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First published online January 13, 2009
doi: 10.1242/10.1242/dev.027599

Development 136, 449-459 (2009)
Published by The Company of Biologists 2009
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no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila

Julie A. Merkle1, Jamie L. Rickmyre1, Aprajita Garg2, Erin B. Loggins1, Jeanne N. Jodoin1, Ethan Lee1, Louisa P. Wu2 and Laura A. Lee1,*

1 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-4200 MRBIII, 465 21st Avenue South, Nashville, TN 37232, USA.
2 Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, MD 20742, USA.


Figure 1
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  		Fig. 1. The nopo phenotype. Representative syncytial embryos and mitotic spindles in embryos from wild-type or nopoZ1447 females. (A,B) Staining of nopo-derived embryos reveals developmental arrest with condensed, unevenly spaced DNA (B) compared with wild type (A). (C-G) Microtubules are in green and DNA in red. (C) Wild-type spindle. (D-F) Shortened, barrel-shaped nopo spindles with detached centrosomes and misaligned chromosomes. Arrowheads indicate detached centrosomes out of focal plane; arrow, DNA at pole. Metaphase-like spindle with two centrosomes per pole (F) reveals an asynchrony of nuclear and centrosome cycles. (G) Similar defects are observed in an nopoExc142/Df(2R)Exel7153-derived embryo. (H-K) Microtubules are in green; centrosomes in blue. (H) Wild-type spindle. (I,J) nopo spindles with detached and/or missing centrosomes. (K) Tripolar nopo spindle. Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. CG5140 is the nopo gene. (A) nopo structure. Coding regions are represented by black boxes, 5'- and 3'-UTRs by white boxes, splicing events by lines. Arrows indicate transcription direction. Asterisk marks position of E11K mutation in nopoZ1447. Triangles represent P-elements. EYG5845 imprecise excision generated nopoExc142 (gap represents deleted region). Dashed line represents genomic region used to create rescue construct. (B) Comparison of Drosophila NOPO and human TRIP. Gray boxes represent RING domains. Asterisk marks mutation in nopoZ1447. Line indicates NOPO region used for antibody production. (C) Alignment of RING domains of putative NOPO/TRIP homologs in Drosophila melanogaster, Homo sapiens, Mus musculus, Danio rerio, Gallus gallus and Anopheles gambiae. Residues 6-46 of Drosophila NOPO are shown. Crucial RING-domain cysteines and histidines are highlighted. Asterisk marks residue mutated in nopoZ1447. (D,E) NOPO immunoblots. (D) NOPO levels in embryos (1-2 hours) of wild-type or nopo females. Anti-NOPO antibodies recognize a specific band the predicted size of NOPO (48 kDa) and a non-specific band (bg). (E) NOPO developmental western. Anti-GAPDH or anti-{alpha}-tubulin was used as a loading control.

 

Figure 3
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  		Fig. 3. Suppression of nopo by mnk. (A-E) Representative mitotic spindles in syncytial embryos from wild-type (A), nopoZ1447 (B) and mnk nopoZ1447 females (C-E). (A-C) Microtubules are in green, DNA in red, and centrosomes in blue. nopo (B) is suppressed by mnk, as evidenced by the restoration of elongated spindles with attached centrosomes (C). (D,E) Microtubules are in green and DNA in red. Aberrant mitotic figures with DNA shared by two spindles are observed in mnk nopo-derived embryos. (F,G) Cellularized embryos (2-3 hours). Actin is in green and DNA in red. Developmental arrest of nopo is suppressed by mnk. Cellularized mnk nopoZ1447-derived embryos show large DNA masses (G) compared to wild type (F). Scale bars: 10 µm in A-E; 20 µm in F,G.

 

Figure 4
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  		Fig. 4. Shortened cycle 11 interphase of mnk nopo-derived embryos. (A,B) Cell-cycle timing during cortical embryonic divisions. Bar graph (A) shows mean cycle 11 interphase lengths for various genotypes. Table (B) summarizes mean cycle 11-13 interphase (I) and mitosis (M) lengths. n, number of embryos. Error bars (A) and ±values (B) represent s.e.m. Single and double asterisks mark interphases significantly shorter than wild type (P<0.01 and <0.001, respectively). (C,D) Immunoblotting reveals normal pY15-CDK1 (C) and Cyclin B (D) levels in mnk nopoZ1447-derived embryos (1-2 hours). Control grp-derived embryos have reduced pY15-CDK1 levels. Anti-{alpha}-tubulin or anti-GAPDH was used as a loading control.

 

Figure 5
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  		Fig. 5. Nuclear localization of NOPO. Immunofluorescence microscopy of transfected HeLa cells. DNA is in blue. (A,B) eGFP is in green and actin in red. eGFP-Drosophila NOPO (B) localizes to nuclear puncta; eGFP (A) is homogeneously distributed. (C-E) eGFP is in green and mCherry in red. eGFP-Drosophila NOPO (C) and mCherry-human TRIP (D) co-localize in nuclear puncta (E, merge). (F-H) eGFP is in green and CREST in red. eGFP-NOPO (F) is not at the centromeres (G; H, merge). (I) Cells with eGFP-NOPO puncta (green) are negative for PCNA puncta (red). (J) Cells with eGFP-NOPO puncta (green) are positive for nuclear Cyclin A (red). Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. NOPO/TRIP, BEN and UEV1A interactions and co-localization. (A) Yeast two-hybrid assay. Yeast cells expressing combinations of NOPO, BEN and UEV1A fused to the Gal4 DNA-binding domain (BD, `bait') or activation domain (AD, `prey') were spotted onto selective media. Growth on SC-Trp-Leu-His media (shown) indicates physical interaction between the fusion proteins. Wild-type and mutant versions of NOPO and BEN (E11K and P97S, respectively) were tested. A representative plate spotted in duplicate is shown; identical results were obtained for three independent Trp+Leu+ colonies per plasmid combination tested. (B-F) Immunofluorescence microscopy of transfected HeLa cells. eGFP-BEN is in green, mCherry-TRIP in red, and DNA in blue. eGFP-BEN (B) and mCherry-TRIP (C) localize distinctly when transfected alone. (D-F) Co-transfection of eGFP-BEN (D) with mCherry-TRIP (E) promotes its localization into nuclear puncta (F, merge). Scale bars: 20 µm.

 

Figure 7
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  		Fig. 7. ben phenocopies nopo. Representative mitotic spindles in syncytial embryos from wild-type, nopo and ben females. (A-J) Microtubules are in green and DNA in red. (A-E) Single mitotic spindle and polar body in a ben1-derived embryo. (A) Dashed line outlines the embryo; arrowhead indicates detached centrosome out of focal plane. (B-E) Magnified images of the polar body (B,C) and mitotic spindle (D,E) from A. (F-K) Mitotic spindles in embryos from wild-type (F), nopoZ1447 (G), ben1 (H,I) and ben1/Df(1)HA92 (J,K) females. ben-derived embryos exhibit nopo phenotypes, including barrel-shaped, acentrosomal spindles and displaced DNA (I, arrow). (K) Microtubules are in green and centrosomes in blue. A ben1/Df(1)HA92 spindle with a detached centrosome (arrowhead). Scale bars: 20 µm in A; 10 µm in B-K.

 

Figure 8
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  		Fig. 8. Model of the function of NOPO in the early embryo. We propose a model in which BEN-UEV1A-NOPO (E2-E3) complexes ubiquitinate unidentified target proteins to ensure the maintenance of genomic integrity in the early Drosophila embryo. In the absence of NOPO-mediated ubiquitination, truncation of S-phase and/or spontaneous DNA damage result in mitotic entry with unreplicated and/or damaged DNA, respectively. A CHK2-mediated DNA checkpoint is then triggered that causes mitotic arrest and a block in embryonic development.

 

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© The Company of Biologists Ltd 2009