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The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised

Wellcome Trust/Cancer Research UK Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (18K): [in a new window]   Fig. 1. GFP-Cdc16 and GFP-Cdc27 appear to be incorporated into the endogenous APC/C. (A) A western blot of wild-type (WT) (lanes 1,3), GFP-Cdc16 (lane 2) and GFP-Cdc27 (lane 4) expressing embryos probed with anti-Cdc16 antibodies (lanes 1,2) or anti-Cdc27 antibodies (lanes 3,4). Twenty 0-4-hour-old embryos were loaded per lane, and blots were stained with anti-tubulin antibodies to confirm an equal loading of protein (not shown). (B) Embryo extracts from wildtype (panels 1,2), GFP-Cdc16- (panel 3) or GFP-Cdc27- (panel 4) expressing embryos were separated on a Superose 6 gel filtration column and fractions were probed with anti-Cdc27 (panel 1), anti-Cdc16 (panel 2) or anti-GFP (panels 3,4) antibodies. The position of standard molecular weight markers is indicated above the blots. (C) GFP antibodies (lanes 1,3) or Random rabbit IgG (lanes 2,4) were used to immunoprecipitate proteins from embryos expressing GFP-Cdc16 (lanes 1,2) or GFP-Cdc27 (lanes 3,4). Blots were probed with anti-GFP (top panels), anti-Cdc16 (middle panels) or anti-Cdc27 antibodies (bottom panels).

View larger version (71K): [in a new window]   Fig. 2. The behaviour of GFP-Cdc16 (top panels) and GFP-Cdc27 (bottom panels) in living syncytial embryos. The time (in minutes) is shown in the top left-hand corner. Arrows indicate the small dots seen in interphase nuclei (0:0) or the position of the spindle midbody (7:00). Bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 3. An analysis of the behaviour of Cdc16RNAi and Cdc27RNAi cells. (A) A western blot showing the levels of Cdc16 (panel 1) and Cdc27 (panel 3) in mock-treated (-) or RNAi-treated (+) cells. These blots were also probed with an anti-tubulin antibody as a loading control (panels 2,4). (B) A FACs analysis of cells at various time points after RNAi treatment. (C) A graph showing the total viable cell numbers in control and RNAi-treated cells at various time points after RNAi treatment. Results were pooled from four independent experiments and error bars indicate the standard deviation. (D) A graph showing the percentage of TUNEL-positive cells after the different RNAi treatments (as indicated under each bar). Only data from the 72 hour time point is shown. Error bars represent the standard deviation.

View larger version (40K): [in a new window]   Fig. 4. The mitotic arrest induced in Cdc16RNAi and Cdc27RNAi cells is morphologically distinct. (A) A graph showing the mitotic index of mock-, Cdc16RNAi-, Cdc27RNAi- or Cdc16RNAi- + Cdc27RNAi-treated S2 cells. This was calculated by counting the percentage of cells in mitosis as judged by phospho-histone H3 staining. Error bars represent the standard deviation. Results were pooled from four different experiments. (B) A low magnification view of Cdc16RNAi- (top panel) or Cdc27RNAi- (bottom panel) treated cells stained to reveal the distribution of DNA (red), phosphohistone H3 (blue) and microtubules (green). Note how most of the mitotic Cdc16RNAi cells have their chromosomes roughly aligned at the equator of a metaphase-like spindle, whereas most of the mitotic Cdc27RNAi cells have elongated spindles that are in a much more disorganised state. Bar, 10 µm. (C) A graph showing the proportions of mitotic cells in different states of mitosis. Typical examples of each mitotic state are shown underneath each graph; the colours are the same as in B: chromosomes aligned on a metaphase-like spindle (panel 1); chromosomes spread throughout an elongated spindle (panel 2); condensed and decondensed chromatin (which appears red, as it is not stained by the anti-phosphohistone H3 antibody) in the same cell (panel 3); a telophase-like cell (panel 4). Error bars represent the standard deviation. Bar, 5 µm.

View larger version (26K): [in a new window]   Fig. 5. The distribution of the anti-centromere antibody CID in Cdc16RNAi and Cdc27RNAi cells. Control (A,B) or Cdc16RNAi or Cdc27RNAi (C-G) cells were stained to reveal the distribution of CID (red), DNA (blue) and microtubules (green). All the images shown are projections of several serial sections that were taken through the entire cell. In control cells during metaphase (A), the centromeres were aligned in closely spaced pairs on the metaphase plate; these pairs were clearly separated by early anaphase (B). In Cdc16RNAi or Cdc27RNAi cells in which the majority of the chromosomes appeared to be roughly aligned on a metaphase plate (C-E), the behaviour of the centromeres fell into three classes (the percentage of cells in each class for each RNAi treatment are shown beneath; C-E). Centromeres were either aligned in closely spaced pairs (C) or were aligned as pairs that appeared to have separated to some extent (D) or were more randomly distributed on the spindle (E). In Cdc16RNAi or Cdc27RNAi cells in which the spindles were elongated (F,G), the centromeres were spread throughout the spindle region. The arrows in F shows a region where it appears that sister chromatids are being pulled apart by tension on the spindle. Bar, 5 µm.

View larger version (94K): [in a new window]   Fig. 6. The distribution of Cyclin A and Cyclin B in Cdc16RNAi and Cdc27RNAi cells. (A) The distribution of Cyclin A (blue in merged image; shown alone in black and white), DNA (red) and microtubules (green). In mock-treated cells, Cyclin A stains the condensing mitotic chromatin in prophase cells (panel 1) but is not detectable on the chromosomes by metaphase when the chromosomes are usually visible as a `shadowed' region in the cyclin A channel; arrow, panel 2). This is also true of Cdc16RNAi cells (panel 3) but is not true of Cdc27RNAi cells where Cyclin A sometimes strongly stains metaphase chromosomes (panel 4), and the DNA `shadow' normally seen in the cyclin A channel is usually not detectable (panel 5). (B) The distribution of Cyclin B (blue in merged image or shown alone in black and white), DNA and microtubules (colours as in A). In mock-treated cells, Cyclin B stains centrosomes in prophase (arrows, panel 1) and centrosomes and spindles in some metaphase cells (panel 2) but not others (panel 3). We failed to detect Cyclin B on centrosomes or spindles in any mock-treated cells in anaphase (panel 4). In Cdc16RNAi or Cdc27RNAi cells, Cyclin B behaved in a similar manner to mock-treated cells, but it was very occasionally detectable on centrosomes and spindles in anaphase-like cells, particularly in Cdc27RNAi-treated cells (panel 5), suggesting that cyclin B is not being degraded properly in at least some of these cells. Bar, 5 µm.

View larger version (57K): [in a new window]   Fig. 7. An analysis of the levels of various cell cycle proteins in Cdc16RNAi and Cdc27RNAi cells. The western blots show the levels of Cdc16 (panel 1), Cdc27 (panel 2), Cyclin A (panel 3), Cyclin B (panel 4) and Fzy (panel 5) in mock-treated (-) or RNAi-treated (+) cells. All blots were also probed with anti-tubulin antibodies to confirm an equal loading (data not shown), and we show the levels of a non-specific band that is variably recognised by the anti-Cdc16 antibodies (asterisk, panel 1). A slower migrating form of Cyclin A is highlighted with an arrowhead in the Cdc27RNAi- and Cdc16RNAi- and Cdc27RNAi-treated cells.