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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02454

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Regulation of mitosis in response to damaged or incompletely replicated DNA require different levels of Grapes (Drosophila Chk1)

Amanda Purdy^*, Lyle Uyetake^*, Melissa Garner Cordeiro and Tin Tin Su

MCD Biology, University of Colorado, Boulder, CO 80309-0347, USA

View larger version (48K): [in a new window]   Fig. 1. After exposure to X-rays, cellularized embryos delay metaphase-anaphase transition by a mei-41-dependent mechanism. (A) Irradiation delayed the metaphase-anaphase transition. Montages of two mitotic cells from the dorsal ectoderm of embryos expressing a GFP-Histone H2Av transgene are shown. Embryos were irradiated with 0 (–IR) or 8.3 Gy (+IR) of X-rays and irradiated embryos were rested for 40 minutes to allow cells to enter mitosis, prior to analysis. Frames were taken every 30 seconds for up to 10 minutes. Arrowheads mark the beginning of metaphase and arrows mark the end of metaphase. (B) The length of metaphase specifically lengthens following irradiation in the 14th, 15th and 16th cellular cycles. Embryos were collected for 30 minutes and aged 130 minutes to reach cycle 14, 240 minutes to reach cycle 15 or 330 minutes to reach cycle 16. Embryos were irradiated with 0 (–IR) or 8.3 Gy (+IR) of X-rays and analyzed 0 minutes (–IR) or 40-60 minutes (+IR) later. For each time point, at least seven nuclei from at least three different embryos were analyzed. Asterisks denote statistically significant data with P<0.0001. P values were generated from the comparison with the non-irradiated control for each cycle (i.e. Cyc14 +IR versus Cyc14–IR). (C) mei-41 is required to delay the metaphase-anaphase transition after irradiation. Wild type (Wt) and mei-41D12 mutant embryos in cycle 16 (330-360 minutes after egg deposition, AED) were irradiated with 0 (–IR) or 8.3Gy (+IR) of X-rays and analyzed 0 minutes (–IR) or 40 minutes (+IR) later. For each time point at least 10 nuclei from four different embryos were analyzed. Asterisks denote statistically significant data with P<0.0001.

View larger version (24K): [in a new window]   Fig. 2. Delay in metaphase-anaphase transition requires prior irradiation at specific parts of the cell cycle. (A) Metaphase index, the ratio of cells in metaphase to those in anaphase and telophase (M/A+T), is maximal at 40 minutes after irradiation. Wild-type embryos in cycle 16 (330-390 minutes AED for non-irradiated controls and 310-370 minutes AED for irradiated embryos) were treated with 8.3 Gy of X-rays (LD80 dose) and fixed 40, 60 or 90 minutes later. Embryos were stained with an antibody to phosphorylated histone H3 (PH3), a mitotic marker, and Hoechst33258 to visualize DNA. The number of metaphases, anaphases and telophases within the dorsal ectoderm was quantified and the metaphase index calculated. Each bar represents approximately 1000 cells from at least 10 embryos. (B) Irradiation with an LD80 dose during S phase, but not G2/M, delayed metaphase-anaphase transition. Schematic of the 15th-16th mitotic cell divisions within the lateral ectoderm. The approximately 60 minutes mitotic cell cycle is separated into M phase (10 minutes), followed directly by S (35-45 minutes) and G2 phase (10 minutes). Numbers 1 and 2 mark the approximate place within the cell cycle in which cells were irradiated (see below). (C) The ability to delay metaphase-anaphase transition is dependent upon the dose of irradiation and cell cycle phase. Wild-type embryos with ventral ectoderm in cycle 15 and dorsal ectoderm in cycle 16 (330-360 minutes AED) were irradiated with 0, 8.3 Gy (LD80) or 83 Gy (10xLD80) of X-rays. Those irradiated with LD80 were allowed to recover for 40 minutes before analysis. Therefore, based on embryonic age, mitotic cells analyzed were in S phase at the time of irradiation (point 1 in B). For irradiation in G2/M, embryos were irradiated and immediately analyzed without an intervening recovery period (point 2 in B approximately 2-10 minutes after IR). Each bar represents at least eight nuclei from six embryos. (D) The mei-41D12 mutants can regulate the metaphase-anaphase transition after exposure to 10xLD80. Wild-type and mei-41D12 mutant embryos were irradiated with a LD80 dose and allowed to recover for 40 minutes before analysis (data reproduced from Fig. 1C for direct comparison), or irradiated with a 10xLD80 dose and analyzed immediately. 10xLD80 sample includes mitotic cells in the ventral ectoderm (cycle 15) and the dorsal ectoderm (cycle 16). For 10xLD80, at least eight nuclei from seven different embryos were analyzed. As in Fig. 1, asterisks denote statistically significant data with P<0.0001. All values were compared with non-irradiated values.

View larger version (46K): [in a new window]   Fig. 3. The role of grapes in regulation of mitosis after DNA damage. (A) grp1 mutants delay entry into mitosis after irradiation. Wild-type (Wt) and grp1 embryos in cycle 16 (325-335 minutes AED) were irradiated with 0 or 8.3 Gy of X-rays and fixed 20 minutes later to visualize the delay in entry into M16. Embryos were stained with an antibody to PH3, Hoechst33258 to visualize DNA, and an antibody to ß-gal to identify homozygous mutants. At the time of fixing, cells of the dorsal ectoderm (enclosed with brackets) enter M16 in untreated embryos (`Wt-IR' and `grp1–IR'), but were delayed in the entry in irradiated embryos (`Wt+IR' and `grp1 +IR'). Embryos are shown with anterior to the left and ventral side down. (B) Quantification of data in A. Mitotic index decreased following irradiation in both wild type and grp1 embryos, indicating that both genotypes could regulate the entry into mitosis in response to damaged DNA. Mitotic index was quantified within the dorsal ectoderm for at least 10 embryos per genotype. (C,D) grp1 mutants show an increase in metaphase index after irradiation. Wild type and grp1 and mutant embryos in cycle 16 (330-360 minutes AED for non-irradiated controls and 310-370 minutes AED for irradiated embryos) were irradiated with 0 or 8.3 Gy of X-rays and fixed 0 (–IR) or 40 minutes (+IR) later. Embryos homozygous for a deficiency (Df/Df) that removes grapes [Df(2)H20] also showed a robust increase in metaphase index after irradiation. Metaphase index in the dorsal ectoderm was quantified as in Fig. 2 for at least 29 embryos per treatment for each genotype (D). PH3 staining of a representative sample is shown in C where arrowheads mark anaphase and telophase nuclei and arrows mark metaphase nuclei. (E) Live analysis confirmed that grp1 mutants delay metaphase-anaphase transition after irradiation as indicated by analysis of fixed embryos in A-D. Wild type and grp1 embryos in cycle 16 (330-360 minutes AED) were irradiated with 0 or 8.3 Gy of X-rays and analyzed 0 (–IR) or 40 minutes (+IR) later. Each bar represents at least 10 nuclei from four different embryos. The data for mei-41D12 mutants is reproduced from Fig. 1 for comparison. As in Fig. 1, asterisks denote statistically significant data with P<0.0001. All values were compared with non-irradiated values.

View larger version (70K): [in a new window]   Fig. 4. grp1 mutants fail to regulate mitosis following incomplete replication. (A) grp1 dupa3 double mutants cannot delay the entry into M16 while dupa3 mutants can. 7-7.5-hours-old embryos were fixed and stained to visualize PH3 and DNA. Ectodermal cells in the dupa3 mutants are still arrested before M16 as indicated by the near absence of PH3-positive cells. By contrast, the corresponding region of grp1 dupa3 mutant embryos show robust PH3 signal, indicating that double mutants are unable to delay the entry into M16. (B) grp1 dupa1 double mutants fail to delay the progress through and eventual exit from M16 in contrast to Df/dupa1. 8.5-9-hour-old embryos were fixed and stained with an antibody to PH3 and with Hoechst33258 to visualize DNA. Hemizygous Df/dupa1 mutants are arrested in M16 and show a robust number of mitotic cells in the lateral ectoderm consistent with published results. By contrast, grp1 dupa1 mutants show a reduced number of mitotic cells. Hemizygous mutants (Df/dupa1) were used due to decreased viability associated with dupa1 chromosome. (C) grp1 mutation restores nuclear density of dupa1 mutants to wild-type levels. Nuclear density in Stage 12 embryos was quantified in thoracic segments 1, 2 and 3 and expressed in arbitrary units. Nuclear density is reduced in dupa1 mutants; this is expected, as they have not completed M16. Nuclear density in grp1 dupa1 mutants resembles that of heterozygous controls, indicating that cells of the double mutant have completed cycle 16. Homozygous mutants were identified by the lack of ß-gal encoded by the balancer chromosome (not shown).

View larger version (35K): [in a new window]   Fig. 5. Grp protein is reduced in grp1 mutants. (A) Developmental profile of Grp protein levels during embryogenesis. Embryo extracts from different times AED (indicated above lanes in A) were blotted for Grp as in experimental procedures. Approximately 50 embryos were loaded per lane. The yolk proteins, visualized by Ponceau staining, confirm equal loading for the initial part of embryogenesis, but become depleted as embryogenesis progresses. (B) The grp1 mutants have severely reduced levels of Grp protein. 6-7-hour-old grp1 homozygous or heterozygous mutant embryos from heterozygous parents were genotyped by the absence or the presence of GFP encoded by the balancer chromosome Cyo-GFP (see experimental procedures). Extracts were prepared and blotted for Grp and for GFP (to confirm genotype). Each lane contained extracts from 25 embryos. Both wild type and grp1/Cyo-GFP heterozygotes have comparable Grp levels, while in grp1 mutants, Grp levels are reduced. (C) Grp protein persists in mutants homozygous for a deficiency that removes grp. 7.5-8.5-hour-old Df/Df homozygous or heterozygous mutant embryos from heterozygous parents were genotyped by the absence or presence of GFP encoded by the balancer chromosome Cyo-GFP. Extracts were prepared and blotted for Grp and for GFP. Each lane contained extracts from 10 embryos. The difference in GFP signal between B and C is due to different antibody dilutions of anti-GFP. (D) Extracts of embryos from grp1 homozygous mothers or wild type (Wt) mothers were analyzed by western blotting. Extracts from approximately 50-75 embryos were loaded in each lane. Maternal genotypes and embryo ages are shown above each lane.

View larger version (86K): [in a new window]   Fig. 6. Subcellular localization of Grp during embryonic cell cycles. Grp localization during syncytial divisions. 1.5-2.5-hour-old wild-type embryos were fixed and stained with an antibody to Grp (-Grp), wheat germ agglutinnin (WGA) to visualize nuclear envelopes and Hoechst33258 to visualize DNA. Grp is nuclear during interphase and disperses from the nucleus as the nuclear envelope breaks down in mitosis. Grp re-accumulates in the nucleus after the nuclear envelope reforms in the next interphase.