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Fission yeast Pds5 is required for accurate chromosome segregation and for survival after DNA damage or metaphase arrest

Imperial Cancer Research Fund Molecular Oncology Laboratory, University of Oxford Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK

View larger version (71K): [in a new window]   Fig. 1. The sole fission yeast Pds5 orthologue is non-essential for viability. (A) The predicted protein product of S. pombe pds5 (SpPds5) with S. cerevisiae Pds5 (Sc Pds5), S. macrospora Spo76 (Sm Spo76), A. nidulans BIMD (An BIMD) and the human AS3 protein (Hs AS3). In each case the overall degree of relatedness to the S. pombe protein is indicated in parentheses (% identity/% similarity). Similarity between all five proteins is concentrated in the four regions filled in white, defined by the amino acid residue numbers shown above the S. pombe protein. The relatedness of each of these regions to the corresponding regions of the orthologous proteins is indicated (numbers below each conserved block; % identity/% similarity), along with the overall number of amino acid residues in each protein and the location of a predicted nuclear localisation sequence (NLS) in S. pombe Pds5. (B) Liquid cultures of HM123 (pds5+) and pds5 strains were grown to mid-exponential phase and were stained with Hoechst 33342 to allow visualisation of DNA and septa by fluorescence microscopy. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 2. pds5 cells are sensitive to DNA damaging agents, but not to inhibition of DNA replication. (A) Strains HM123 (pds5+), pds5-GFP and pds5 were tested for sensitivity to UV. Colony numbers were counted after four days growth at 30°C following exposure to the indicated UV doses on YE agar plates, and were expressed as a percentage of the number of colonies seen after mock irradiation (upper panel). Cellular and nuclear morphologies of HM123 (pds5+) and pds5 strains were determined by fluorescence microscopy of DAPI-stained cells 12 hours after exposure to 150 Jm-2 UV (lower panels; Bar, 10 µm). (B) Tenfold serial dilutions of HM123 (pds5+) and the other strains indicated were spotted onto YE agar plates containing 0.005% MMS, 5 µg/ml bleomycin or neither drug (control). Plates were photographed after 3-5 days incubation at 30°C. (C) The strains indicated were streaked in the positions shown onto YE agar (control) or YE agar containing 10 mM HU. Plates were photographed after 4 days growth at 30°C.

View larger version (33K): [in a new window]   Fig. 3. pds5 cells are sensitive to delayed progression through mitosis, but do not undergo precocious sister chromatid separation during metaphase arrest. (A) Tenfold serial dilutions of HM123 (pds5+), pds5 and pds5-GFP strains were spotted onto YE agar containing 20 µg/ml TBZ or no drug (control). Plates were photographed after 3-5 days incubation at 30°C. (B) HM123 (pds5+) and the other strains indicated were streaked in parallel onto YE agar plates and were photographed after 5 days incubation at 23°C or 30°C, as indicated. (C) Micrographs of formaldehyde fixed, DAPI stained nda3-KM311 (pds5+) and nda3-KM311 pds5 (pds5) cells before (0 minutes) and 9 minutes after shifting to 36°C to release from a metaphase arrest, imposed previously by holding the cells at 20°C for 8 hours. Examples of anaphase cells with lagging chromosomes are indicated by arrowheads. The percentages of total anaphase cells and anaphase cells with lagging chromosomes 9 minutes after release are indicated graphically on the right. (D) The nda3-KM311 ndc80-GFP pds5 strain was incubated in EMM2 medium at 30°C or shifted to 18°C for 12 hours. Fluorescence micrographs of living cells were acquired after staining with Hoechst 33342, revealing green fluorescence (Ndc80-GFP) and DNA (Hoechst) in the same field, as indicated. Bars, 10 µm.

View larger version (40K): [in a new window]   Fig. 4. Deletion of pds5 leads to an increased chromosome segregation failure rate. (A) Rates of mini-chromosome loss were calculated for strains HM248 (pds5+) and pds5 Ch16 (pds5) as described in Materials and Methods and are expressed as mean chromosome loss per generation. Arrowheads in the right-hand panels indicate examples of ade- sectored colonies indicative of chromosome loss. (B) Living GFP-swi6 (pds5+) and GFP-swi6 pds5 (pds5) cells were observed by green fluorescence microscopy. In each case a series of three images of a single cell progressing through early anaphase is shown; images were acquired at the time points indicated. (C) Visualisation of lagging chromosomes in pds5 cells. Individual GFP-swi6 pds5 cells were observed as in (B), over a 5 minute period, with images collected every 30 seconds. Bar, 10 µm. (D) Genetic interaction between pds5 and bub1. Ten tetrads derived from a diploid h+/h- pds5:ura4+/pds5+ bub1::LEU2/bub1+ ura4-D18/ura4-DS/E leu1-32/leu1-32 strain were microdissected onto YE agar and the resulting colonies were photographed after 4 days growth at 30°C. The genotypes of the segregants were determined by replica plating and are indicated schematically below (wt, pds5+ bub1+; p, pds5:ura4+; b, bub1::LEU2; pb, pds5:ura4+ bub1::LEU2; the latter is also indicated by white boxes in the upper panel). The inset panel shows a micrograph of a representative pds5:ura4+ bub1::LEU2 colony taken at the same time. Bar, 20 µm. (E) Slow growth of pds5 bub1 cells is associated with chromosome segregation defects. Fluorescence micrographs of formaldehyde fixed, DAPI-stained cells from exponentially growing YE liquid cultures of HM123 (pds5+) and the indicated strains. Cells exhibiting abnormal chromosome segregation are indicated (arrowheads).

View larger version (51K): [in a new window]   Fig. 5. Meiotic defects in the absence of Pds5. (A) Phase-contrast micrographs of living cells (upper panels) and fluorescence micrographs of DAPI-stained, formaldehyde-fixed cells (lower panels) of sporulating 428h/429h (pds5+/pds5+) and pds5/pds5 strains are shown, as indicated. Bars, 10 µm. (B) Quantification of the sporulation defect in pds5/pds5 progeny. The percentages of all asci containing four, three, two or one spores, and the overall spore viabilities of 428h/429h (pds5+) and pds5/pds5 (pds5) strains are shown, as indicated (error bars represent one standard deviation in the right-hand panel; the mean of three separate measurements is shown).

View larger version (27K): [in a new window]   Fig. 6. Rad21 and Pds5 are present in high molecular weight complexes of similar sizes. The sizes of soluble Rad21-HA and Pds5-HA were estimated in parallel by gel filtration chromatography as described in Materials and Methods and cell extracts from the rad21-HA and pds5-HA strains. Samples of each 0.5 ml Superose 6 column fraction were processed for immunoblotting with an anti-HA antibody. The elution volume (ml) of each fraction is shown above the blots. The positions at which dextran blue (void volume; 2000 kDa) and thyroglobin (670 kDa) migrated are indicated. The remaining smaller size fractions did not contain significant amounts of either HA-tagged protein.

View larger version (44K): [in a new window]   Fig. 7. Nuclear localisation of Pds5 is cohesin-dependent, and cohesin localisation is altered in the absence of Pds5. (A,B) Fluorescence micrographs showing Pds5-GFP and DNA (Hoechst 33342) localisation in living cells. (A) Left panels show examples from an asynchronous culture of pds5-GFP cells at the cell cycle stages indicated. Right panels show metaphase arrested nda3-KM311 pds5-GFP cells after 12 hours incubation at 18°C. (B) pds5-GFP (rad21+) and rad21-K1 pds5-GFP (rad21-K1) cells grown at 25°C or shifted to 36°C for 3 hours, as indicated. (C) Fluorescence micrographs showing Rad21-GFP and DNA (Hoechst 33342) in living rad21-GFP (pds5+) and pds5 rad21-GFP cells (pds5), as indicated. Bars, 10 µm.

View larger version (47K): [in a new window]   Fig. 8. Genetic interactions between pds5, rad21 and mis4. (A) Ten tetrads derived from the diploid strain rad21-K1 pds5 (h+ h- rad21-K1:ura4/rad21+ pds5::LEU2/pds5+ ade6-M210/ade6-M216 leu1-32/leu1-32 ura4-D18/ura4-D18) were microdissected onto YE agar, and the resulting colonies were photographed after 5 days growth at 30°C. The genotypes of the segregants were determined by replica plating and are indicated schematically below (wt, pds5+ rad21+; p, pds5::LEU2; r, rad21-K1:ura4+; rp, rad21-K1:ura4+ pds5::LEU2; the latter are also indicated by white boxes in the upper panel). The terminal phenotype of rad21-K1:ura4+ pds5::LEU2 segregants was determined by microscopic examination of the cells in situ after 5 days growth at 30°C. Four representative examples are shown in the inset panel on the right. (B) HM123 (pds5+ mis4+) and the other strains indicated were streaked in parallel onto YE agar plates and were photographed after incubation for 3-5 days at the temperatures indicated. (C) fluorescence micrographs of formaldehyde fixed, DAPI stained pds5 mis4-242 and mis4-242 cells grown at 25°C. Bars, 10 µm.

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