC images of the same fields of cells. Strong H3SP staining is clearly visible in the mitotic wild-type nuclei (W) but undetectable in the stretched ark1.1 mitotic nuclei (A).

View larger version (31K): [in a new window]   Fig. 5. Ark1.PkC levels do not fluctuate as cells progress through the cell cycle. A cell-cycle synchronised population of strain IH2036 was generated by elutrient centrifugation at 25°C in rich YES medium. The small G2 cells were harvested at the beginning of the experiment and samples were taken every 20 minutes to score the septation index (A) and for western blot analysis of whole cell extracts (B). The blot was cut in two and the top portion was probed with antibodies to a tubulin (TAT1) to act as a loading control for the Ark1.PkC blot (below). The position of the peaks seen in the septation index are indicated by asterisks above the septation profile and the western blot. Quantitation of the ratio of the TAT1 to Ark1.PkC signals for two independent blots led to the conclusion that there is no great fluctuation in Ark1.PkC levels as cells progress through the cell cycle (data not shown). (C) Ark1 protein levels remain constant after arrest in the G1 phase of the cell cycle. Extracts were prepared from cells which were manipulated to be in distinct cell cycle stages. IH2117 was blocked at 37°C for 4 hours 15 minutes and released to 25°C for 45 minutes (mitotic sample). In this mitotic sample 60% of the cells had either condensed chromosomes or were binuclear without septa. A culture of IH2116 was split in two and one half was incubated at 37°C for 5 hours 30 minutes to arrest cell cycle progression at the cdc10 execution point (G1-phase sample). (D) Quantification of the bands shown in C. In each case the intensity of the Tat1 signal in the upper panel was divided by the intensity of the Ark1.PkC signal in the lower panel.

View larger version (42K): [in a new window]   Fig. 6. Ark1-associated phosphorylation of histone H3 at serine 10 is highest in mitosis. This figure shows a cold kinase assay that uses anti-phospho-serine 10-histone H3 antibodies to detect phosphorylation of serine 10 of histone H3 in immunoprecipitates. Extracts were prepared from cells that were manipulated to be the equivalent of mid-log phase growth state but be enriched in one of four distinct cell cycle stages. IH2117 was blocked at 37°C for 4 hours 15 minutes (G2-phase sample) and released to 25°C for 45 minutes (mitotic sample). In this mitotic sample, 60% of the cells had either condensed chromosomes or were binuclear without septa. IH2116 was blocked at 37°C for 5 hours 30minutes (G1-phase sample). 100 mM hydroxyurea was added to IH2036 and the culture was incubated for 5 hours at 36°C (S-phase sample). The presence of -Pk antibodies on the beads or Ark1.PKC protein in the strain used to make the extract is indicated above each lane. The band corresponding to the full length protein is weaker than in Fig. 5B and a number of degradation products appear below Ark1.PkC in the Ark1.PkC panel. Despite this uniform degradation there is dramatic increase in H3SP reactivity in mitotic extracts over the interphase extracts (right-hand panel).

View larger version (75K): [in a new window]   Fig. 7. The kinetochores can wander some distance from the spindle as it forms. IH2035 cells were grown to mid-log phase in rich medium at 30°C and processed for anti-Sad1 immunofluorescence. In each case the top panel is a merge of the middle Nuf2.GFP signal and the lower Sad1 staining. It is shown at twice the magnification of the images of the individual channels to show the juxtaposition of the signals most clearly. During interphase a single spot of Nuf2.GFP always associated with the Sad1 SPB stain. Upon spindle formation the association of Nuf2.GFP with Sad1 staining became much more varied. In some cases (B) association was maintained, in others (C) it was not. The two lower kinetochores in B have wandered a long way from the spindle that is forming in the upper right portion of the nucleus. At later stages of spindle formation the kinetochore marker was always seen on a line that marked the shortest route between the two Sad1-reactive SPB spots (D-E) until the kinetochores reached the poles at the end of anaphase B (F).

View larger version (92K): [in a new window]   Fig. 8. Ark1.PkC and Nuf2.GFP staining as cells transit through the cell cycle. IH2035 was grown to mid-log phase before preparation for anti-Pk immunofluorescence microscopy. In each case the panels show (from right to left): DAPI/DIC, DAPI, Nuf2.GFP fluorescence, Ark1.PkC staining and a merge of the two middle panels on the left. For cells 1-14 the merged image is double the size of the other images of these cells, whereas the merged image in 15-17* is the same scale as all the other panels. For full details see the text. The figure shows images of cells following 2 minutes fixation. The same patterns were seen if fixation time was extended to 30 minutes with the exception that the intensity diminished and the interphase nuclear spots and mitotic nuclear accumulation were no longer detected.

View larger version (88K): [in a new window]   Fig. 9. Ark1.PkC and Nuf2.GFP staining from pro-metaphase to early anaphase B. Images from panels 8-12 of Fig. 8 processed to superimpose (top) the distribution of Nuf2.GFP fluorescence (bottom) upon the Ark1.PkC staining pattern (middle) rather than give the best representation of the signal from each individual channel. The merged images are twice the size of the single signals. For full details see text. Note the two green Nuf2.GFP signals (arrowheads) either side of the red Ark1 spot (arrow) at the bottom of the image in A.

View larger version (65K): [in a new window]   Fig. 10. Cut7 and Ark1.PkC do not show appreciable co-localisation as the spindle elongates during anaphase B. IH2035 cells were grown to mid-log phase in rich medium at 30°C and processed for Cut7 and Ark1.PkC staining. Cut7 staining of the termini of the metaphase spindle does not show a strong overlap with Ark1.PkC staining in the same cells (A). Similarly the distribution of the two molecules along the anaphase B spindle is very different (B,C). The restricted nature of the co-localisation of the two signals is graphically shown by the predominance of green and red signals over the co-localisation yellow signals in the merged image.

View larger version (39K): [in a new window]   Fig. 11. Ark1 distribution in meiosis. A mid log culture of IH2124 was spread on MSA plates and incubated overnight to induce mating. Cells were washed off and processed for Pk staining. In each case the panels show (from right to left): DAPI/DIC, DAPI, Nuf2.GFP fluorescence, Ark1.PkC immunofluorescence and a merge of the two middle panels on the left. Ark1.PkC distribution is highly reminiscent of that in mitosis. As the spores form Ark1.PkC reactivity accumulates.

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