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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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Varicose is required for correct tracheal tube and epidermis formation. (A) Cuticle preparations of variMD109 mutant embryos exhibit convoluted tracheae (white arrows). (B, B') Wild-type embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari localises at the SJ, basal of Crb. Wild-type tracheae appear straight in contrast to the convoluted tracheae in A. (C) variMD109 mutant embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari is lost from the tracheae and the epidermis, while apical Crb is not affected. Tracheae appear convoluted. (D) Stage 15 embryo with targeted knockdown of vari in the tracheae of embryos by using btlGal4 (btlGal4>UAS vari-RNAi), stained with anti-Vari (green) and anti-Crb (red). Vari is reduced to background levels in the tracheae, but not affected in the epidermis. Apical localisation of Crb is not affected in the tracheae. (E) Dorsal tracheal trunk of a wild-type embryo of stage 15, stained with anti-Coracle (Cora; green) and anti-Crb (red). Cora localises in the SJ, basal to Crb. (F) Dorsal tracheal trunk of a variMD109 mutant embryo of stage 15, stained with anti-Cora (green) and Crb (red). Cora is delocalised to apical and basal sites (white arrows), whereas Crb remains in its apical position. (G) Dorsal tracheal trunk of a Df(3L)BK9 mutant embryo of stage 15, in which the NrxIV locus is deleted, stained with anti-Cora (green) and anti-Crb (red). As in variMD109 mutant embryos, Cora becomes mislocalised to apical and basal positions (white arrows) in the absence of NrxIV, while apical localisation of Crb is not affected. (H) Epidermis of a wild-type embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). Both proteins are co-localised at the SJ. (I) variMD109 mutant embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). The amount of Cora is reduced and the remaining Cora protein is mislocalised along the whole lateral membrane. In B-D and H-I apical is up. White dotted lines in H' and I' mark the apical and basal side of the epithelial cells, respectively.
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Figure 3: Varicose is required for correct tracheal tube and epidermis formation. (A) Cuticle preparations of variMD109 mutant embryos exhibit convoluted tracheae (white arrows). (B, B') Wild-type embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari localises at the SJ, basal of Crb. Wild-type tracheae appear straight in contrast to the convoluted tracheae in A. (C) variMD109 mutant embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari is lost from the tracheae and the epidermis, while apical Crb is not affected. Tracheae appear convoluted. (D) Stage 15 embryo with targeted knockdown of vari in the tracheae of embryos by using btlGal4 (btlGal4>UAS vari-RNAi), stained with anti-Vari (green) and anti-Crb (red). Vari is reduced to background levels in the tracheae, but not affected in the epidermis. Apical localisation of Crb is not affected in the tracheae. (E) Dorsal tracheal trunk of a wild-type embryo of stage 15, stained with anti-Coracle (Cora; green) and anti-Crb (red). Cora localises in the SJ, basal to Crb. (F) Dorsal tracheal trunk of a variMD109 mutant embryo of stage 15, stained with anti-Cora (green) and Crb (red). Cora is delocalised to apical and basal sites (white arrows), whereas Crb remains in its apical position. (G) Dorsal tracheal trunk of a Df(3L)BK9 mutant embryo of stage 15, in which the NrxIV locus is deleted, stained with anti-Cora (green) and anti-Crb (red). As in variMD109 mutant embryos, Cora becomes mislocalised to apical and basal positions (white arrows) in the absence of NrxIV, while apical localisation of Crb is not affected. (H) Epidermis of a wild-type embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). Both proteins are co-localised at the SJ. (I) variMD109 mutant embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). The amount of Cora is reduced and the remaining Cora protein is mislocalised along the whole lateral membrane. In B-D and H-I apical is up. White dotted lines in H' and I' mark the apical and basal side of the epithelial cells, respectively.

Mentions: Embryos homozygous mutant for variMD109 or transheterozygous for variMD109/vari03953b died at the end of embryogenesis with convoluted tracheal tubes (Fig. 3A), a phenotype similar to that described for vari03953b [13,26]. Apical-basal polarity was not affected, as revealed by proper apical localisation of Crb (Fig. 3B,C). The same phenotype was achieved by specifically knocking down vari function in the tracheae by expressing vari-RNAi by means of btlGal4 (Fig. 3D). Septate junction components, such as Coracle or Neurexin IV, which are restricted to the lateral membrane in wild-type embryos, became distributed to all membranes in vari mutant embryos, both in the tracheae (Fig. 3E,F) and the epidermis (Fig. 3H,I). This phenotype was reminiscent to that achieved in the absence of Neurexin IV function (Fig. 3G).


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)

Varicose is required for correct tracheal tube and epidermis formation. (A) Cuticle preparations of variMD109 mutant embryos exhibit convoluted tracheae (white arrows). (B, B') Wild-type embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari localises at the SJ, basal of Crb. Wild-type tracheae appear straight in contrast to the convoluted tracheae in A. (C) variMD109 mutant embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari is lost from the tracheae and the epidermis, while apical Crb is not affected. Tracheae appear convoluted. (D) Stage 15 embryo with targeted knockdown of vari in the tracheae of embryos by using btlGal4 (btlGal4>UAS vari-RNAi), stained with anti-Vari (green) and anti-Crb (red). Vari is reduced to background levels in the tracheae, but not affected in the epidermis. Apical localisation of Crb is not affected in the tracheae. (E) Dorsal tracheal trunk of a wild-type embryo of stage 15, stained with anti-Coracle (Cora; green) and anti-Crb (red). Cora localises in the SJ, basal to Crb. (F) Dorsal tracheal trunk of a variMD109 mutant embryo of stage 15, stained with anti-Cora (green) and Crb (red). Cora is delocalised to apical and basal sites (white arrows), whereas Crb remains in its apical position. (G) Dorsal tracheal trunk of a Df(3L)BK9 mutant embryo of stage 15, in which the NrxIV locus is deleted, stained with anti-Cora (green) and anti-Crb (red). As in variMD109 mutant embryos, Cora becomes mislocalised to apical and basal positions (white arrows) in the absence of NrxIV, while apical localisation of Crb is not affected. (H) Epidermis of a wild-type embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). Both proteins are co-localised at the SJ. (I) variMD109 mutant embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). The amount of Cora is reduced and the remaining Cora protein is mislocalised along the whole lateral membrane. In B-D and H-I apical is up. White dotted lines in H' and I' mark the apical and basal side of the epithelial cells, respectively.
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Figure 3: Varicose is required for correct tracheal tube and epidermis formation. (A) Cuticle preparations of variMD109 mutant embryos exhibit convoluted tracheae (white arrows). (B, B') Wild-type embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari localises at the SJ, basal of Crb. Wild-type tracheae appear straight in contrast to the convoluted tracheae in A. (C) variMD109 mutant embryo of stage 15 stained with anti-Vari (green) and anti-Crb (red). Vari is lost from the tracheae and the epidermis, while apical Crb is not affected. Tracheae appear convoluted. (D) Stage 15 embryo with targeted knockdown of vari in the tracheae of embryos by using btlGal4 (btlGal4>UAS vari-RNAi), stained with anti-Vari (green) and anti-Crb (red). Vari is reduced to background levels in the tracheae, but not affected in the epidermis. Apical localisation of Crb is not affected in the tracheae. (E) Dorsal tracheal trunk of a wild-type embryo of stage 15, stained with anti-Coracle (Cora; green) and anti-Crb (red). Cora localises in the SJ, basal to Crb. (F) Dorsal tracheal trunk of a variMD109 mutant embryo of stage 15, stained with anti-Cora (green) and Crb (red). Cora is delocalised to apical and basal sites (white arrows), whereas Crb remains in its apical position. (G) Dorsal tracheal trunk of a Df(3L)BK9 mutant embryo of stage 15, in which the NrxIV locus is deleted, stained with anti-Cora (green) and anti-Crb (red). As in variMD109 mutant embryos, Cora becomes mislocalised to apical and basal positions (white arrows) in the absence of NrxIV, while apical localisation of Crb is not affected. (H) Epidermis of a wild-type embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). Both proteins are co-localised at the SJ. (I) variMD109 mutant embryo of stage 15, stained with anti-Vari (green) and anti-Cora (red). The amount of Cora is reduced and the remaining Cora protein is mislocalised along the whole lateral membrane. In B-D and H-I apical is up. White dotted lines in H' and I' mark the apical and basal side of the epithelial cells, respectively.
Mentions: Embryos homozygous mutant for variMD109 or transheterozygous for variMD109/vari03953b died at the end of embryogenesis with convoluted tracheal tubes (Fig. 3A), a phenotype similar to that described for vari03953b [13,26]. Apical-basal polarity was not affected, as revealed by proper apical localisation of Crb (Fig. 3B,C). The same phenotype was achieved by specifically knocking down vari function in the tracheae by expressing vari-RNAi by means of btlGal4 (Fig. 3D). Septate junction components, such as Coracle or Neurexin IV, which are restricted to the lateral membrane in wild-type embryos, became distributed to all membranes in vari mutant embryos, both in the tracheae (Fig. 3E,F) and the epidermis (Fig. 3H,I). This phenotype was reminiscent to that achieved in the absence of Neurexin IV function (Fig. 3G).

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