Menu Close

Taken together, these results argue that Cortex encodes a functional member of the Cdc20/Cdh1 family

Taken together, these results argue that Cortex encodes a functional member of the Cdc20/Cdh1 family. Fzy and Cort Co-operate to promote cyclin destruction and meiotic progression While the Drosophila genome has 4 genes that encode Cdc20/Cdh1 proteins, only two of these proteins, Fzy and Cort are expressed at detectable levels in the female germ-line (Raff et al., 2002) (Jacobs et al., 2002) (Chu AZ 10417808 et al., 2001). show that Cort functions together with the canonical mitotic APC adaptor, Cdc20/Fzy to target the three mitotic cyclins for destruction in the egg and drive anaphase progression in both meiotic divisions. AZ 10417808 In addition to controlling cyclin destruction globally in the egg, Cort and Fzy appear to both be required for local destruction of cyclin B on spindles. We find that cyclin B associates with spindle microtubules throughout meiosis I and meiosis II, and dissociates from your meiotic spindle in anaphase II. Fzy and Cort are required for this loss of cyclin B from your meiotic spindle. Our results lead to a model in which the germline specific APCCort cooperates with the more general APCFzy both locally around the meiotic spindle, and globally in the egg cytoplasm to target cyclins for destruction and drive progression through the two meiotic divisions. encodes a Cdc20/Cdh1 related protein that appears to be required specifically in female meiosis (Chu et al., 2001; Lieberfarb et al., 1996; Page and Orr-Weaver, 1996), and functions with a germ-line specific gene, to mediate the destruction of cyclin A (Swan et al., 2005; Swan and Schupbach, 2005). Here we show that this canonical APC adaptor, Fzy, functions together with Cort to target mitotic cyclins for destruction and drive anaphase in both meiosis I and meiosis II. Female meiosis, like the subsequent syncytial mitotic cell cycles, appears to involve local destruction of cyclin B, and we find that both Cort and Fzy are required for this process. Materials and Methods Drosophila stocks The two mutants (Schupbach and Wieschaus, 1989) have comparable meiotic phenotypes (Page and Orr-Weaver, 1996)and were analyzed as transheterozygotes. has a conserved Y303C switch and encodes a truncated protein lacking the 7th WD repeat. corresponds to the remnants gene and was generated by site directed mutagenesis and is AZ 10417808 a molecular null, lacking a start codon (Swan et al., 2005). and (Dawson et al., 1995) are heat sensitive lethal alleles that are female sterile at 22C. These were analyzed as transheterozygotes. Double mutant chromosomes were generated by recombining with and recombining with mutants and double mutants were performed on eggs from females kept at 29C for 3 to 5 5 days. was made by PCR amplification of genomic (including introns), followed by cloning into pUASp with a 2X Hemaglutin (HA) tag AZ 10417808 at the N-terminus. Expression of UAS-HA-Cort using the driver results in rescue of the female sterility of mutants (data not shown). UAS-Fzy and UAS-Fzr were obtained from Christian Lehner (Sigrist and Lehner, 1997). UAS-CyclinB-TPM-GFP was obtained from Jordan Raff (Raff et al., 2002). Wing expression of UAS-HA-Cort, UAS-Fzy and UAS-Fzr was driven by ptcGal4 and enGal4. With these drivers, UAS-Fzy and UAS-Fzr expression results in pupal lethality. To observe the wing phenotype in enGal4/UAS-Fzy, flies were raised at 18C to reduce expression levels and permit survival to adult. Antibody stainings To observe early meiotic events in wild-type, mature AZ 10417808 eggs were activated to undergo meiosis in vitro as explained (Page and Orr-Weaver, 1997). For later meiotic events in wild-type, eggs were collected from 0 to 20 minute selections. To detect cyclin B, eggs from 0-2 hour selections or from activated oocytes were fixed in 100% Methanol, rehydrated gradually, blocked in PBST, 1%BSA and incubated with rabbit anti-cyclin B antiserum (from Jordan Raff) at 1/500. Rat anti–Tubulin (Cappel) was used at 1/500 and DNA was labeled either with mouse anti-Histones (Chemicon) at 1/1000 or Oligreen (Molecular Probes) at 1/500. Rat anti-Sub antibody (Jang et al., 2005) was used at 1/3000. FISH was performed on 0-2 hour eggs or dissected oocytes, using a probe to a repeated 359 bp repeat sequence unique to the centromeric region of the X-chromosome (Dernburg, 2000). For immunostaining of wing discs, 3rd instar larvae were collected from crosses of UAS-HA-Cort, UAS-Fzr or UAS-Fzy to ptcGal 4. Discs were fixed 30 minutes in 3.7% formaldehyde/PBST, extracted 1 hour in PBST + .3% Triton X-100, and labeled with rabbit anti cyclin B, B3 (from Christian INF2 antibody Lehner) at 1/500, or rabbit anti cyclin A (from David Glover) at 1/500. Discs were also labeled with rat anti HA antiserum (Roche) at 1/500 and mouse anti B-gal antiserum (Promega) at 1/500. Western analysis Extracts were prepared in 2X sample buffer from wild-type and mutant eggs collected over a 2-hour period. The wild-type eggs derive from unfertilized females (crossed to XO males). Western blotting was performed by standard techniques. Antibodies were mouse anti Cyclin A and mouse anti cyclin B (both from Developmental Studies Hybridoma Lender), rabbit anti cyclin B3 (Sigrist et al., 1995)and rabbit anti Pim (Stratmann and Lehner, 1996), and rabbit anti PSTAIR (Santa Cruz). Results Cort and Fzy are required for the completion of meiosis I and II The Drosophila genome contains 4 Cdc20/Cdh1 genes. Fzr2 appears to be exclusively transcribed in.