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An A-kinase anchoring protein is required for Protein kinase A regulatory subunit localization and morphology of actin structures during oogenesis in Drosophila

Stephen M. Jackson* and Celeste A. Berg

Department of Genome Sciences, Box 357730, University of Washington, 1705 Pacific Street, Seattle, WA 98195-7730, USA



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  		Fig. 1. Akap200 and Pka-RII localization in Drosophila wild-type ovaries. (A-C) Akap200 localization. (A) Akap200 protein was present on membranes of follicle cells and nurse cells at all stages of oogenesis. It was also a component of ring canals (arrows). (B) Akap200 expression diminished after stage ten in the follicle cells over the oocyte (arrow), but persisted in the nurse cell-associated follicle cells, nurse cells and ring canals. (C) Akap200 immunoreactivity (red) did not colocalize with Hu-li tai shao immunoreactivity (green). Akap200 was enriched on the outer rim of the ring canal (arrows). (D-F) Pka-RII localization. (D) Germarium and early-staged egg chambers. Like Akap200, Pka-RII protein localized to membranes and ring canals (arrows). (E) Later-staged egg chambers, including a portion of a stage nine egg chamber (arrowhead). Pka-RII was also enriched in a group of follicle cells at the anterior of egg chambers (bracket). (F) Pka-RII immunoreactivity (red) did not colocalize with Hu-li tai shao immunoreactivity (green), suggesting that it was enriched on the outer rim of ring canals.

 


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  		Fig. 2. Molecular characterization of Akap200 alleles. Exons are depicted as black bars at the top. The BDGP nucleotide number of particular landmarks within contig AE003621 are shown on the bar below, including the P-element insertion site into Akap200 (8327022), start codon (8337328) and polyadenylation site (8341381). The site of the PlacW insertion in the first intron is indicated at the confluence of the dashed lines. The P element alleles are denoted with blue bars indicating lacZ sequences, red bars as miniwhite sequences. Sequences removed in the excision lines are indicated with jagged lines.

 


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  		Fig. 3. Pka-RII localization in Akap200 mutants. (A,B) Control germaria. Akap200 (A) and Pka-RII (B) localized to membranes of germline and follicle cells in the germarium (arrowhead in B). (C-H) Akap200 mutant ovaries. In Akap200k07118/Df(2L)N22-14 germaria, most of the Akap200 immunoreactivity was lost (C), resulting in a decrease of Pka-RII membrane localization in germline cells (D, arrowhead). Females hemizygous for the excision allele Akap200ix4/Df(2L)N22-14 exhibited a partial restoration of Akap200 immunoreactivity in the germarium (E). As a result, Pka-RII membrane association was partially restored (F, arrowhead). Females hemizygous for the strongest allele (Akap200{Delta}7/Df(2L)N22-14) had almost undetectable Akap200 immunoreactivity (G) and decreased Pka-RII membrane localization (H, arrowhead). Pka-RII membrane localization was not completely eliminated by this allele, however. Note that the total amount of Pka-RII protein appeared to be unchanged in all Akap200 mutants; only the fraction associated with membranes and the fraction found in the cytoplasm changed.

 


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  		Fig. 4. Akap200 mutants produce binucleate nurse cells. (A) Wild-type stage ten egg chamber, stained with DAPI (blue) and rhodamine-conjugated phalloidin (red). Note the ring canal (arrowhead). (B) Similarly staged Akap200k07118/Df(2L)N22-14 egg chamber, stained with DAPI and rhodamine phalloidin. Two binucleate nurse cells with ring canal remnants are indicated with arrowheads. (C-E) Expressing Pka-C throughout the germline with the GAL4/UAS system produces binucleate nurse cells. (C) Expression pattern of GAL4 line NGT40, visualized in NGT40/UAS-GFPnls ovaries. GAL4 expression was weak in this line, and was only detectable in germline cells in the germarium. (D) Expression pattern of GAL4 line nos-GAL4-VP16, visualized in nos-GAL4-VP16/UAS-GFPnls females. GAL4 expression was much stronger in this line, and highest in the germline cells in the germarium and after approximately stage seven. (E) Egg chambers from NGT40/UAS-PkaC* females, stained with DAPI to visualize nuclei (blue) and rhodamine phalloidin (red). Note the binucleate nurse cell with a ring canal remnant (arrowhead). Binucleate cells with identical morphologies were observed in nos-GAL4-VP16/UAS-Pka-C* ovaries. In C,D, the membrane and ring canal staining is due to bleed-through from the rhodamine-phalloidin channel.

 


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  		Fig. 5. Altering Akap200 expression levels changes ring canal size. (A) Confocal projection of wild-type stage-ten egg chamber stained with rhodamine phalloidin. (B) Similar staged Akap200k07118/Df(2L)N22-14 egg chamber. Note the larger, thinner ring canals. (C) Similar-staged nos-GAL4-VP16/UAS-Akap200 egg chamber. Females containing these transgenes overexpressed Akap200 throughout the germline, resulting in smaller, thicker ring canals. Insets show a representative ring canal from each egg chamber, magnified a further four times.

 


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  		Fig. 6. An Akap200 mutation dominantly suppresses the Src64B ring canal size phenotype. (A-D) Confocal projection of egg chambers stained with phosphotyrosine antibodies. (A) Wild-type stage-ten egg chamber. (B) Src64BPI/Src64BPI stage-ten egg chamber. Note small ring canals and misshapen ring canal remnant in the fused, binucleate nurse cell (arrow). (C) Akap200k07118/+; Src64BPI/Src64BPI stage-ten egg chamber. The ring canals are near wild-type-sized. (D) Akap200k07118/Akap200k07118; Src64BPI/+ stage-ten egg chamber. Ring canals are near normal sized; a ring canal remnant is indicated with an arrowhead. (E) Src64BPI/Src64BPI early stage-ten egg chamber stained with Akap200 antiserum. Except for the small ring canals, Akap200 localization in these mutants appears normal.

 


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  		Fig. 7. Model for localized regulation of Protein kinase A activity by Akap200. See Discussion for details. Red depicts filamentous actin in subcortical regions of nurse cells and ring canals, green symbolizes Akap200, dark blue is Pka-RII, pink is Pka-C; yellow circles and orange blobs are other proteins that regulate actin filament polymerization or function. Phosphorylation by Pka-C inhibits the actin-regulating activity of the yellow circle proteins in this diagram, whereas Pka-C phosphorylation is required for the orange blob proteins to regulate actin function properly. Pka-C* is a mutant isoform of catalytic subunit that is incapable of binding to Pka-RII.

 

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© The Company of Biologists Ltd 2002