QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 September 2004
doi: 10.1242/dev.01348

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Thomer, M. Articles by Calvi, B. R. Search for Related Content PubMed PubMed Citation Articles by Thomer, M. Articles by Calvi, B. R.

Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2

Marguerite Thomer^*, Noah R. May^*, Bhagwan D. Aggarwal, Garrick Kwok and Brian R. Calvi

Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145, USA

View larger version (37K): [in a new window]   Fig. 4. Phosphorylation of the Dup N terminus in vivo depends on cyclin E/CDK2. (A) Immunoprecipitation (IP) of embryo extracts with polyclonal guinea pig Dup antibody followed by western blot with affinity-purified polyclonal rabbit Dup antibody detects 106 kDa, 105 kDa and 82 kDa isoforms (lane 2). (Lane 4) IP of these three isoforms with Geminin. (Lane 1) Input, (lane 3) pre-immune. (B) The 106 kDa isoform differs from the 105 kDa by a cyclin E/CDK2-dependent phosphorylation. Western blot labeled with Dup antibody of extracts from wild-type third instar larval brains (lanes 1, 2), hsp70:GAL4; UAS:cyclin E (lanes 3, 4, 5), or hsp70:GAL4; UAS: Dacapo (lanes 6, 7, 8). Lanes 4, 5, 7, 8: induced expression of UAS trangenes; lane 2, 5, 8: lambda phosphatase (PPase) treated. (C) Cyclin E/CDK2 alters migration of Myc:FL-Dup but not Myc:Dup 10(A). Transgene expression from Myc:FL-Dup (lanes 1-4) and Myc:Dup 10(A) (lanes 5-8) was detected with Myc antibodies. Over-expression of cyclin E (lanes 3, 4, 7, 8). Lanes 1, 3, 6, 8: phosphatase treated. Blots were reprobed for lamin C as a loading control. (D) Abundance of FL-Dup (lanes 1-6) and Dup 10(A) (lanes 7-12) at different times after a 30-minute heat pulse of expression without (lanes 1-3, 7-9) or with (lanes 4-6, 10-12) coexpression of cyclin E. (E) Quantification of Myc-tagged protein from the experiment shown in D. Each point shows the average value for three replicates normalized against the lamin C loading control and a linear regression curve.

View larger version (53K): [in a new window]   Fig. 1. Dup rapidly declines at G1/S in the eye disc. Synchronized cell cycles of the eye morphogenetic furrow (MF) in a third instar larva are indicated above (posterior to the right). (A) Dup antibody labeling (red), BrdU (green). (B) Cyclin E (red) and Dup (green) (overlap appears yellow). (C) Cyclin B (red) Dup (green). Images are composites of confocal sections. Scale bar: 10 µm (A-C).

View larger version (81K): [in a new window]   Fig. 5. The N terminus is necessary and sufficient for Dup degradation in follicle cells of the ovary. (A) Schematic of Myc-tagged wild-type and mutant Dup transgenes expressed using the hsp70 promoter. The N terminus has ten putative phosphorylation sites (ball and stick, consensus shown above). (B-E) Follicle cells of stage 8 egg chambers expressing different Myc-tagged Dup proteins double labeled for Myc (green) and BrdU (red). (B) Myc:FL-Dup, (C) Myc:Dup 10(A), (D) Myc:N-Dup, (E) Myc:C-Dup. Only C-Dup had significant overlap between Myc and BrdU labeling (yellow). Images are composites of confocal sections. Scale bar: 10 µm (B-E).

View larger version (134K): [in a new window]   Fig. 2. Cyclin E/CDK2 is required for Dup degradation. Dup antibody labeling (red) in the morphogenetic furrow (MF) of (A) wild-type eye disc, (B) GMR:p21 expressing eye disc. Posterior is to the right. Image is a composite of confocal sections. Scale bar: 20 µm (A,B).

View larger version (33K): [in a new window]   Fig. 3. Dup is associated with CDK2 protein and activity in embryo extracts. (A) Dup immunoprecipitates Myc:CDK2 from embryo extracts. Extracts from da:GAL4; UAS:6XMyc:CDK2 embryos were used for immunoprecipitation with antibodies to Dup (lane 1), Myc (lane 2) or with pre-immune sera (lane 3) and western blots were subsequently probed for Myc. The asterisk indicates the mouse immunoglobulin band. (B) Dup is associated with a CDK kinase activity. Dup antibody (lanes 1 and 2) or pre-immune serum (lane 3) was used for immunoprecipitation from wild-type embryos (lane 1) or those expressing 6XMyc:CDK2 (lanes 2 and 3), and the pellet was subsequently used in an in vitro kinase assay with 32P[ATP] and histone H1 as substrate.

View larger version (114K): [in a new window]   Fig. 6. Prolonged mis-expression of Dup is sufficient to induce re-replication and cell death in the ovary. Follicle cells labeled with Toto-3 (blue) and BrdU (red). (A) Stage 6 wild-type egg chamber. (B) FL-Dup expression in stage 5 and 6 follicle cells results in enlarged nuclei, some of which are actively replicating (two indicated by arrows), while others are pycnotic (arrowhead). The punctate BrdU incorporation is late replication of heterochromatin. (C) Myc:Dup 10(A) expression also results in enlarged nuclei that incorporate BrdU (arrow), and pycnotic nuclei (arrowhead). Images are composites of confocal sections. Scale bar: 10 µm.

View larger version (61K): [in a new window]   Fig. 7. Mis-expression of Dup is sufficient to induce polyploidy of wing disc cells. Larvae were subjected to three heat pulses over 30 hours and wing discs were labeled with DAPI and examined by fluorescence microscopy (A,C,E,G) or by Hoechst 33342 staining and analyzed by FACS (B,D,F). Arrows indicate giant polyploid cells and arrowheads, cell death. (A,B) Wild-type. (C,D) Myc:FL-Dup. (E,F) Myc:Dup 10(A). (G) High magnification of one polytene nucleus from a wing disc over-expressing FL-Dup. Polytene chromosome arms can be seen radiating from a heterochromatic chromocenter that labels brightly with DAPI. Scale bar: 10 µm.

View larger version (56K): [in a new window]   Fig. 8. Mis-expression of Dup enhances genomic replication within S phase. BrdU labeling in third instar eye imaginal discs. (A) Wild-type (B) FL-Dup mis-expression. (C) Dup 10(A) mis-expression. Anterior is to the left. Scale bar: 20 µm (A-C).

View larger version (147K): [in a new window]   Fig. 9. Mis-expression of Dup has distinct effects on amplification of chorion genes. Confocal images of BrdU labeling (red) of amplification foci within mid-stage 10B follicle cell nuclei (Toto-3, blue) 3 hours after expressing different Dup proteins. (A) Wild-type, (B) FL-Dup, (C) C-Dup, (D) Dup 10(A). All images were taken at the same exposure. Scale bar: 10 µm (A-D).