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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 specifically required for Notch-dependent processes and interacts genetically with Notch mutants. (A) Schematic illustration of the lateral inhibition process mediated by Notch signaling. (A') Schematic illustration of inductive signaling. The “N's” in the cells represent high Notch activity. The wing margin cell fate (blue) is induced by Notch signaling between dorsal and ventral compartments (yellow and orange). In B–E and J–K, homozygous mutant regions lack GFP expression (green). (B) Lateral inhibition is impaired in homozygous kiga mutant clones. The SOPs of the pupal notum at 12 h APF are labeled for Sens (red). (B and B') Note that SOPs in the wild-type region are spaced regularly between epithelial cells, whereas no epithelial cells are present between mutant SOPs. (C and C') Binary fate decision at 24 h APF in kiga mutant clones. Cut (blue) marks all of the cells of the SOP progeny. Elav (red) stains the neuronal cells. In the wild-type domain, there are well-spaced clusters of four Cut-positive cells and one Elav-positive cell. In kiga mutant clones, Cut expression is expanded, and it is difficult to identify single clusters, indicating a lateral inhibition defect. However, the presence of neuronal cells suggests that cell fate specification and differentiation are likely normal. (D and D') Defective wing margin formation is associated with kiga mutants. Wing imaginal disc from a third instar larva stained for Cut (red). Cells in the large homozygous mutant clones lack expression of Cut at the DV boundary. A higher magnification of a small portion of D is shown in E and E'. Note that in large clones, mutant cells at the boundary of the clone are not Cut positive (arrow), whereas in some smaller mutant clones and near the edge of wild-type cells, some margin cells are Cut positive (*). (F and G) Removal of one copy of kiga suppresses a gain of function phenotype of Notch. A male adult wing from an NAxE-2/Y fly is shown (F) with a reduced length of veins (F, arrows). The loss of wing veins is suppressed (G, arrows) when one copy of kiga is removed in NAx E-2/Y; kiga/+ male wings (G). (H and I) Removal of one copy of kiga does not suppress a gain of function phenotype of Egfr. A male adult wing from an Elp/+ fly is shown (H) with an ectopic wing vein (H, arrow). The gain of wing vein (I, arrow) is not suppressed when one copy of kiga is removed. (J–K') Dpp and Hh signaling are unaffected in kiga mutant clones. Wing imaginal disc with kiga mutant clones stained for phospho-Mad (J and J', red) and Ci (K and K', red). Bars: (B–C') 10 μm; (D–E' and J–K') 40 μm.
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fig2: kiga is specifically required for Notch-dependent processes and interacts genetically with Notch mutants. (A) Schematic illustration of the lateral inhibition process mediated by Notch signaling. (A') Schematic illustration of inductive signaling. The “N's” in the cells represent high Notch activity. The wing margin cell fate (blue) is induced by Notch signaling between dorsal and ventral compartments (yellow and orange). In B–E and J–K, homozygous mutant regions lack GFP expression (green). (B) Lateral inhibition is impaired in homozygous kiga mutant clones. The SOPs of the pupal notum at 12 h APF are labeled for Sens (red). (B and B') Note that SOPs in the wild-type region are spaced regularly between epithelial cells, whereas no epithelial cells are present between mutant SOPs. (C and C') Binary fate decision at 24 h APF in kiga mutant clones. Cut (blue) marks all of the cells of the SOP progeny. Elav (red) stains the neuronal cells. In the wild-type domain, there are well-spaced clusters of four Cut-positive cells and one Elav-positive cell. In kiga mutant clones, Cut expression is expanded, and it is difficult to identify single clusters, indicating a lateral inhibition defect. However, the presence of neuronal cells suggests that cell fate specification and differentiation are likely normal. (D and D') Defective wing margin formation is associated with kiga mutants. Wing imaginal disc from a third instar larva stained for Cut (red). Cells in the large homozygous mutant clones lack expression of Cut at the DV boundary. A higher magnification of a small portion of D is shown in E and E'. Note that in large clones, mutant cells at the boundary of the clone are not Cut positive (arrow), whereas in some smaller mutant clones and near the edge of wild-type cells, some margin cells are Cut positive (*). (F and G) Removal of one copy of kiga suppresses a gain of function phenotype of Notch. A male adult wing from an NAxE-2/Y fly is shown (F) with a reduced length of veins (F, arrows). The loss of wing veins is suppressed (G, arrows) when one copy of kiga is removed in NAx E-2/Y; kiga/+ male wings (G). (H and I) Removal of one copy of kiga does not suppress a gain of function phenotype of Egfr. A male adult wing from an Elp/+ fly is shown (H) with an ectopic wing vein (H, arrow). The gain of wing vein (I, arrow) is not suppressed when one copy of kiga is removed. (J–K') Dpp and Hh signaling are unaffected in kiga mutant clones. Wing imaginal disc with kiga mutant clones stained for phospho-Mad (J and J', red) and Ci (K and K', red). Bars: (B–C') 10 μm; (D–E' and J–K') 40 μm.

Mentions: The tufting phenotype associated with kiga mutant clones suggests a defect in lateral inhibition (Fig. 2 A; Ghysen et al., 1993). Loss of Notch signaling in this process causes transformation of most or all epithelial cells within a proneural cluster into SOPs, resulting in a bristle-tufting phenotype (Hartenstein and Posakony, 1990). To probe whether lateral inhibition is affected in kiga mutant clones, we performed immunostaining using anti-Sens (Nolo et al., 2000) to label the SOPs in pupal nota at 12 h after puparium formation (APF) when the primary SOPs of the bristles are specified. As shown in Fig. 2 (B and B'), all cells within this clone are labeled with Sens, whereas only a few regularly spaced cells outside of the mutant clone are labeled with Sens. This indicates that lateral inhibition is indeed affected in the absence of kiga.


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 specifically required for Notch-dependent processes and interacts genetically with Notch mutants. (A) Schematic illustration of the lateral inhibition process mediated by Notch signaling. (A') Schematic illustration of inductive signaling. The “N's” in the cells represent high Notch activity. The wing margin cell fate (blue) is induced by Notch signaling between dorsal and ventral compartments (yellow and orange). In B–E and J–K, homozygous mutant regions lack GFP expression (green). (B) Lateral inhibition is impaired in homozygous kiga mutant clones. The SOPs of the pupal notum at 12 h APF are labeled for Sens (red). (B and B') Note that SOPs in the wild-type region are spaced regularly between epithelial cells, whereas no epithelial cells are present between mutant SOPs. (C and C') Binary fate decision at 24 h APF in kiga mutant clones. Cut (blue) marks all of the cells of the SOP progeny. Elav (red) stains the neuronal cells. In the wild-type domain, there are well-spaced clusters of four Cut-positive cells and one Elav-positive cell. In kiga mutant clones, Cut expression is expanded, and it is difficult to identify single clusters, indicating a lateral inhibition defect. However, the presence of neuronal cells suggests that cell fate specification and differentiation are likely normal. (D and D') Defective wing margin formation is associated with kiga mutants. Wing imaginal disc from a third instar larva stained for Cut (red). Cells in the large homozygous mutant clones lack expression of Cut at the DV boundary. A higher magnification of a small portion of D is shown in E and E'. Note that in large clones, mutant cells at the boundary of the clone are not Cut positive (arrow), whereas in some smaller mutant clones and near the edge of wild-type cells, some margin cells are Cut positive (*). (F and G) Removal of one copy of kiga suppresses a gain of function phenotype of Notch. A male adult wing from an NAxE-2/Y fly is shown (F) with a reduced length of veins (F, arrows). The loss of wing veins is suppressed (G, arrows) when one copy of kiga is removed in NAx E-2/Y; kiga/+ male wings (G). (H and I) Removal of one copy of kiga does not suppress a gain of function phenotype of Egfr. A male adult wing from an Elp/+ fly is shown (H) with an ectopic wing vein (H, arrow). The gain of wing vein (I, arrow) is not suppressed when one copy of kiga is removed. (J–K') Dpp and Hh signaling are unaffected in kiga mutant clones. Wing imaginal disc with kiga mutant clones stained for phospho-Mad (J and J', red) and Ci (K and K', red). Bars: (B–C') 10 μm; (D–E' and J–K') 40 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2542473&req=5

fig2: kiga is specifically required for Notch-dependent processes and interacts genetically with Notch mutants. (A) Schematic illustration of the lateral inhibition process mediated by Notch signaling. (A') Schematic illustration of inductive signaling. The “N's” in the cells represent high Notch activity. The wing margin cell fate (blue) is induced by Notch signaling between dorsal and ventral compartments (yellow and orange). In B–E and J–K, homozygous mutant regions lack GFP expression (green). (B) Lateral inhibition is impaired in homozygous kiga mutant clones. The SOPs of the pupal notum at 12 h APF are labeled for Sens (red). (B and B') Note that SOPs in the wild-type region are spaced regularly between epithelial cells, whereas no epithelial cells are present between mutant SOPs. (C and C') Binary fate decision at 24 h APF in kiga mutant clones. Cut (blue) marks all of the cells of the SOP progeny. Elav (red) stains the neuronal cells. In the wild-type domain, there are well-spaced clusters of four Cut-positive cells and one Elav-positive cell. In kiga mutant clones, Cut expression is expanded, and it is difficult to identify single clusters, indicating a lateral inhibition defect. However, the presence of neuronal cells suggests that cell fate specification and differentiation are likely normal. (D and D') Defective wing margin formation is associated with kiga mutants. Wing imaginal disc from a third instar larva stained for Cut (red). Cells in the large homozygous mutant clones lack expression of Cut at the DV boundary. A higher magnification of a small portion of D is shown in E and E'. Note that in large clones, mutant cells at the boundary of the clone are not Cut positive (arrow), whereas in some smaller mutant clones and near the edge of wild-type cells, some margin cells are Cut positive (*). (F and G) Removal of one copy of kiga suppresses a gain of function phenotype of Notch. A male adult wing from an NAxE-2/Y fly is shown (F) with a reduced length of veins (F, arrows). The loss of wing veins is suppressed (G, arrows) when one copy of kiga is removed in NAx E-2/Y; kiga/+ male wings (G). (H and I) Removal of one copy of kiga does not suppress a gain of function phenotype of Egfr. A male adult wing from an Elp/+ fly is shown (H) with an ectopic wing vein (H, arrow). The gain of wing vein (I, arrow) is not suppressed when one copy of kiga is removed. (J–K') Dpp and Hh signaling are unaffected in kiga mutant clones. Wing imaginal disc with kiga mutant clones stained for phospho-Mad (J and J', red) and Ci (K and K', red). Bars: (B–C') 10 μm; (D–E' and J–K') 40 μm.
Mentions: The tufting phenotype associated with kiga mutant clones suggests a defect in lateral inhibition (Fig. 2 A; Ghysen et al., 1993). Loss of Notch signaling in this process causes transformation of most or all epithelial cells within a proneural cluster into SOPs, resulting in a bristle-tufting phenotype (Hartenstein and Posakony, 1990). To probe whether lateral inhibition is affected in kiga mutant clones, we performed immunostaining using anti-Sens (Nolo et al., 2000) to label the SOPs in pupal nota at 12 h after puparium formation (APF) when the primary SOPs of the bristles are specified. As shown in Fig. 2 (B and B'), all cells within this clone are labeled with Sens, whereas only a few regularly spaced cells outside of the mutant clone are labeled with Sens. This indicates that lateral inhibition is indeed affected in the absence of kiga.

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
Related in: MedlinePlus