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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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Phalloidin staining reveals a strong F-actin cable at the A/P boundary corresponding with reduced E-Cadherin in ΔNArm1–155 and Myr-ΔNArm1–155, but not in ArmS10.The extent of the overexpression domain is indicated with dimension lines, as assessed by staining (not shown here). (A) Although F-Actin staining clearly indicates the aligned cells at the A/P boundary with ArmS10, no change in levels was observed. (B) In contrast, overexpression of ΔNArm1–155 results in stretching of the A/P cells and an increase of F-Actin (red arrowhead), corresponding to a region of low E-Cadherin expression (blue arrowhead). Note the folding of the epithelium which reveals peripodial membrane cells. (C) Similarly, Myr-ΔNArm1–155 causes stretching and F-Actin accumulation at the A/P boundary where E-Cadherin is lower (red and blue arrowheads), and even appears to induce boundary cell-like behaviour at the anterior extent of mutant overexpression (yellow asterisk). (D) Basally, Myr-ΔNArm1–155 uniquely causes the formation of filopodia, the base of which are associated with F-Actin bundles (inset).
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pone-0002893-g014: Phalloidin staining reveals a strong F-actin cable at the A/P boundary corresponding with reduced E-Cadherin in ΔNArm1–155 and Myr-ΔNArm1–155, but not in ArmS10.The extent of the overexpression domain is indicated with dimension lines, as assessed by staining (not shown here). (A) Although F-Actin staining clearly indicates the aligned cells at the A/P boundary with ArmS10, no change in levels was observed. (B) In contrast, overexpression of ΔNArm1–155 results in stretching of the A/P cells and an increase of F-Actin (red arrowhead), corresponding to a region of low E-Cadherin expression (blue arrowhead). Note the folding of the epithelium which reveals peripodial membrane cells. (C) Similarly, Myr-ΔNArm1–155 causes stretching and F-Actin accumulation at the A/P boundary where E-Cadherin is lower (red and blue arrowheads), and even appears to induce boundary cell-like behaviour at the anterior extent of mutant overexpression (yellow asterisk). (D) Basally, Myr-ΔNArm1–155 uniquely causes the formation of filopodia, the base of which are associated with F-Actin bundles (inset).

Mentions: Recently, the dogma that α-catenin can simultaneously bind the E-Cadherin/β-catenin complex and the cytoskeleton has been challenged [49], [50]. Further, Major and Irvine [57] have reported that actin, in response to Notch signalling, is responsible for cell sorting and shape changes at the D/V boundary in wing imaginal discs. Our results indicating that Armadillo mutants differentially alter the subcellular distribution of endogenous Armadillo and E-Cadherin, in addition to the observation that cells align at the A/P boundary, and that the mutants likely differ in their ability to bind α-catenin (Figure 1), led us to examine the distribution of F-actin. We found that the changes in tension of cells along the A/P boundary upon overexpression of ArmS10, ΔNArm1–155 and Myr-ΔNArm1–155 were correlated with differing levels of actin accumulation both at the AJ (Figure 14), and basally (not shown). In particular, while ArmS10 produced little effect (Figure 14A), both ΔNArm1–155 and Myr-ΔNArm1–155 caused a strong accumulation of actin, as assessed by phalloidin staining, along the A/P boundary. What resembled an actin “cable” corresponded most strongly with reduced E-Cadherin levels in the boundary cells (Figure 14B, C). Further, this phenomenon was also observed at the anterior extent of the Myr-ΔNArm1–155 expression domain, as though cells were adopting a boundary-like fate there (Figure 14C, yellow asterisk).


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)

Phalloidin staining reveals a strong F-actin cable at the A/P boundary corresponding with reduced E-Cadherin in ΔNArm1–155 and Myr-ΔNArm1–155, but not in ArmS10.The extent of the overexpression domain is indicated with dimension lines, as assessed by staining (not shown here). (A) Although F-Actin staining clearly indicates the aligned cells at the A/P boundary with ArmS10, no change in levels was observed. (B) In contrast, overexpression of ΔNArm1–155 results in stretching of the A/P cells and an increase of F-Actin (red arrowhead), corresponding to a region of low E-Cadherin expression (blue arrowhead). Note the folding of the epithelium which reveals peripodial membrane cells. (C) Similarly, Myr-ΔNArm1–155 causes stretching and F-Actin accumulation at the A/P boundary where E-Cadherin is lower (red and blue arrowheads), and even appears to induce boundary cell-like behaviour at the anterior extent of mutant overexpression (yellow asterisk). (D) Basally, Myr-ΔNArm1–155 uniquely causes the formation of filopodia, the base of which are associated with F-Actin bundles (inset).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483348&req=5

pone-0002893-g014: Phalloidin staining reveals a strong F-actin cable at the A/P boundary corresponding with reduced E-Cadherin in ΔNArm1–155 and Myr-ΔNArm1–155, but not in ArmS10.The extent of the overexpression domain is indicated with dimension lines, as assessed by staining (not shown here). (A) Although F-Actin staining clearly indicates the aligned cells at the A/P boundary with ArmS10, no change in levels was observed. (B) In contrast, overexpression of ΔNArm1–155 results in stretching of the A/P cells and an increase of F-Actin (red arrowhead), corresponding to a region of low E-Cadherin expression (blue arrowhead). Note the folding of the epithelium which reveals peripodial membrane cells. (C) Similarly, Myr-ΔNArm1–155 causes stretching and F-Actin accumulation at the A/P boundary where E-Cadherin is lower (red and blue arrowheads), and even appears to induce boundary cell-like behaviour at the anterior extent of mutant overexpression (yellow asterisk). (D) Basally, Myr-ΔNArm1–155 uniquely causes the formation of filopodia, the base of which are associated with F-Actin bundles (inset).
Mentions: Recently, the dogma that α-catenin can simultaneously bind the E-Cadherin/β-catenin complex and the cytoskeleton has been challenged [49], [50]. Further, Major and Irvine [57] have reported that actin, in response to Notch signalling, is responsible for cell sorting and shape changes at the D/V boundary in wing imaginal discs. Our results indicating that Armadillo mutants differentially alter the subcellular distribution of endogenous Armadillo and E-Cadherin, in addition to the observation that cells align at the A/P boundary, and that the mutants likely differ in their ability to bind α-catenin (Figure 1), led us to examine the distribution of F-actin. We found that the changes in tension of cells along the A/P boundary upon overexpression of ArmS10, ΔNArm1–155 and Myr-ΔNArm1–155 were correlated with differing levels of actin accumulation both at the AJ (Figure 14), and basally (not shown). In particular, while ArmS10 produced little effect (Figure 14A), both ΔNArm1–155 and Myr-ΔNArm1–155 caused a strong accumulation of actin, as assessed by phalloidin staining, along the A/P boundary. What resembled an actin “cable” corresponded most strongly with reduced E-Cadherin levels in the boundary cells (Figure 14B, C). Further, this phenomenon was also observed at the anterior extent of the Myr-ΔNArm1–155 expression domain, as though cells were adopting a boundary-like fate there (Figure 14C, yellow asterisk).

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