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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

Example illustrating the quantification method developed to compare changes in Armadillo protein levels in the nucleus across experiments.(A, A′, A″) UAS Myr-ΔNArm1–155 is overexpressed in the dppGAL4 domain, which drives expression in a stripe at the A/P boundary. Red, green, and blue channels; representing (A) endogenous Armadillo, (A′) E-Cadherin-GFP under a ubiquitous promoter, and (A″) Myr-ΔNArm1–155, respectively; are assessed separately from the same confocal section, here through the cytoplasm approximately 10% below the AJ (60×2 magnification). The red, green and blue spots represent the 10 data points selected from which to calculate median levels within the domain of expression (B, B′, B″). The white spots highlight the data points outside the domain of expression used to remove “background noise”, as the nuclei are expected to have zero pixel intensity here. Thus for each channel, p1 is calculated as μ2 subtracted from μ1, and normalised to a maximal pixel intensity of 255.
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pone-0002893-g003: Example illustrating the quantification method developed to compare changes in Armadillo protein levels in the nucleus across experiments.(A, A′, A″) UAS Myr-ΔNArm1–155 is overexpressed in the dppGAL4 domain, which drives expression in a stripe at the A/P boundary. Red, green, and blue channels; representing (A) endogenous Armadillo, (A′) E-Cadherin-GFP under a ubiquitous promoter, and (A″) Myr-ΔNArm1–155, respectively; are assessed separately from the same confocal section, here through the cytoplasm approximately 10% below the AJ (60×2 magnification). The red, green and blue spots represent the 10 data points selected from which to calculate median levels within the domain of expression (B, B′, B″). The white spots highlight the data points outside the domain of expression used to remove “background noise”, as the nuclei are expected to have zero pixel intensity here. Thus for each channel, p1 is calculated as μ2 subtracted from μ1, and normalised to a maximal pixel intensity of 255.

Mentions: Since the cytoplasm is difficult to distinguish from the basolateral membrane in wing discs, and the section will include a large fraction composed of the nucleus, an additional method was used to help clarify changes in levels and subcellular location of proteins (Figure 3). For both the basolateral membrane and the nucleus, 10 points were chosen randomly both in flanking wild type cells and in the central or lateral parts of the expression domain. As with the AJ, a ratio of median pixel intensity values of expression domain over wild type cells was used to quantify changes in Armadillo and E-Cadherin levels. In the case of the construct, “background values” from the wild type domain were subtracted from levels in the expression domain to remove noise from the dataset. The relative intensity of the construct in the lateral and central domains can then be compared. As E-Cadherin does not enter the nucleus (not shown), it was omitted from the nuclear analysis. When levels calculated for the “basolateral” and “nuclear” fractions deviate appreciably from the “cellular” component, it may be possible to infer changes in the cytoplasmic levels of proteins (see results).


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)

Example illustrating the quantification method developed to compare changes in Armadillo protein levels in the nucleus across experiments.(A, A′, A″) UAS Myr-ΔNArm1–155 is overexpressed in the dppGAL4 domain, which drives expression in a stripe at the A/P boundary. Red, green, and blue channels; representing (A) endogenous Armadillo, (A′) E-Cadherin-GFP under a ubiquitous promoter, and (A″) Myr-ΔNArm1–155, respectively; are assessed separately from the same confocal section, here through the cytoplasm approximately 10% below the AJ (60×2 magnification). The red, green and blue spots represent the 10 data points selected from which to calculate median levels within the domain of expression (B, B′, B″). The white spots highlight the data points outside the domain of expression used to remove “background noise”, as the nuclei are expected to have zero pixel intensity here. Thus for each channel, p1 is calculated as μ2 subtracted from μ1, and normalised to a maximal pixel intensity of 255.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002893-g003: Example illustrating the quantification method developed to compare changes in Armadillo protein levels in the nucleus across experiments.(A, A′, A″) UAS Myr-ΔNArm1–155 is overexpressed in the dppGAL4 domain, which drives expression in a stripe at the A/P boundary. Red, green, and blue channels; representing (A) endogenous Armadillo, (A′) E-Cadherin-GFP under a ubiquitous promoter, and (A″) Myr-ΔNArm1–155, respectively; are assessed separately from the same confocal section, here through the cytoplasm approximately 10% below the AJ (60×2 magnification). The red, green and blue spots represent the 10 data points selected from which to calculate median levels within the domain of expression (B, B′, B″). The white spots highlight the data points outside the domain of expression used to remove “background noise”, as the nuclei are expected to have zero pixel intensity here. Thus for each channel, p1 is calculated as μ2 subtracted from μ1, and normalised to a maximal pixel intensity of 255.
Mentions: Since the cytoplasm is difficult to distinguish from the basolateral membrane in wing discs, and the section will include a large fraction composed of the nucleus, an additional method was used to help clarify changes in levels and subcellular location of proteins (Figure 3). For both the basolateral membrane and the nucleus, 10 points were chosen randomly both in flanking wild type cells and in the central or lateral parts of the expression domain. As with the AJ, a ratio of median pixel intensity values of expression domain over wild type cells was used to quantify changes in Armadillo and E-Cadherin levels. In the case of the construct, “background values” from the wild type domain were subtracted from levels in the expression domain to remove noise from the dataset. The relative intensity of the construct in the lateral and central domains can then be compared. As E-Cadherin does not enter the nucleus (not shown), it was omitted from the nuclear analysis. When levels calculated for the “basolateral” and “nuclear” fractions deviate appreciably from the “cellular” component, it may be possible to infer changes in the cytoplasmic levels of proteins (see results).

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