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First published online 30 September 2008
doi: 10.1242/jcs.035220

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The protein phosphatase PP2A-B' subunit Widerborst is a negative regulator of cytoplasmic activated Akt and lipid metabolism in Drosophila

Natalia Vereshchagina^1,*, Marie-Christine Ramel^1,,, Emmanuelle Bitoun^2, and Clive Wilson^1,¶

¹ Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, UK
² MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, Oxford Centre for Gene Function, South Parks Road, Oxford OX1 3QX, UK

View larger version (138K): [in this window] [in a new window]   Fig. 1. Wdb modulates the IIS-regulated effects of FOXO in the differentiating eye. Overexpression of foxo in the differentiating eye from the foxoGS9928 insertion using the GMR-GAL4 driver produces a characteristic reduction in the adult eye (D) (Goberdhan et al., 2005) compared with the wild type (A). This effect is completely suppressed by co-overexpressing a UAS-Akt1 construct (G). Overexpressing wdb from the wdbGS9548 insertion produces a disorganised and slightly reduced eye (B). Co-overexpression of FOXO and wdbGS9548 drastically enhances the FOXO phenotype (E), an effect that is completely suppressed by Akt1 (H; compare with I, where Akt1 and Wdb are expressed in the absence of FOXO). An EP insertion in wdb, wdbEP3559, has very little effect on its own (C), but enhances the FOXO phenotype (F).

View larger version (77K): [in this window] [in a new window]   Fig. 2. Wdb exerts its effects on FOXO in the eye via its PP2A-B' regulatory activity. The reduced eye phenotype produced by overexpression of foxoGS9928 under GMR-GAL4 control (F) is enhanced by co-overexpressing wdbGS9548 (G). When wdbGS9548 is expressed alone (B), the eye is reduced and disorganised relative to wild-type controls (A). Flies heterozygous for the recessive lethal mts allele mts02496 show no obvious eye defects (C). This allele also appears to have little, if any, effect on the FOXO phenotype (H). Although mts02496 does not noticeably modify the phenotype produced by overexpressing wdbGS9548 (D), it does suppress the reduced eye phenotype caused by co-overexpression of wdbGS9548 and foxoGS9928 (I). Overexpression of a dominant-negative form of Mts with GMR-GAL4 induces overgrowth (bulged eye in E) and completely suppresses the FOXO phenotype (J).

View larger version (59K): [in this window] [in a new window]   Fig. 3. wdb regulates animal size in Akt1 mutant flies. Figure shows female and male flies of the following genotypes (from top to bottom): wild-type Canton-S, wdbIP Akt11/Akt104226 and Akt11/Akt104226. Note that the Akt1 mutant growth phenotype is strongly suppressed by heterozygous wdbIP.

View larger version (93K): [in this window] [in a new window]   Fig. 4. Wdb is required for photoreceptor differentiation and survival in the developing eye. Eyflp-generated eye imaginal discs that are almost entirely homozygous for the wdbIP mutation fail to differentiate adult eye structures (B), unlike discs from their heterozygous siblings (A). (C-V). Mosaic eyes generated in combination with a normal GFP-labelled third chromosome, were stained with phalloidin to detect the actin cytoskeleton (E,I,M,Q,U), and with two antibodies against the neuronal antigens 24B10 (D,H,L) and 22C10 (P,T), and wdb mutant clones identified by lack of GFP expression (C,G,K,O,S and merge in F,J,N,R,V). Arrows indicate position of mutant clones, which lack neuron-specific staining, and only show low levels of disorganised phalloidin staining. In the large clone shown in G-J, mutant tissue folds down and much of it is basal to the rest of the epithelium. For all eye discs, posterior is to the right. Scale bar: 20 µm.

View larger version (87K): [in this window] [in a new window]   Fig. 5. Wdb regulates lipid droplet size in nurse cells by inhibiting accumulation of cytoplasmic pAkt1. Clones mutant for wdbIP (A-D,I-K,O-V), for wdbdw (E-H), and for both wdbIP and Akt11 (L-N,W-Z) were generated in combination with a normal GFP-labelled third chromosome, and stained with phalloidin to detect the actin cytoskeleton (C,G), with an antibody against activated pAkt1 (B,F), with Nile Red to detect neutral lipid (J,M,P), with Hoechst 33258 to detect nuclei (Q,U,Y) and with an anti-LSD2 antibody (T,X). Mutant clones were identified by lack of GFP expression (A,E,I,L,O,S,W and merges in D,H,K,N,R,V,Z). wdb mutant clones contain cells with normal nuclei, elevated levels of cytoplasmic pAkt1 and large lipid droplets (arrows in J,P). LSD2 protein levels are also increased (T). The lipid droplet phenotype is completely suppressed by the Akt11 hypomorphic allele (M), but the LSD2 phenotype is only partially suppressed (X). Most egg chambers are at stage 10, but E is at stage 9 and O is at stage 11 of oogenesis. Scale bars: 50 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 6. Wdb physically interacts with Akt1 in Drosophila ovaries. Total protein extracts from wild-type whole larvae (640 µg) or adult ovaries (340 µg) were immunoprecipitated (IP) with antibodies against Wdb, pan-Akt1, pAkt1 or Flag epitope, resolved by SDS-PAGE and analysed by western blot (WB) using anti-Wdb and anti-pan-Akt1 antibodies. A faint signal is observed roughly in the location of Akt1 in the immunoprecipitation from larval extracts with Wdb antibody. However, Wdb is not immunoprecipitated from these extracts by anti-Akt1 antibodies. The proteins only show clear coimmunoprecipitation from ovary extracts. Arrows indicate the position of the Wdb protein doublet and Akt1 proteins. Asterisks indicate heavy IgG antibody chains recognised by the secondary antibodies. Molecular size markers are given on the left in kDa.

View larger version (91K): [in this window] [in a new window]   Fig. 7. Wdb regulates levels of cytoplasmic pAkt1 and LSD2 in ovarian follicle cells. Clones mutant for wdbIP (A-L), for wdbIP and Akt11 (M-P) and PTEN1 (Q-B') were generated in combination with a normal GFP-labelled third chromosome, and stained with phalloidin to detect the actin cytoskeleton (C,S), with an antibody against activated pAkt1 (B,F,R,V), with anti-LSD2 (J,N,Z; image in Z is at higher gain to show normal low levels of LSD2 staining) and with Hoechst 33258 to detect nuclei (G,K,O,W,A'). Although pAkt1 levels and LSD2 expression are upregulated in all wdb mutant clones (B,F), only a minority of PTEN mutant clones exhibit these phenotypes (compare R with V,Z). Note that the LSD2 phenotype in wdb mutant clones (J) is partially suppressed by the Akt11 mutation (N). When this Akt1 mutation is combined with a wdb mutation, mutant follicle cells are not markedly different in size from their heterozygous neighbours (O), supporting the idea that the Akt11 allele is not a null and can be suppressed by wdb. Egg chambers are at stage 10. Scale bars: 50 µm (E,U), 25 µm (A,I,M,Q,Y).

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