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Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila.

Gluderer S, Brunner E, Germann M, Jovaisaite V, Li C, Rentsch CA, Hafen E, Stocker H - J. Biol. (2010)

Bottom Line: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function.The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved.Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

ABSTRACT

Background: The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-beta1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure.

Results: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth.

Conclusions: The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

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Co-overexpression of Madm and bunA causes overgrowth. (a-d) Scanning electron micrographs of adult eyes as a readout for the consequences of overexpression of bunA and Madm under the control of the GMR-Gal4 driver line late during eye development. Whereas expression of (b) bunA or (c) Madm singly does not cause a size alteration compared to the control (a), overexpression of both leads to increased eye size (d). (e) The size increase on bunA and Madm coexpression is due to larger ommatidia (Student's t-test, n = 9, ***p < 0.001). (f-i) The growth-promoting effect of bunA and Madm co-overexpression is also observed in the wing. Single expression of either (g, g') bunA or (h, h') Madm during wing development (by means of C10-Gal4) does not change wing size or curvature visibly. However, their combined expression causes a slight overgrowth of the tissue between the wing veins, resulting in a wavy wing surface and wing bending (i'), manifested as folds between wing veins in (i) (arrows). Genotypes are: (a) y, w; GMR-Gal4/UAS-eGFP; UAS-lacZ/+; (b) y, w; GMR-Gal4/UAS-eGFP; UAS-bunA/+; (c) y, w; GMR-Gal4/UAS-Madm; UAS-lacZ/+; (d) y, w; GMR-Gal4/UAS-Madm; UAS-bunA/+; (f) y, w; UAS-eGFP/+; C10-Gal4/UAS-lacZ; (g) y, w; UAS-eGFP/+; C10-Gal4/UAS-bunA; (h) y, w; UAS-Madm/+; C10-Gal4/UAS-lacZ; (i) y, w; UAS-Madm/+; C10-Gal4/UAS-bunA.
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Figure 5: Co-overexpression of Madm and bunA causes overgrowth. (a-d) Scanning electron micrographs of adult eyes as a readout for the consequences of overexpression of bunA and Madm under the control of the GMR-Gal4 driver line late during eye development. Whereas expression of (b) bunA or (c) Madm singly does not cause a size alteration compared to the control (a), overexpression of both leads to increased eye size (d). (e) The size increase on bunA and Madm coexpression is due to larger ommatidia (Student's t-test, n = 9, ***p < 0.001). (f-i) The growth-promoting effect of bunA and Madm co-overexpression is also observed in the wing. Single expression of either (g, g') bunA or (h, h') Madm during wing development (by means of C10-Gal4) does not change wing size or curvature visibly. However, their combined expression causes a slight overgrowth of the tissue between the wing veins, resulting in a wavy wing surface and wing bending (i'), manifested as folds between wing veins in (i) (arrows). Genotypes are: (a) y, w; GMR-Gal4/UAS-eGFP; UAS-lacZ/+; (b) y, w; GMR-Gal4/UAS-eGFP; UAS-bunA/+; (c) y, w; GMR-Gal4/UAS-Madm; UAS-lacZ/+; (d) y, w; GMR-Gal4/UAS-Madm; UAS-bunA/+; (f) y, w; UAS-eGFP/+; C10-Gal4/UAS-lacZ; (g) y, w; UAS-eGFP/+; C10-Gal4/UAS-bunA; (h) y, w; UAS-Madm/+; C10-Gal4/UAS-lacZ; (i) y, w; UAS-Madm/+; C10-Gal4/UAS-bunA.

Mentions: Madm is a growth-promoting gene producing phenotypes reminiscent of bunA phenotypes and its gene product physically interacts with BunA. It is thus conceivable that the two proteins participate in the same complex to enhance growth. We tested for dominant genetic interactions between Madm and bunA in vivo. However, we did not detect dominant interactions in hypomorphic mutant tissues or flies (data not shown). Thus, we hypothesized that Madm and BunA form a molecular complex and, as a consequence, the phenotype of the limiting complex component is displayed. This hypothesis also implies that overexpression of Madm or BunA alone would not be sufficient to enhance the activity of the complex. As previously reported, overexpression of bunA from a UAS-bunA construct did not produce any overgrowth phenotypes, unless co-overexpressed with dS6K in a sensitized system in the wing [12] (Figure 5b, g). Similarly, with a UAS-Madm transgenic line, no obvious overgrowth phenotypes were observed (Figure 5c, h; Madm overexpression caused patterning defects, Materials and methods). However, co-overexpression of bunA and Madm by means of GMR-Gal4 resulted in larger eyes due to larger ommatidia (Figure 5d, e). Consistently, co-overexpression of UAS-Madm together with UAS-bunA using a wing driver (C10-Gal4) caused an overgrowth phenotype in the wing (Figure 5i, j). We observed additional tissue between the wing veins, resulting in crinkled wings. Thus, Madm and BunA cooperate to increase organ growth when overexpressed during eye and wing development.


Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila.

Gluderer S, Brunner E, Germann M, Jovaisaite V, Li C, Rentsch CA, Hafen E, Stocker H - J. Biol. (2010)

Co-overexpression of Madm and bunA causes overgrowth. (a-d) Scanning electron micrographs of adult eyes as a readout for the consequences of overexpression of bunA and Madm under the control of the GMR-Gal4 driver line late during eye development. Whereas expression of (b) bunA or (c) Madm singly does not cause a size alteration compared to the control (a), overexpression of both leads to increased eye size (d). (e) The size increase on bunA and Madm coexpression is due to larger ommatidia (Student's t-test, n = 9, ***p < 0.001). (f-i) The growth-promoting effect of bunA and Madm co-overexpression is also observed in the wing. Single expression of either (g, g') bunA or (h, h') Madm during wing development (by means of C10-Gal4) does not change wing size or curvature visibly. However, their combined expression causes a slight overgrowth of the tissue between the wing veins, resulting in a wavy wing surface and wing bending (i'), manifested as folds between wing veins in (i) (arrows). Genotypes are: (a) y, w; GMR-Gal4/UAS-eGFP; UAS-lacZ/+; (b) y, w; GMR-Gal4/UAS-eGFP; UAS-bunA/+; (c) y, w; GMR-Gal4/UAS-Madm; UAS-lacZ/+; (d) y, w; GMR-Gal4/UAS-Madm; UAS-bunA/+; (f) y, w; UAS-eGFP/+; C10-Gal4/UAS-lacZ; (g) y, w; UAS-eGFP/+; C10-Gal4/UAS-bunA; (h) y, w; UAS-Madm/+; C10-Gal4/UAS-lacZ; (i) y, w; UAS-Madm/+; C10-Gal4/UAS-bunA.
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Figure 5: Co-overexpression of Madm and bunA causes overgrowth. (a-d) Scanning electron micrographs of adult eyes as a readout for the consequences of overexpression of bunA and Madm under the control of the GMR-Gal4 driver line late during eye development. Whereas expression of (b) bunA or (c) Madm singly does not cause a size alteration compared to the control (a), overexpression of both leads to increased eye size (d). (e) The size increase on bunA and Madm coexpression is due to larger ommatidia (Student's t-test, n = 9, ***p < 0.001). (f-i) The growth-promoting effect of bunA and Madm co-overexpression is also observed in the wing. Single expression of either (g, g') bunA or (h, h') Madm during wing development (by means of C10-Gal4) does not change wing size or curvature visibly. However, their combined expression causes a slight overgrowth of the tissue between the wing veins, resulting in a wavy wing surface and wing bending (i'), manifested as folds between wing veins in (i) (arrows). Genotypes are: (a) y, w; GMR-Gal4/UAS-eGFP; UAS-lacZ/+; (b) y, w; GMR-Gal4/UAS-eGFP; UAS-bunA/+; (c) y, w; GMR-Gal4/UAS-Madm; UAS-lacZ/+; (d) y, w; GMR-Gal4/UAS-Madm; UAS-bunA/+; (f) y, w; UAS-eGFP/+; C10-Gal4/UAS-lacZ; (g) y, w; UAS-eGFP/+; C10-Gal4/UAS-bunA; (h) y, w; UAS-Madm/+; C10-Gal4/UAS-lacZ; (i) y, w; UAS-Madm/+; C10-Gal4/UAS-bunA.
Mentions: Madm is a growth-promoting gene producing phenotypes reminiscent of bunA phenotypes and its gene product physically interacts with BunA. It is thus conceivable that the two proteins participate in the same complex to enhance growth. We tested for dominant genetic interactions between Madm and bunA in vivo. However, we did not detect dominant interactions in hypomorphic mutant tissues or flies (data not shown). Thus, we hypothesized that Madm and BunA form a molecular complex and, as a consequence, the phenotype of the limiting complex component is displayed. This hypothesis also implies that overexpression of Madm or BunA alone would not be sufficient to enhance the activity of the complex. As previously reported, overexpression of bunA from a UAS-bunA construct did not produce any overgrowth phenotypes, unless co-overexpressed with dS6K in a sensitized system in the wing [12] (Figure 5b, g). Similarly, with a UAS-Madm transgenic line, no obvious overgrowth phenotypes were observed (Figure 5c, h; Madm overexpression caused patterning defects, Materials and methods). However, co-overexpression of bunA and Madm by means of GMR-Gal4 resulted in larger eyes due to larger ommatidia (Figure 5d, e). Consistently, co-overexpression of UAS-Madm together with UAS-bunA using a wing driver (C10-Gal4) caused an overgrowth phenotype in the wing (Figure 5i, j). We observed additional tissue between the wing veins, resulting in crinkled wings. Thus, Madm and BunA cooperate to increase organ growth when overexpressed during eye and wing development.

Bottom Line: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function.The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved.Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland.

ABSTRACT

Background: The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-beta1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure.

Results: In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth.

Conclusions: The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm.

Show MeSH
Related in: MedlinePlus