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First published online 27 February 2008
doi: 10.1242/dev.019075

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PLK-1 asymmetry contributes to asynchronous cell division of C. elegans embryos

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.

View larger version (51K): [in this window] [in a new window]   Fig. 1. PLK-1 is distributed asymmetrically in early C. elegans embryos. (A) Schematic representation of C. elegans PLK-1. The kinase (KD) and polo boxes (PB1 and PB2) are indicated, as is the region against which the C-terminal antibodies were raised (amino acids 589-648; underlined in red). (B) Western blot of wild-type (lane 1) and strong plk-1(RNAi) (lane 2) embryonic extracts probed with PLK-1 antibodies, which recognize a specific band at the expected size of 70 kDa. The blot was reprobed with -tubulin antibodies as a loading control. (C-H) Wild-type (C-F), strong plk-1(RNAi) (G) or GFP-PLK-1 (H) embryos stained for PLK-1 (C-G) or GFP (H) (shown alone in the left panels and in red in the merged panels), -tubulin (green) and DNA (blue). In all figures, anterior is towards the left and scale bars correspond to 10 µm. Line scans of PLK-1 intensity of C-F (yellow rectangles) are shown on the right, with 1.0 corresponding to the average pixel intensity within the rectangle. (C) Shortly after the completion of meiosis, PLK-1 distribution in the cytoplasm is uniform. (D) In early prophase, cytoplasmic PLK-1 becomes asymmetric, with more protein present in the anterior. (E,F) PLK-1 asymmetry is maintained in anaphase (E) and in the early two-cell stage (F). PLK-1 is enriched at centrosomes (E, arrowheads) and the midbody (E,F, arrows). The signal detected by PLK-1 antibodies is specific, as it is essentially absent in strong plk-1(RNAi) embryos (G), which nevertheless allowed passage through the meiotic divisions in this particular embryo. Finally, asymmetric distribution in two-cell stage embryos is also apparent with GFP-PLK-1 (H). The fact that asymmetry with this particular transgenic line is less pronounced than that of endogenous PLK-1 may reflect the fact that excess GFP-PLK-1 cannot be efficiently retained at the anterior in the presence of endogenous PLK-1. Compatible with this view, anterior enrichment of GFP-PLK-1 to an extent comparable to that of endogenous PLK-1 was observed with a distinct transgenic line (a gift from Asako Sugimoto) that expresses lower protein levels (data not shown).

View larger version (77K): [in this window] [in a new window]   Fig. 2. Achieving PLK-1 asymmetry. (A-D) Wild-type (A), zen-4(RNAi) (B), tba-2(RNAi) (C) or nmy-2(RNAi) (D) embryos stained for PLK-1 (shown alone in the left panels and in red in the merged panels), -tubulin (green) and DNA (blue). PLK-1 asymmetry is maintained despite cytokinesis failure (B). Whereas microtubules are not essential for PLK-1 asymmetry as the asymmetry is maintained and even slightly increased upon -tubulin depletion (C), the actomyosin network is needed (D). (E,F) Wild-type (E) and partial plk-1(RNAi) (F) embryos stained for PLK-1 (shown alone in the left panels and in red in the merged panels), -tubulin (green) and DNA (blue). Overall PLK-1 levels are diminished in partial plk-1(RNAi) embryos (F, top panel). To better illustrate the alteration in ratio between AB and P1, a second image of the same partial plk-1(RNAi) embryo is shown (F, bottom panel), in which intensities were increased so as to match those in AB in the wild-type embryo. (G) Average ratios, along with s.e.m., of PLK-1 in AB versus P1 in wild-type and partial plk-1(RNAi) embryos stained as in E and F. Wild type, 1.52±0.05, n=64; partial plk-1(RNAi), 1.97±0.13, n=16. Student's t-test, P=2.6x10-11. The star indicates that the difference with wild type is statistically significant.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Anterior retention of PLK-1. (A,C) Confocal images of GFP-PLK-1 embryos before photobleaching (first panels), immediately after photobleaching the anterior half (A, second panel) or the posterior half (C, second panel) and three time intervals afterwards (third to fifth panels). Time elapsed is indicated in seconds with 0 corresponding to the end of photobleaching. (B,D) GFP-PLK-1 relative intensity ±s.e.m. plotted as a function of time, both for anterior (B, n=5) and posterior (D, n=7) photobleaching. Five images before photobleaching and 20 images afterwards were captured every 10 seconds, except for the images captured immediately after photobleaching, which were captured 12 seconds after the previous one. GFP-PLK-1 movement from the posterior to the anterior rapidly results in uniform GFP-PLK-1 distribution (B), whereas movement from the anterior to the posterior does not, leaving GFP-PLK-1 distribution asymmetric (D).

View larger version (100K): [in this window] [in a new window]   Fig. 4. PLK-1 asymmetry is controlled by A-P polarity cues. (A,B) GFP-PAR-2 embryos stained for PLK-1 (shown alone in the left panels and in red in the merged panels), GFP (shown alone in the middle panels and in green in the merged panels) and DNA (blue). The posterior domain of GFP-PAR-2 is established before PLK-1 distribution becomes asymmetric. (C-J) Embryos of the indicated genotypes stained for PLK-1 (shown alone in the left panels and in red in the merged panels), -tubulin (green) and DNA (blue). PLK-1 asymmetry is dependent on the PAR proteins (D-H), but not on GOA-1/GPA-16 (collectively referred to as G) or PIE-1 (I,J).

View larger version (72K): [in this window] [in a new window]   Fig. 5. PLK-1 levels correlate with the timing of mitotic entry after par-2 or par-3 inactivation. (A,B,D,E) Images from DIC time-lapse microscopy of wild-type (A,D), par-2(RNAi) (B) or par-3(it71) embryos (E) (see corresponding Movies 1-4). Black arrowheads indicate cleavage furrow ingression; white arrowheads with intact outline indicate intact nuclei; white arrowheads with broken outline indicate nuclei undergoing NEBD, which is apparent by loss of the smooth line corresponding to the nuclear envelope; circles indicate centrosomes in AB, the separation of which gives an indication of the advancement of mitosis. Cleavage furrow ingression at the onset of cytokinesis in P0 is defined as t=0, and the time after that is indicated in seconds. (C,F) Average duration of interphase ±s.e.m. in AB and P1 in wild-type and par-2(RNAi) (C) or wild-type and par-3(it71) (F) embryos. See Table S1 in the supplementary material for numerical values and statistical analysis. (G) Relative PLK-1 levels ±s.e.m. in anterior and posterior of one-cell stage wild-type, par-2(RNAi) and par-3(it71) embryos. Wild-type anterior, 0.68±0.04, n=12; wild-type posterior, 0.45±0.03, n=12; par-2(RNAi), 0.61±0.08, n=9; par-3(it71), 0.47±0.06, n=7. Student's t-test for wild-type anterior and par-2(RNAi), P=0.1; for wild-type posterior and par-2(RNAi), P=1.7x10-4; for wild-type anterior and par-3(it71), P=7.36x10-7; for wild-type posterior and par-3(it71), P=0.42. PLK-1 levels in par-2(RNAi) embryos are similar to those of the wild-type anterior, whereas PLK-1 levels in par-3(it71) are similar to those of the wild-type posterior.

View larger version (70K): [in this window] [in a new window]   Fig. 6. PLK-1 contributes to differential timing of mitotic entry in AB and P1. (A-D) Images from DIC time-lapse microscopy of wild-type (A), mild plk-1(RNAi) (B), ncc-1(RNAi) (C) and atp-2(RNAi) (D) (see corresponding Movies 5-8 in the supplementary material). See Fig. 5 legend for explanation of symbols and timing. (E) Average ratios ±s.e.m. of the duration of interphase in P1 over the duration of interphase in AB [(IP1)/(IAB)] (RI) in embryos of the indicated genotypes. The star indicates that the difference with wild type is statistically significant. See Tables S1, S2 in the supplementary material for numerical values and statistical analysis.

View larger version (54K): [in this window] [in a new window]   Fig. 7. PLK-1 mediated differential timing is independent of ATL-1. (A-C) Wild-type (A), atl-1(tm853) (B) or div-1(RNAi) (C) two-cell stage embryo stained for PLK-1 (shown alone in left panels and in red in the merged panels), -tubulin (green) and DNA (blue). PLK-1 distribution is asymmetric as in the wild type. (D) Average ratios, along with s.e.m., of PLK-1 in AB versus P1 determined on fixed wild-type, atl-1(tm853) and div-1(RNAi) embryos. Wild type, 1.52±0.05, n=64; atl-1(tm853), 1.48±0.12, n=9, P=0.58, compared with the wild type (two-tailed Student's t-test); div-1(RNAi), 1.45±0.17, n=8, P=0.32, compared with the wild type (two-tailed Student's t-test). (E-G) Images from DIC time-lapse microscopy of wild-type (E), atl-1(tm853) (F) or atl-1(tm853) embryos treated with mild plk-1(RNAi) (G) (see corresponding Movies 5, 9-10 in the supplementary material). See legend of Fig. 5 for explanation of symbols and timing. (H) Average ratios ±s.e.m. of the duration of interphase in P1 over the duration of interphase in AB [(IP1)/(IAB)] (RI) in embryos of the indicated genotypes. The star denotes that the difference with wild type is statistically significant. See Table S2 in the supplementary material for numerical values and statistical analysis. (I) Working model. Preferential promotion of mitotic entry in AB through the presence of more PLK-1, together with preferential retardation of mitotic entry in P1 through engagement of an ATL-1/CHK-1-dependent checkpoint, ensure differential cell cycle duration in two-cell stage C. elegans embryos.