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Cis-interactions between Notch and its ligands block ligand-independent Notch activity.

Palmer WH, Jia D, Deng WM - Elife (2014)

Bottom Line: Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling.Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling.We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, United States.

ABSTRACT
The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

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Addition of Ser dsRNA had no effect on the Notch activation in S2 cells in comparison with cells treated with control green fluorescent protein (GFP) dsRNA, indicating that the small amount of Ser expression is either not translated or does not significantly contribute to Notch activation upon transfection with pMT-NFL.This validates our assumption that the Notch activation which occurs in S2 cells is by a DSL-ligand-independent mechanism. Dl was not tested, as studies have already shown a lack of Dl mRNA and protein in S2 cells (Fehon et al., 1990; Graveley et al., 2011). Again, experiments were carried out with two technical replicates and three biological replicates, with means of the technical replicates used for a paired t-test to assess significance. Error bars represent SD.DOI:http://dx.doi.org/10.7554/eLife.04415.009
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fig3s1: Addition of Ser dsRNA had no effect on the Notch activation in S2 cells in comparison with cells treated with control green fluorescent protein (GFP) dsRNA, indicating that the small amount of Ser expression is either not translated or does not significantly contribute to Notch activation upon transfection with pMT-NFL.This validates our assumption that the Notch activation which occurs in S2 cells is by a DSL-ligand-independent mechanism. Dl was not tested, as studies have already shown a lack of Dl mRNA and protein in S2 cells (Fehon et al., 1990; Graveley et al., 2011). Again, experiments were carried out with two technical replicates and three biological replicates, with means of the technical replicates used for a paired t-test to assess significance. Error bars represent SD.DOI:http://dx.doi.org/10.7554/eLife.04415.009

Mentions: Drosophila S2 cells are reported to have no Dl expression and a very low level of Ser expression, which had no effect on Notch signaling (Fehon et al., 1990; Graveley et al., 2011) (Figure 3—figure supplement 1), and have been used as a model to study ligand-independent Notch activity (Hori et al., 2011). Upon transfection with pMT-NFL, a CuSO4-inducible full-length Notch construct, Notch activation was increased by a factor of 5.13 compared with the control cells, as indicated by a NRE-firefly luciferase reporter gene (p < 0.0001) (Figure 3C). Notch activation in S2 cells is at least partially dependent on endosomal trafficking, as double-stranded (ds) RNA against early endosome component, Rab5, or multivesicular body sorting protein, hrs, reduced the levels of Notch activation (Figure 3A,B). This is consistent with the in vivo studies indicating that ligand-independent Notch activation relies heavily on receptor trafficking (Hori et al., 2012) (Rab5 p = 0.00623, hrs p = 0.0159), and our observation that Notch accumulates in Dl-/Dl- clones (Figure 3—figure supplement 2). A requirement for trafficking is consistent with the results of others who have demonstrated aberrant Notch activation in follicle cell mutants for trafficking components (Wilkin et al., 2004; Vaccari et al., 2008; Schneider et al., 2013), such as tsg101 mutant clones, which show early Notch activation in the follicle cells (Figure 3—figure supplement 3). Furthermore, co-transfecting pMT-NFL with pMT-GAL4 and pUASt-Serdel3, a form of Ser that cannot activate Notch, but only cis-inhibit, (Fleming et al., 2013) almost entirely abolished the Notch activation detected when NFL was transfected alone (p = 0.0048) (Figure 3C). These results suggest that if Notch is expressed in a cell free of cis- and trans-ligands, DSL ligand-independent activity will occur and that cis-inhibition is extremely efficient in preventing this ‘accidental’ Notch activity as it travels through the endosomal pathway en route to degradation.10.7554/eLife.04415.008Figure 3.DSL-ligand-independent Notch activity in S2 cells is buffered by cis-ligand.


Cis-interactions between Notch and its ligands block ligand-independent Notch activity.

Palmer WH, Jia D, Deng WM - Elife (2014)

Addition of Ser dsRNA had no effect on the Notch activation in S2 cells in comparison with cells treated with control green fluorescent protein (GFP) dsRNA, indicating that the small amount of Ser expression is either not translated or does not significantly contribute to Notch activation upon transfection with pMT-NFL.This validates our assumption that the Notch activation which occurs in S2 cells is by a DSL-ligand-independent mechanism. Dl was not tested, as studies have already shown a lack of Dl mRNA and protein in S2 cells (Fehon et al., 1990; Graveley et al., 2011). Again, experiments were carried out with two technical replicates and three biological replicates, with means of the technical replicates used for a paired t-test to assess significance. Error bars represent SD.DOI:http://dx.doi.org/10.7554/eLife.04415.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4286723&req=5

fig3s1: Addition of Ser dsRNA had no effect on the Notch activation in S2 cells in comparison with cells treated with control green fluorescent protein (GFP) dsRNA, indicating that the small amount of Ser expression is either not translated or does not significantly contribute to Notch activation upon transfection with pMT-NFL.This validates our assumption that the Notch activation which occurs in S2 cells is by a DSL-ligand-independent mechanism. Dl was not tested, as studies have already shown a lack of Dl mRNA and protein in S2 cells (Fehon et al., 1990; Graveley et al., 2011). Again, experiments were carried out with two technical replicates and three biological replicates, with means of the technical replicates used for a paired t-test to assess significance. Error bars represent SD.DOI:http://dx.doi.org/10.7554/eLife.04415.009
Mentions: Drosophila S2 cells are reported to have no Dl expression and a very low level of Ser expression, which had no effect on Notch signaling (Fehon et al., 1990; Graveley et al., 2011) (Figure 3—figure supplement 1), and have been used as a model to study ligand-independent Notch activity (Hori et al., 2011). Upon transfection with pMT-NFL, a CuSO4-inducible full-length Notch construct, Notch activation was increased by a factor of 5.13 compared with the control cells, as indicated by a NRE-firefly luciferase reporter gene (p < 0.0001) (Figure 3C). Notch activation in S2 cells is at least partially dependent on endosomal trafficking, as double-stranded (ds) RNA against early endosome component, Rab5, or multivesicular body sorting protein, hrs, reduced the levels of Notch activation (Figure 3A,B). This is consistent with the in vivo studies indicating that ligand-independent Notch activation relies heavily on receptor trafficking (Hori et al., 2012) (Rab5 p = 0.00623, hrs p = 0.0159), and our observation that Notch accumulates in Dl-/Dl- clones (Figure 3—figure supplement 2). A requirement for trafficking is consistent with the results of others who have demonstrated aberrant Notch activation in follicle cell mutants for trafficking components (Wilkin et al., 2004; Vaccari et al., 2008; Schneider et al., 2013), such as tsg101 mutant clones, which show early Notch activation in the follicle cells (Figure 3—figure supplement 3). Furthermore, co-transfecting pMT-NFL with pMT-GAL4 and pUASt-Serdel3, a form of Ser that cannot activate Notch, but only cis-inhibit, (Fleming et al., 2013) almost entirely abolished the Notch activation detected when NFL was transfected alone (p = 0.0048) (Figure 3C). These results suggest that if Notch is expressed in a cell free of cis- and trans-ligands, DSL ligand-independent activity will occur and that cis-inhibition is extremely efficient in preventing this ‘accidental’ Notch activity as it travels through the endosomal pathway en route to degradation.10.7554/eLife.04415.008Figure 3.DSL-ligand-independent Notch activity in S2 cells is buffered by cis-ligand.

Bottom Line: Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling.Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling.We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, United States.

ABSTRACT
The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a twofold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. We show that cells without both cis- and trans-ligands can mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis- and trans-ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically induced situations of elevated trans-ligand-independent Notch signaling. We conclude that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand-independent activation, and propose a new function of cis-inhibition which buffers cells against accidental Notch activity.

Show MeSH
Related in: MedlinePlus