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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 at the level of the AJ across experiments.(A, A′, A″) UAS ArmS10 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″) ArmS10, respectively; are assessed separately from the same confocal section, here at the level of the AJ (60×2 magnification). The coloured lines through the images represent the cross-section at which intensity levels were measured. (B, B′, B″) Using NIH ImageJ software, a histogram is produced in which pixel intensity for each pixel is calculated across the confocal section for each channel. Median values are calculated from both wild type tissue (μ1) and the expression domains (μ2). (B, B′) μ1 is used as the baseline value for endogenous protein levels, and is used to set the proportion of protein in the AJ at p1 = 1. The proportion p2 of junctional protein in the expression domain is then calculated as the median value μ2/μ1 and is a fraction of p1. (B″) p1 is set to 0 as no protein is expected outside of the expression domain, while p2 is set to 1 as it is assumed that the maximal amount of ArmS10 will reside in the junction within the expression domain. This allows a distinction between zones of high and low expression levels, the latter being a fraction of p2, such that changes in endogenous protein levels can be monitored (not shown).
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pone-0002893-g002: Example illustrating the quantification method developed to compare changes in Armadillo protein levels at the level of the AJ across experiments.(A, A′, A″) UAS ArmS10 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″) ArmS10, respectively; are assessed separately from the same confocal section, here at the level of the AJ (60×2 magnification). The coloured lines through the images represent the cross-section at which intensity levels were measured. (B, B′, B″) Using NIH ImageJ software, a histogram is produced in which pixel intensity for each pixel is calculated across the confocal section for each channel. Median values are calculated from both wild type tissue (μ1) and the expression domains (μ2). (B, B′) μ1 is used as the baseline value for endogenous protein levels, and is used to set the proportion of protein in the AJ at p1 = 1. The proportion p2 of junctional protein in the expression domain is then calculated as the median value μ2/μ1 and is a fraction of p1. (B″) p1 is set to 0 as no protein is expected outside of the expression domain, while p2 is set to 1 as it is assumed that the maximal amount of ArmS10 will reside in the junction within the expression domain. This allows a distinction between zones of high and low expression levels, the latter being a fraction of p2, such that changes in endogenous protein levels can be monitored (not shown).

Mentions: To illustrate the method, an example of the quantification system used at the level of the AJ in a wing disc overexpressing UAS ArmS10 is presented in Figure 2. Confocal images were separated into the component red, green and blue channels (Figure 2A, A′ and A″). A longitudinal section across the dppGAL4 domain was chosen, and pixel intensity (gray value) plotted against the distance along the X-axis (pixels). Thus, for each channel it was possible to generate a profile plot in which endogenous Armadillo, E-Cadherin and construct levels were assessed (Figure 2B, B′ and B″). In the case of Armadillo and E-Cadherin, levels in the “wild type” cells outside the domain of expression were used as controls with which to compare changes in levels within the dppGAL4 expression stripe. The expression domain was also separated into “central” and “lateral” domains representing high and low level dpp expression, respectively (not shown). The median intensity value for “wild type” and “expression domain” was then calculated for endogenous Armadillo, E-Cadherin and the construct. Median pixel intensity values were used to represent protein levels instead of mean values as the latter are affected by both outliers and departure from normality.


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 at the level of the AJ across experiments.(A, A′, A″) UAS ArmS10 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″) ArmS10, respectively; are assessed separately from the same confocal section, here at the level of the AJ (60×2 magnification). The coloured lines through the images represent the cross-section at which intensity levels were measured. (B, B′, B″) Using NIH ImageJ software, a histogram is produced in which pixel intensity for each pixel is calculated across the confocal section for each channel. Median values are calculated from both wild type tissue (μ1) and the expression domains (μ2). (B, B′) μ1 is used as the baseline value for endogenous protein levels, and is used to set the proportion of protein in the AJ at p1 = 1. The proportion p2 of junctional protein in the expression domain is then calculated as the median value μ2/μ1 and is a fraction of p1. (B″) p1 is set to 0 as no protein is expected outside of the expression domain, while p2 is set to 1 as it is assumed that the maximal amount of ArmS10 will reside in the junction within the expression domain. This allows a distinction between zones of high and low expression levels, the latter being a fraction of p2, such that changes in endogenous protein levels can be monitored (not shown).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002893-g002: Example illustrating the quantification method developed to compare changes in Armadillo protein levels at the level of the AJ across experiments.(A, A′, A″) UAS ArmS10 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″) ArmS10, respectively; are assessed separately from the same confocal section, here at the level of the AJ (60×2 magnification). The coloured lines through the images represent the cross-section at which intensity levels were measured. (B, B′, B″) Using NIH ImageJ software, a histogram is produced in which pixel intensity for each pixel is calculated across the confocal section for each channel. Median values are calculated from both wild type tissue (μ1) and the expression domains (μ2). (B, B′) μ1 is used as the baseline value for endogenous protein levels, and is used to set the proportion of protein in the AJ at p1 = 1. The proportion p2 of junctional protein in the expression domain is then calculated as the median value μ2/μ1 and is a fraction of p1. (B″) p1 is set to 0 as no protein is expected outside of the expression domain, while p2 is set to 1 as it is assumed that the maximal amount of ArmS10 will reside in the junction within the expression domain. This allows a distinction between zones of high and low expression levels, the latter being a fraction of p2, such that changes in endogenous protein levels can be monitored (not shown).
Mentions: To illustrate the method, an example of the quantification system used at the level of the AJ in a wing disc overexpressing UAS ArmS10 is presented in Figure 2. Confocal images were separated into the component red, green and blue channels (Figure 2A, A′ and A″). A longitudinal section across the dppGAL4 domain was chosen, and pixel intensity (gray value) plotted against the distance along the X-axis (pixels). Thus, for each channel it was possible to generate a profile plot in which endogenous Armadillo, E-Cadherin and construct levels were assessed (Figure 2B, B′ and B″). In the case of Armadillo and E-Cadherin, levels in the “wild type” cells outside the domain of expression were used as controls with which to compare changes in levels within the dppGAL4 expression stripe. The expression domain was also separated into “central” and “lateral” domains representing high and low level dpp expression, respectively (not shown). The median intensity value for “wild type” and “expression domain” was then calculated for endogenous Armadillo, E-Cadherin and the construct. Median pixel intensity values were used to represent protein levels instead of mean values as the latter are affected by both outliers and departure from normality.

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