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First published online April 28, 2005
doi: 10.1242/10.1242/jcs.02309

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Grp/DChk1 is required for G₂-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

¹ Department of Radiation and Stress Cell Biology, Division Cell Biology, Faculty of Medical Sciences, University of Gröningen, Ant. Deusinglaan 1, Building 3215, 9713 AV Groningen, The Netherlands
² Department Molecular, Cellular and Developmental Biology, 347 UCB, University of Colorado, Boulder, CO 80309-0347, USA

View larger version (5K): [in a new window]   Fig. 1. S2 cells display cell-cycle arrest in response to HU or IR. S2 cells were treated with HU for 15 hours or treated with 150 Gy IR followed by 2 hours of recovery. Cells positively stained for PH3 were counted using flow cytometry. S2 cells showed a decrease in the proportion of mitotic cells when treated with HU (1.5%) or IR (1.6%) compared with untreated cells (4.3%). Error bars represent s.e.m. (n=13).

View larger version (35K): [in a new window]   Fig. 2. HU- and IR-induced modifications of Cdc25Stg and Cdc2 in control S2 cells but not in Grp/DChk1-depleted S2 cells. Wild-type embryos at 0-2 hours of age (lane 1) expressed the active phosphorylated isoform (form 1) and the unphosphorylated neutral isoform (form 2) of Cdc2. Wild-type embryos at 2-4 hours of age (lane 2) showed accumulation of inhibitory phosphorylated isoforms (forms 3 and 4) of Cdc2. In early embryos (lane 1) Cdc25Stg was expressed, whereas Cdc25Stg protein was almost absent from in older embryos (lane 2). In S2 cells (lanes 3 and 5), Cdc2 isoforms 2-4 were detected and Cdc25Stg was present. In S2 cells treated with 10 mM HU during 15 hours (lane 4) the Cdc2 isoforms 3 and 4 accumulated, Cdc2 isoform 2 was almost absent and Cdc25Stg levels decreased. S2 cells treated with 150 Gy IR followed by 2 hours of recovery (lane 6) showed accumulation of Cdc2 isoform 4 and presence of Cdc2 isoforms 2 and 3. Cdc25Stg levels decreased moderately in response to IR. S2 cells depleted for Grp/DChk1 by RNAi (lanes 7 and 9) expressed Cdc25Stg and all Cdc2 isoforms (forms 2-4). Grp/DChk1-depleted cells treated with 10 mM HU (lane 8) or treated with 150 Gy IR followed by 2 hours recovery (lane 10) did not show modifications of Cdc25Stg and Cdc2. Equal numbers (n=20) of Drosophila embryos were loaded in lanes 1 and 2. For lanes 3-10, a background band (Bg) recognized by the Cdc25Stg antibody was used as a loading control.

View larger version (43K): [in a new window]   Fig. 3. Grp/DChk1 is phosphorylated in S2 cells in response to HU or IR. Western-blot analysis of Grp/DChk1 of S2 cells treated with 10 mM HU during 15 hours or with 150 Gy IR followed by 2 hours recovery. Grp/DChk1 was detected as a slower migrating protein in S2 cells treated with HU (A, lane 2) or treated with IR (B, lane 2) compared with untreated S2 cells (A,B, lanes 1,3). A shift in mobility of Grp/DChk1 was not detected in S2 cells treated with HU or IR and calf-intestine alkaline phosphatase (CIP; A,B, lane 4). In both A and B, CP190 protein levels were detected as a loading control.

View larger version (13K): [in a new window]   Fig. 4. (A) Downregulation of Grp/DChk1 protein levels in S2 cells using RNAi. Grp/DChk1 expression in S2 cells (Fig. 4A, lane 1) and S2 cells treated with dsRNA of Grp/DChk1 (Fig. 4A, lane 2). Incubation of S2 cells with dsRNA of Grp/DChk1 for 96 hours reduced Grp/Dchk1 expression to below detection levels. As a loading control, protein levels of -tubulin were detected. (B) Grp/DChk1-depleted cells are defective in accomplishing cell-cycle arrest in response to HU or IR. Grp/DChk1 levels were downregulated by RNAi before cells were treated with 10 mM HU for 15 hours or treated with 150 Gy ionizing radiation followed by 2 hours of recovery. Grp/DChk1-depleted cells positively stained for PH3 were counted using flow cytometry. In Grp/DChk1-depleted cells left untreated 5.1% of the cells was in mitosis. In Grp/DChk1-depleted cells treated with HU also 5.1% of the cells was in mitosis and, in IR-treated Grp/DChk1-depleted cells, 4.4% of the cells were in mitosis. Error bars represent s.e.m. (n=13).

View larger version (25K): [in a new window]   Fig. 5. Phosphorylation of Grp/DChk1 in response to HU or IR is dependent on the expression of Mei-41/DATR. Western-blot analysis of Grp/DChk1 in control S2 cells and in Mei-41/DATR-depleted S2 cells treated with 10 mM HU during 15 hours or treated with 150 Gy IR followed by 2 hours of recovery. Compared with untreated cells (lanes 1,3,5,7), phosphorylated Grp/DChk1 was detected as a slower-migrating protein in control S2 cells treated with HU (lane 2) or IR (lane 4) and in Mei-41/ATR-depleted S2 cells treated with HU (lane 6) or IR (lane 8), no phosphorylation of Grp/DChk1 was observed. As a loading control, CP190 protein levels were detected.

View larger version (78K): [in a new window]   Fig. 6. Analysis of mitotic chromosomes from wild-type S2 cells and Grp/DChk1-depleted S2 cells in response to HU or IR. S2 cells were grown on cover slips and mitotic chromosomes were labelled with the anti-PH3 antibody. Images were made by confocal microscopy. (A-C) Mitotic chromosomes of Grp/DChk1-depleted S2 cells under control conditions in prophase (A), metaphase (B) and anaphase (C). (D,E) Fragmentation of mitotic chromosomes in Grp/DChk1-depleted cells treated with 10 mM HU (15 hours) (D) or IR (150 Gy, 2 hours recovery) (E). Scale bar, 1 µm.

View larger version (63K): [in a new window]   Fig. 7. Grp/DChk1-depleted cells display mitotic catastrophe in the presence of incompletely replicated and damaged DNA. (A) Time-lapse confocal analysis allowed normal cell-cycle progression of S2 cells transiently expressing histone H2B-GFP under control conditions. (B,C) Time-lapse confocal analysis of Grp/DChk1-depleted cells transiently expressing histone H2B-GFP and treated with 10 mM HU (B) for 15 hours or with 150 Gy (C) IR followed by 2 hours of recovery. Mitotic chromosomes were irregularly condensed and did not align in the metaphase plate. After prolonged mitosis, chromosomes separated disproportionally and micronuclei were formed. Times are indicated in minutes. Scale bars, 1 µm.

View larger version (55K): [in a new window]   Fig. 8. (A) No mobility shift is observed for Dmnk/DChk2 in response to HU or IR, irrespective of the presence of Grp/DChk1. Western-blot analysis of Dmnk/DChk2 protein in S2 cells and Grp/DChk1-depleted S2 cells treated with 10 mM HU for 15 hours or treated with 150 Gy IR followed by 2 hours of recovery. In S2 cells, Dmnk/DChk2 protein was detected (lane 1) and, in response to HU (lane 2) or IR (lane 3), no shift in mobility of Dmnk/DChk2 was observed. (lane 4) Downregulation of Dmnk/DChk2 protein levels using RNAi. Depletion of Grp/DChk1 using RNAi did not affect the mobility of Dmnk/DChk2 protein in untreated (lanes 5,7), HU-treated (lane 6) or IR-treated (lane 8) S2 cells. As a loading control, -tubulin protein levels were detected. (B) Phosphorylation of Grp/DChk1 in response to HU or IR is unaffected in Dmnk/DChk2-depleted cells. Western-blot analysis of Grp/DChk1 in S2 cells and Dmnk/DChk2-depleted cells treated with HU (10 mM, 15 hours) or IR (150 Gy, 2 hours recovery). In S2 cells and in Dmnk/DChk2-depleted cells, a slower-migrating form of Grp/DChk1 was detected in response to HU (lanes 2,6) or IR (lanes 4,8). As a loading control, -tubulin protein levels were detected.