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Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

Somorjai IM, Martinez-Arias A - PLoS ONE (2008)

Bottom Line: Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification.Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling.Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom. ildiko.somorjai@obs-banyuls.fr

ABSTRACT

Background: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets.

Methodology/principal findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin.

Conclusions/significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

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Related in: MedlinePlus

Distribution of Armadillo-GFP relative to basolateral markers.Armadillo-GFP (ArmGFP) and E-CadherinGFP (E-CadGFP) are expressed under endogenous Armadillo ubiquitous promoters, respectively. (A–C) Distribution of ArmGFP (A) and E-CadGFP (B) at AJs (red channels) are coincident; Wingless (Wg) and Patched (Ptc) outline the D/V and A/P boundaries, respectively, which are characterised by aligned cells (arrowheads, blue channel). (C) Mitotic cells express ArmGFP at the AJs, but lack septate junction markers immediately basally (red arrows, Scribble Scrb shown). (D) E-Cadherin (E-Cad, blue) and ArmGFP colocalise at membranes and in puncta (red, blue and white oultines). (E) Fasciclin III (FasIII, blue) crisply and exclusively marks basolateral membranes, with which ArmGFP puncta are closely associated (compare red and blue channels). The downregulation of FasIII at the A/P boundary (blue arrowheads) clearly outlines the ArmGFP puncta there (outlined in red).
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pone-0002893-g005: Distribution of Armadillo-GFP relative to basolateral markers.Armadillo-GFP (ArmGFP) and E-CadherinGFP (E-CadGFP) are expressed under endogenous Armadillo ubiquitous promoters, respectively. (A–C) Distribution of ArmGFP (A) and E-CadGFP (B) at AJs (red channels) are coincident; Wingless (Wg) and Patched (Ptc) outline the D/V and A/P boundaries, respectively, which are characterised by aligned cells (arrowheads, blue channel). (C) Mitotic cells express ArmGFP at the AJs, but lack septate junction markers immediately basally (red arrows, Scribble Scrb shown). (D) E-Cadherin (E-Cad, blue) and ArmGFP colocalise at membranes and in puncta (red, blue and white oultines). (E) Fasciclin III (FasIII, blue) crisply and exclusively marks basolateral membranes, with which ArmGFP puncta are closely associated (compare red and blue channels). The downregulation of FasIII at the A/P boundary (blue arrowheads) clearly outlines the ArmGFP puncta there (outlined in red).

Mentions: At 0%, Armadillo clearly outlines the AJs, showing the arrangement of the imaginal cells with respect to the D/V and A/P axes from early third instar (Figure 4C′). This alignment of cells at the boundaries corresponds to domains of Wingless and Hedgehog signalling (Figure 5A and B), though by late third instar, the increase in cell number makes it difficult to perceive the delineation of cells along the A/P axis. However, by this stage high levels of Armadillo can be observed in two stripes at the D/V boundary (Figure 4F′) on either side of the Wingless expression domain (not shown).


Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

Somorjai IM, Martinez-Arias A - PLoS ONE (2008)

Distribution of Armadillo-GFP relative to basolateral markers.Armadillo-GFP (ArmGFP) and E-CadherinGFP (E-CadGFP) are expressed under endogenous Armadillo ubiquitous promoters, respectively. (A–C) Distribution of ArmGFP (A) and E-CadGFP (B) at AJs (red channels) are coincident; Wingless (Wg) and Patched (Ptc) outline the D/V and A/P boundaries, respectively, which are characterised by aligned cells (arrowheads, blue channel). (C) Mitotic cells express ArmGFP at the AJs, but lack septate junction markers immediately basally (red arrows, Scribble Scrb shown). (D) E-Cadherin (E-Cad, blue) and ArmGFP colocalise at membranes and in puncta (red, blue and white oultines). (E) Fasciclin III (FasIII, blue) crisply and exclusively marks basolateral membranes, with which ArmGFP puncta are closely associated (compare red and blue channels). The downregulation of FasIII at the A/P boundary (blue arrowheads) clearly outlines the ArmGFP puncta there (outlined in red).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2483348&req=5

pone-0002893-g005: Distribution of Armadillo-GFP relative to basolateral markers.Armadillo-GFP (ArmGFP) and E-CadherinGFP (E-CadGFP) are expressed under endogenous Armadillo ubiquitous promoters, respectively. (A–C) Distribution of ArmGFP (A) and E-CadGFP (B) at AJs (red channels) are coincident; Wingless (Wg) and Patched (Ptc) outline the D/V and A/P boundaries, respectively, which are characterised by aligned cells (arrowheads, blue channel). (C) Mitotic cells express ArmGFP at the AJs, but lack septate junction markers immediately basally (red arrows, Scribble Scrb shown). (D) E-Cadherin (E-Cad, blue) and ArmGFP colocalise at membranes and in puncta (red, blue and white oultines). (E) Fasciclin III (FasIII, blue) crisply and exclusively marks basolateral membranes, with which ArmGFP puncta are closely associated (compare red and blue channels). The downregulation of FasIII at the A/P boundary (blue arrowheads) clearly outlines the ArmGFP puncta there (outlined in red).
Mentions: At 0%, Armadillo clearly outlines the AJs, showing the arrangement of the imaginal cells with respect to the D/V and A/P axes from early third instar (Figure 4C′). This alignment of cells at the boundaries corresponds to domains of Wingless and Hedgehog signalling (Figure 5A and B), though by late third instar, the increase in cell number makes it difficult to perceive the delineation of cells along the A/P axis. However, by this stage high levels of Armadillo can be observed in two stripes at the D/V boundary (Figure 4F′) on either side of the Wingless expression domain (not shown).

Bottom Line: Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification.Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling.Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom. ildiko.somorjai@obs-banyuls.fr

ABSTRACT

Background: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets.

Methodology/principal findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin.

Conclusions/significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

Show MeSH
Related in: MedlinePlus