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On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development.

Bachmann A, Draga M, Grawe F, Knust E - BMC Dev. Biol. (2008)

Bottom Line: Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains.Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. bachmana@uni-duesseldorf.de

ABSTRACT

Background: Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes.

Results: Here we show by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.

Conclusion: Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

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Vari function in the developing eye. (A, A') Third instar wild-type eye imaginal discs stained with anti-Vari (green). Vari is predominantly expressed behind the morphogenetic furrow (white arrow in A) in the developing ommatidia. The close-up view on ommatidia of the 4-cone cell stage (A') demonstrates that Vari localises to the lateral membranes of the photoreceptor and cone cells. (B, B') Close-up views of ommatidia, stained with antibodies against Cora (B, green) and NrxIV (B', red), respectively. Cora and NrxIV are expressed in the photoreceptor and cone cells. (C-C') Head of a wild-type fly (C) and a fly overexpressing vari-RNAi with eyGal4 (C'). Reduction of vari induces roughening, downsizing and malformation of the eye. (D-D') Eye imaginal discs of third instar wild-type larvae (D) and larvae overexpressing vari-RNAi with eyGal4 (D'), stained with anti-Futsch/22C10 antibody to mark the photoreceptor cells. Reduction of vari function (D') leads to aberrant folding of the eye imaginal disc and additional tissue formation at the antennal disc (arrow). Black arrowheads mark the position of the morphogenetic furrow in D. bw = Bolwig's nerve, ad = antennal disc, ed = eye disc. (E-E") X-Gal staining of eyGal4; Gbe+Su(H)lacZ (E) and eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi (E') third instar eye imaginal discs to visualize activity of the Notch signaling pathway. Despite morphological aberrations Notch activity does not seem to be strikingly affected by RNAi-mediated knockdown of vari. eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi eye imaginal discs from second instar larvae already display abnormal folding (E", arrow). The eye imaginal disc in E" is shown in a higher magnification than those in E and E'. (F, F') Semi-thin sections of eyes from wild-type flies (F) and flies overexpressing vari-RNAi with eyGal4 (F'). In ommatidia with reduced vari function (F') the seven photoreceptor cells visible do not exhibit major morphogenetic defects and are still organised in a trapezoid pattern as in wild-type (F). In A, D and E anterior is left.
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Figure 8: Vari function in the developing eye. (A, A') Third instar wild-type eye imaginal discs stained with anti-Vari (green). Vari is predominantly expressed behind the morphogenetic furrow (white arrow in A) in the developing ommatidia. The close-up view on ommatidia of the 4-cone cell stage (A') demonstrates that Vari localises to the lateral membranes of the photoreceptor and cone cells. (B, B') Close-up views of ommatidia, stained with antibodies against Cora (B, green) and NrxIV (B', red), respectively. Cora and NrxIV are expressed in the photoreceptor and cone cells. (C-C') Head of a wild-type fly (C) and a fly overexpressing vari-RNAi with eyGal4 (C'). Reduction of vari induces roughening, downsizing and malformation of the eye. (D-D') Eye imaginal discs of third instar wild-type larvae (D) and larvae overexpressing vari-RNAi with eyGal4 (D'), stained with anti-Futsch/22C10 antibody to mark the photoreceptor cells. Reduction of vari function (D') leads to aberrant folding of the eye imaginal disc and additional tissue formation at the antennal disc (arrow). Black arrowheads mark the position of the morphogenetic furrow in D. bw = Bolwig's nerve, ad = antennal disc, ed = eye disc. (E-E") X-Gal staining of eyGal4; Gbe+Su(H)lacZ (E) and eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi (E') third instar eye imaginal discs to visualize activity of the Notch signaling pathway. Despite morphological aberrations Notch activity does not seem to be strikingly affected by RNAi-mediated knockdown of vari. eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi eye imaginal discs from second instar larvae already display abnormal folding (E", arrow). The eye imaginal disc in E" is shown in a higher magnification than those in E and E'. (F, F') Semi-thin sections of eyes from wild-type flies (F) and flies overexpressing vari-RNAi with eyGal4 (F'). In ommatidia with reduced vari function (F') the seven photoreceptor cells visible do not exhibit major morphogenetic defects and are still organised in a trapezoid pattern as in wild-type (F). In A, D and E anterior is left.

Mentions: The data show that varicose has an essential function during development of the embryo. Vari protein is also expressed at later stages, in the wing and the eye imaginal discs. In the wing imaginal disc, Vari is expressed throughout the disc epithelium, where it localises basal to Crumbs, which marks the apical pole (Fig. 7A,B). In the eye disc of third instar larvae, there is enriched expression of Vari behind the morphogenetic furrow (Fig. 8A). NrxIV and Coracle, two other components of the septate junction, exhibit the same expression pattern (8B, B') [27], suggesting that Vari is associated with SJ in differentiating ommatidia (Fig. 8A). In both the eye and the wing discs, both isoforms could be detected, while ovaries expressed only Vari-short, but not Vari-long (Fig. 7F).


On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development.

Bachmann A, Draga M, Grawe F, Knust E - BMC Dev. Biol. (2008)

Vari function in the developing eye. (A, A') Third instar wild-type eye imaginal discs stained with anti-Vari (green). Vari is predominantly expressed behind the morphogenetic furrow (white arrow in A) in the developing ommatidia. The close-up view on ommatidia of the 4-cone cell stage (A') demonstrates that Vari localises to the lateral membranes of the photoreceptor and cone cells. (B, B') Close-up views of ommatidia, stained with antibodies against Cora (B, green) and NrxIV (B', red), respectively. Cora and NrxIV are expressed in the photoreceptor and cone cells. (C-C') Head of a wild-type fly (C) and a fly overexpressing vari-RNAi with eyGal4 (C'). Reduction of vari induces roughening, downsizing and malformation of the eye. (D-D') Eye imaginal discs of third instar wild-type larvae (D) and larvae overexpressing vari-RNAi with eyGal4 (D'), stained with anti-Futsch/22C10 antibody to mark the photoreceptor cells. Reduction of vari function (D') leads to aberrant folding of the eye imaginal disc and additional tissue formation at the antennal disc (arrow). Black arrowheads mark the position of the morphogenetic furrow in D. bw = Bolwig's nerve, ad = antennal disc, ed = eye disc. (E-E") X-Gal staining of eyGal4; Gbe+Su(H)lacZ (E) and eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi (E') third instar eye imaginal discs to visualize activity of the Notch signaling pathway. Despite morphological aberrations Notch activity does not seem to be strikingly affected by RNAi-mediated knockdown of vari. eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi eye imaginal discs from second instar larvae already display abnormal folding (E", arrow). The eye imaginal disc in E" is shown in a higher magnification than those in E and E'. (F, F') Semi-thin sections of eyes from wild-type flies (F) and flies overexpressing vari-RNAi with eyGal4 (F'). In ommatidia with reduced vari function (F') the seven photoreceptor cells visible do not exhibit major morphogenetic defects and are still organised in a trapezoid pattern as in wild-type (F). In A, D and E anterior is left.
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Figure 8: Vari function in the developing eye. (A, A') Third instar wild-type eye imaginal discs stained with anti-Vari (green). Vari is predominantly expressed behind the morphogenetic furrow (white arrow in A) in the developing ommatidia. The close-up view on ommatidia of the 4-cone cell stage (A') demonstrates that Vari localises to the lateral membranes of the photoreceptor and cone cells. (B, B') Close-up views of ommatidia, stained with antibodies against Cora (B, green) and NrxIV (B', red), respectively. Cora and NrxIV are expressed in the photoreceptor and cone cells. (C-C') Head of a wild-type fly (C) and a fly overexpressing vari-RNAi with eyGal4 (C'). Reduction of vari induces roughening, downsizing and malformation of the eye. (D-D') Eye imaginal discs of third instar wild-type larvae (D) and larvae overexpressing vari-RNAi with eyGal4 (D'), stained with anti-Futsch/22C10 antibody to mark the photoreceptor cells. Reduction of vari function (D') leads to aberrant folding of the eye imaginal disc and additional tissue formation at the antennal disc (arrow). Black arrowheads mark the position of the morphogenetic furrow in D. bw = Bolwig's nerve, ad = antennal disc, ed = eye disc. (E-E") X-Gal staining of eyGal4; Gbe+Su(H)lacZ (E) and eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi (E') third instar eye imaginal discs to visualize activity of the Notch signaling pathway. Despite morphological aberrations Notch activity does not seem to be strikingly affected by RNAi-mediated knockdown of vari. eyGal4; Gbe+Su(H)lacZ>UAS vari-RNAi eye imaginal discs from second instar larvae already display abnormal folding (E", arrow). The eye imaginal disc in E" is shown in a higher magnification than those in E and E'. (F, F') Semi-thin sections of eyes from wild-type flies (F) and flies overexpressing vari-RNAi with eyGal4 (F'). In ommatidia with reduced vari function (F') the seven photoreceptor cells visible do not exhibit major morphogenetic defects and are still organised in a trapezoid pattern as in wild-type (F). In A, D and E anterior is left.
Mentions: The data show that varicose has an essential function during development of the embryo. Vari protein is also expressed at later stages, in the wing and the eye imaginal discs. In the wing imaginal disc, Vari is expressed throughout the disc epithelium, where it localises basal to Crumbs, which marks the apical pole (Fig. 7A,B). In the eye disc of third instar larvae, there is enriched expression of Vari behind the morphogenetic furrow (Fig. 8A). NrxIV and Coracle, two other components of the septate junction, exhibit the same expression pattern (8B, B') [27], suggesting that Vari is associated with SJ in differentiating ommatidia (Fig. 8A). In both the eye and the wing discs, both isoforms could be detected, while ovaries expressed only Vari-short, but not Vari-long (Fig. 7F).

Bottom Line: Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains.Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. bachmana@uni-duesseldorf.de

ABSTRACT

Background: Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes.

Results: Here we show by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes.

Conclusion: Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

Show MeSH
Related in: MedlinePlus