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Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster.

Tien AC, Rajan A, Schulze KL, Ryoo HD, Acar M, Steller H, Bellen HJ - J. Cell Biol. (2008)

Bottom Line: Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch.These LNRs are unique to the Notch family of proteins.Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Notch-mediated cell-cell communication regulates numerous developmental processes and cell fate decisions. Through a mosaic genetic screen in Drosophila melanogaster, we identified a role in Notch signaling for a conserved thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1-like (Ero1L). Although Ero1L is reported to play a widespread role in protein folding in yeast, in flies Ero1L mutant clones show specific defects in lateral inhibition and inductive signaling, two characteristic processes regulated by Notch signaling. Ero1L mutant cells accumulate high levels of Notch protein in the ER and induce the unfolded protein response, suggesting that Notch is misfolded and fails to be exported from the ER. Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch. These LNRs are unique to the Notch family of proteins. Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor.

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kiga is required in signal-receiving cells for Notch signaling. (A–B') Wild-type (WT) ovary (A and A') and ovary with kiga mutant clones (B and B') were stained with phalloidin (red) to outline the cell boundary. (B and B') A large kiga mutant clone marked by loss of GFP expression (green) results in a fused egg chamber phenotype. (C–F') Notch signaling activity reported by Cut expression at the DV boundary using the MARCM technique. Part of the wing discs stained for Cut (red) is shown, and the mutant region is marked positively with GFP expression (green). (C and C') Overexpression of Dl in kiga mutant cells can induce Cut expression in the adjacent cells near the DV boundary at the dorsal compartment. (D and D') Overexpression of Notch in wild-type cells can induce Cut expression cell autonomously near the DV boundary. (E and E') Overexpression of Notch in kiga mutant cells fails to induce Cut expression near the DV boundary. Note that when the clones are crossing the boundary, Cut expression is lost in the mutant cells. (F and F') Overexpression of NEXT in kiga mutant cells can induce Cut expression cell autonomously. Bars: (A–B') 20 μm; (C–F') 10 μm.
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fig3: kiga is required in signal-receiving cells for Notch signaling. (A–B') Wild-type (WT) ovary (A and A') and ovary with kiga mutant clones (B and B') were stained with phalloidin (red) to outline the cell boundary. (B and B') A large kiga mutant clone marked by loss of GFP expression (green) results in a fused egg chamber phenotype. (C–F') Notch signaling activity reported by Cut expression at the DV boundary using the MARCM technique. Part of the wing discs stained for Cut (red) is shown, and the mutant region is marked positively with GFP expression (green). (C and C') Overexpression of Dl in kiga mutant cells can induce Cut expression in the adjacent cells near the DV boundary at the dorsal compartment. (D and D') Overexpression of Notch in wild-type cells can induce Cut expression cell autonomously near the DV boundary. (E and E') Overexpression of Notch in kiga mutant cells fails to induce Cut expression near the DV boundary. Note that when the clones are crossing the boundary, Cut expression is lost in the mutant cells. (F and F') Overexpression of NEXT in kiga mutant cells can induce Cut expression cell autonomously. Bars: (A–B') 20 μm; (C–F') 10 μm.

Mentions: To determine whether kiga is required for the Notch signaling in the signal-sending or signal-receiving cell, we analyzed the role of kiga in ovary development. In ovaries, there is a distinct requirement for the ligand (Dl) and the receptor (Notch); Dl is expressed in the germ cells to activate Notch in the surrounding somatic follicle cells (Lopez-Schier and St Johnston, 2001). Loss of Notch in the follicle cells results in failure of follicle cell differentiation and leads to formation of giant compound egg chambers in which multiple germline cysts are surrounded by a single follicular epithelium (Xu et al., 1992). Loss of Dl in the follicle cells does not affect encapsulation (Lopez-Schier and St Johnston, 2001). We found that clones of kiga in follicle cells result in formation of giant compound egg chambers (Fig. 3, A and B), indicating that loss of kiga phenocopies loss of Notch in this developmental context.


Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster.

Tien AC, Rajan A, Schulze KL, Ryoo HD, Acar M, Steller H, Bellen HJ - J. Cell Biol. (2008)

kiga is required in signal-receiving cells for Notch signaling. (A–B') Wild-type (WT) ovary (A and A') and ovary with kiga mutant clones (B and B') were stained with phalloidin (red) to outline the cell boundary. (B and B') A large kiga mutant clone marked by loss of GFP expression (green) results in a fused egg chamber phenotype. (C–F') Notch signaling activity reported by Cut expression at the DV boundary using the MARCM technique. Part of the wing discs stained for Cut (red) is shown, and the mutant region is marked positively with GFP expression (green). (C and C') Overexpression of Dl in kiga mutant cells can induce Cut expression in the adjacent cells near the DV boundary at the dorsal compartment. (D and D') Overexpression of Notch in wild-type cells can induce Cut expression cell autonomously near the DV boundary. (E and E') Overexpression of Notch in kiga mutant cells fails to induce Cut expression near the DV boundary. Note that when the clones are crossing the boundary, Cut expression is lost in the mutant cells. (F and F') Overexpression of NEXT in kiga mutant cells can induce Cut expression cell autonomously. Bars: (A–B') 20 μm; (C–F') 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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fig3: kiga is required in signal-receiving cells for Notch signaling. (A–B') Wild-type (WT) ovary (A and A') and ovary with kiga mutant clones (B and B') were stained with phalloidin (red) to outline the cell boundary. (B and B') A large kiga mutant clone marked by loss of GFP expression (green) results in a fused egg chamber phenotype. (C–F') Notch signaling activity reported by Cut expression at the DV boundary using the MARCM technique. Part of the wing discs stained for Cut (red) is shown, and the mutant region is marked positively with GFP expression (green). (C and C') Overexpression of Dl in kiga mutant cells can induce Cut expression in the adjacent cells near the DV boundary at the dorsal compartment. (D and D') Overexpression of Notch in wild-type cells can induce Cut expression cell autonomously near the DV boundary. (E and E') Overexpression of Notch in kiga mutant cells fails to induce Cut expression near the DV boundary. Note that when the clones are crossing the boundary, Cut expression is lost in the mutant cells. (F and F') Overexpression of NEXT in kiga mutant cells can induce Cut expression cell autonomously. Bars: (A–B') 20 μm; (C–F') 10 μm.
Mentions: To determine whether kiga is required for the Notch signaling in the signal-sending or signal-receiving cell, we analyzed the role of kiga in ovary development. In ovaries, there is a distinct requirement for the ligand (Dl) and the receptor (Notch); Dl is expressed in the germ cells to activate Notch in the surrounding somatic follicle cells (Lopez-Schier and St Johnston, 2001). Loss of Notch in the follicle cells results in failure of follicle cell differentiation and leads to formation of giant compound egg chambers in which multiple germline cysts are surrounded by a single follicular epithelium (Xu et al., 1992). Loss of Dl in the follicle cells does not affect encapsulation (Lopez-Schier and St Johnston, 2001). We found that clones of kiga in follicle cells result in formation of giant compound egg chambers (Fig. 3, A and B), indicating that loss of kiga phenocopies loss of Notch in this developmental context.

Bottom Line: Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch.These LNRs are unique to the Notch family of proteins.Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Notch-mediated cell-cell communication regulates numerous developmental processes and cell fate decisions. Through a mosaic genetic screen in Drosophila melanogaster, we identified a role in Notch signaling for a conserved thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1-like (Ero1L). Although Ero1L is reported to play a widespread role in protein folding in yeast, in flies Ero1L mutant clones show specific defects in lateral inhibition and inductive signaling, two characteristic processes regulated by Notch signaling. Ero1L mutant cells accumulate high levels of Notch protein in the ER and induce the unfolded protein response, suggesting that Notch is misfolded and fails to be exported from the ER. Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch. These LNRs are unique to the Notch family of proteins. Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor.

Show MeSH