GFP, green fluorescent protein; Sd, Scalloped. (TIFF) Click here for more data file.(13M, tiff) S3 FigLoss of Sd prevents Yki nuclear localisation and causes arrest of egg chamber development at stage 10. cell epithelium throughout oogenesis. A) An SdCGFP knockin collection localises to the nucleus in all follicle cells at early stages of oogenesis. F-actin is definitely costained in reddish. B) An SdCGFP knockin collection localises to the nucleus in all follicle cells at phases 6C10 of oogenesis. F-actin is definitely costained in reddish. DAPI marks nuclei in blue. C) An SdCGFP knockin collection localises to the nucleus in all follicle cells at stage 14 of oogenesis. DAPI marks nuclei in blue. GFP, green fluorescent protein; Sd, Scalloped.(TIFF) pbio.3000509.s002.tiff (13M) GUID:?19C623DF-8A14-49F9-9C58-59F72B13C031 S3 Fig: Loss of Sd prevents Yki nuclear localisation and causes arrest of egg chamber development at stage 10. A) Manifestation of SdCRNAi helps prevent nuclear localisation of YkiCGFP in early-stage egg chambers. Compare with Fig 1B. PRDM1 B) Manifestation of SdCRNAi helps prevent nuclear localisation of YkiCGFP in late-stage egg chambers, including stretch cells at stage 10. C) Apoptosis, noticeable by Dcp1-positive cells, happens in stage 10 germline cells affected by insufficiency in follicle cell figures upon manifestation of SdCRNAi. The Sd loss-of-function phenotype is definitely a weaker version of the Yki loss-of-function phenotype; compare with Fig 1D. Dcp1, Death Caspase 1; GFP, green fluorescent protein; RNAi, RNA interference; Sd, Scalloped; Yki, Yorkie.(TIFF) pbio.3000509.s003.tiff (11M) GUID:?1CD2C93F-6EC6-4677-B3C8-8BD79F7D4188 S4 Fig: Tor-driven germline cell growth is required for flattening of stretch cells at stage 9 of oogenesis at which Yki becomes strongly nuclear. A) YkiCGFP localises to the nucleus in stretch cells and to the cytoplasm in columnar cells of the follicular epithelium at stage 9 of oogenesis. DAPI marks nuclei in blue. F-actin is definitely costained in Ginkgetin reddish. B) YkiCGFP localises to the cytoplasm in all cells when germline growth is definitely arrested by silencing of Tor by manifestation of specifically in germline cells with the maternal driver line. Note failure of stretch cells to become flattened with this stage 9 egg chamber. C) YkiCGFP localises to the cytoplasm in all cells when germline growth is definitely arrested by silencing of Tor by manifestation of specifically in germline cells with the maternal driver line. Note failure of stretch cells to become flattened with this stage 8 egg chamber. GFP, green fluorescent protein; RNAi, RNA interference; TOR, Target of Rapamycin; (Hpo and human being MST1/2, but not in the non-Hippo pathway kinases MST3/4. A pan-Akt substrate phosphospecific antibody recognises monomeric immunoprecipitated Hpo kinase but not the dimeric form, suggesting that Ginkgetin Akt phosphorylation may inhibit Hpo dimerisation in S2 cells. C) Diagram of the Hpo kinase structure showing the surface accessibility of the Akt phosphorylation site adjacent to the ATP binding cleft. D) Close-up of the loop linking the Akt phosphorylation site with the catalytic aspartate residue. E) Manifestation of wild-type Hpo from a third chromosome landing site causes a slight reduction in the number of follicle cells, with occasional gaps in the epithelium(*). Manifestation of phosphomutant HpoT132A from your same landing site causes a strong Ginkgetin reduction in the number of follicle cells, with frequent gaps in the epithelium(*) and a failure of posterior cells to columnarise (arrow). YkiCGFP remains cytoplasmic, actually in highly stretched cells, upon manifestation of HpoT132A. F) Manifestation of wild-type Hpo from a third chromosome landing site causes a slight reduction in wing size, while manifestation of phosphomutant HpoT132 from your same landing site causes a dramatic reduction in wing size. G) Quantification of F. Observe supplementary file S1_Data.xlsx for underlying data. GFP, green fluorescent protein; Hpo, Hippo; MST, Mammalian Sterile 20 kinase; Yki, Ginkgetin Yorkie.(TIFF) pbio.3000509.s009.tiff (14M) GUID:?6676DC55-9F53-4778-842F-2149BFCE9276 S10 Fig: Genetic epistasis between overexpressed active Akt and overexpressed Hpo kinases. A) Wing-specific induces wing overgrowth. Overexpression of strongly active helps Ginkgetin prevent wing growth and also helps prevent coexpressed from traveling growth. B) Quantification of wing area from A. Observe supplementary file S1_Data.xlsx for underlying data. Hpo, Hippo; has a solitary YAP/TAZ homolog named Yorkie (Yki) that is controlled by Hippo pathway signalling in response to epithelial polarity and cells mechanics during development. Here, we display that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth element 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. We find similar results in the developing wing, where Yki becomes nuclear in the mechanically stretched cells of the wing.
