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First published online 24 January 2007
doi: 10.1242/dev.02784

Development 134, 891-899 (2007)
Published by The Company of Biologists 2007
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The Cdc20 (Fzy)/Cdh1-related protein, Cort, cooperates with Fzy in cyclin destruction and anaphase progression in meiosis I and II in Drosophila

Andrew Swan and Trudi Schüpbach*

Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.


Figure 1
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  		Fig. 1. Cort and Fzy are required for the completion of meiosis I and meiosis II. (A-L') Microtubules are red, a 359 bp centromeric repeat unique to the X-chromosome is labeled green and DNA is blue. Smaller panels (A'-L') are higher-magnification views of one of the spindles in A-L, respectively. Mitotic spindles derived from the male pronucleus (when present) are more internal and are not shown in these images. (A-C) Meiosis in wild-type eggs obtained by in vitro activation (A) or from 0-20 minute egg collections (B,C). In metaphase of meiosis I (A), the egg contains a single spindle with two discrete X-chromosome signals representing two pairs of sister chromatids. In metaphase II (B), there are two tandemly arranged spindles, each containing a single X-chromosome signal. In anaphase II (C), sister chromatids separate and both spindles contain two discrete X-chromosome signals. (D-L) Eggs at 0-2 hours from females of the genotypes indicated. (D) In cortQW55/cortRH65 (females kept at 29°C for 3-5 days), eggs contain two spindles, each with one X-chromosome, indicating arrest in metaphase II. (E,F) Cks30AKO/Cks30AKO eggs with two spindles and either one (E) or two (F) X-chromosomes, indicating a delay as early as metaphase of meiosis II. (G) In fzy6/fzy7 kept at 29°C, the majority of eggs contain two spindles, both with two X-chromosome signals, indicating arrest in anaphase II. Arrow indicates a small spindle and associated chromatin. (H,I) In cortQW55,fzy6/cortRH65,fzy7 double mutants kept at 29°C, eggs contain either two spindles with one X-chromosome each (H), indicating a metaphase II arrest, or a single spindle with two X-chromosomes (I), indicating a metaphase I arrest. (J-L) Eggs from nosGal4VP16/UAS-cyclinB-TPM-GFP arrest with multiple spindles (J) or with two spindles (K), suggesting a meiosis II arrest, or with a single spindle (L), indicative of a meiosis I arrest.

 

Figure 2
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  		Fig. 2. Cks30A, Cort and Fzy regulate overall cyclin levels in the egg. Western blots of eggs aged 0-2 hours from unfertilized (females crossed to X0 males) wild type (yw), Cks30AKO/Cks30AKO (cks), cortQW55/cortRH65 (cort), fzy6/fzy7 (fzy), and cortQW55,fzy6/cortRH65,fzy7 (cort,fzy) kept at 29°C and probed for cyclin B3, cyclin A and cyclin B. Cdk1, a stable cell-cycle regulator, serves as a loading control. Cyclin B is slightly elevated in Cks30A, more so in fzy and cort, and is highly elevated in fzy; cort double mutants. Cyclin A and cyclin B3 are both elevated in cort and fzy, but elevation is higher in cks30A and fzy; cort double mutants.

 

Figure 3
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  		Fig. 3. Cort can mediate the destruction of mitotic regulators and its activity depends on Cks30A. (A-C') UAS-HA-Cort expressed under ptcGal4 and detected with anti-HA antibodies. Cyclin A (A'), cyclin B (B') and cyclin B3 (C') are reduced in the stripe, corresponding to Cort expression. (D,D') UAS-Fzy expressed under ptcGal4 (detected with anti-ß-gal antibodies; D) results in a corresponding reduction in cyclin A levels (D'). (E-H) Magnified area of an adult wing from Drosophila expressing HA-Cort (E,G,H) or UAS-Fzy (F) in the posteriorhalf of the wing (P) using enGal4. The anterior-half of the wing (A) serves as a control. Expression of UAS-HA-Cort (E) or UAS-Fzy (F) in the posterior wing results in a wider spacing of wing hairs. (G) Wing-hair-spacing phenotype is suppressed when HA-Cort is expressed in a Cks30AKO/Cks30AKO background. (H) Co-expression of UAS-Flag-Cks30A with HA-Cort in a Cks30AKO/Cks30AKO background restores the wing-hair-spacing phenotype. (I) Expression of Flag-Cks30A alone does not affect bristle spacing. Scale bar: 20 µm.

 

Figure 4
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  		Fig. 4. Cyclin B associates with the spindle in female meiosis. Wild-type (yw) eggs obtained by in vitro activation of oocytes (A-D') or from 0-20 minute egg collections (E-G'). In all panels except B,B', microtubules are red, DNA is green and cyclin B is blue. The cyclin B channel is shown by itself in grayscale in right-hand panels (labeled with '). (A) In metaphase I, high levels of cyclin B accumulate on the spindle mid-zone. (B,B') Metaphase I spindle labeled with antibodies to Sub (B) and cyclin B (B') reveals colocalization at the spindle mid-zone. (C,D) In anaphase I, cyclin B persists at the spindle mid-zone. (E) In metaphase II, cyclin B accumulates at the mid-zone on both spindles. (F) Cyclin B persists at lower levels at the mid-zone in anaphase II. (G) Late in anaphase II, cyclin B is no longer detected on the meiotic spindle. Scale bars: 5 µm in A for A,A',C-G'; 2 µm in B for B,B'.

 

Figure 5
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  		Fig. 5. Cyclin B is stabilized on the meiotic spindle in cort, fzy and Cks30A. (A-B',D-H') Tubulin is red, DNA is green and cyclin B is blue. Cyclin B is shown by itself in grayscale in right hand panels (labeled with '). (A) In wild type, cyclin B accumulates on the spindle mid-zone in metaphase of meiosis II. (B) Arrested meiosis II spindle from cortQW55/cortRH65 at 29°C, with high levels of cyclin B associated with the mid-zone of the spindle. (C,C') Co-labeling of a metaphase II spindle from cortQW55/cortRH65 with antibodies to Sub (C) and cyclin B (C') reveals colocalization at the spindle mid-zone. (D) Meiosis II spindle from fzy6/fzy7, showing the accumulation of cyclin B along the length of the spindle. (E) Metaphase-arrested meiotic spindle from cortQW55,fzy6/cortRH65,fzy7 with a high level of cyclin B at the spindle mid-zone. (F) Meiotic spindle from Cks30AKO/Cks30AKO, with elevated levels of cyclin B on the spindle mid-zone and along the spindle. (G,H) Mitotic spindles in yw (G) and fzy6/fzy7 (H). In wild type, cyclin B associates with the spindle mid-zone late in metaphase, before disappearing from the spindle in anaphase. In fzy, cyclin B accumulates along the length of the arrested mitotic spindle. Scale bars: 5 µm in A for A-B',D-H';. 2 µm in C for C,C'.

 




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