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Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry.

Brauchle M, Hansen S, Caussinus E, Lenard A, Ochoa-Espinosa A, Scholz O, Sprecher SG, Plückthun A, Affolter M - Biol Open (2014)

Bottom Line: For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM.Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations.Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland Department of Zoology, University of Fribourg, Chemi du Musée 10, 1700 Fribourg, Switzerland.

No MeSH data available.


Tissue specific expression of a Slmb-anti-GFP-DARPin fusion in Drosophila phenocopies a non-muscle myosin II mutant phenotype.(A) Shown is an embryo with the following genotype: sqhAX3; sqhSqh::GFP. The arrow points to the normal dorsal closure. (B) Shown is an embryo with the following genotype: sqhAX3/Y; sqhSqh::GFP/Gal4; NSlmb-3G86.32/+. The dotted line outlines the “dorsal open” phenotype exposing the amnioserosa. Scale bars are 20 µm.
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f05: Tissue specific expression of a Slmb-anti-GFP-DARPin fusion in Drosophila phenocopies a non-muscle myosin II mutant phenotype.(A) Shown is an embryo with the following genotype: sqhAX3; sqhSqh::GFP. The arrow points to the normal dorsal closure. (B) Shown is an embryo with the following genotype: sqhAX3/Y; sqhSqh::GFP/Gal4; NSlmb-3G86.32/+. The dotted line outlines the “dorsal open” phenotype exposing the amnioserosa. Scale bars are 20 µm.

Mentions: Next, we attempted to degrade an endogenously GFP-tagged protein in Drosophila in order to assess whether we were able to phenocopy the mutant phenotype of the gene of interest. For this purpose, we used a fly line expressing Spaghetti squash (Sqh) (a subunit of the non-muscle myosin II complex) as a GFP fusion protein under the control of its own promoter, hereafter called sqhSqh::GFP, thereby rescuing the sqh mutant (Fig. 5A) (Royou et al., 2004; also see Caussinus et al., 2012). In such embryos, we expressed the F-Box anti-GFP DARPin fusion protein, Slmb-3G86.32, using an amnioserosa-specific GAL4 driver. These flies exhibited a clear phenotype where the lateral epidermis could not close at the dorsal side, similar to what is observed in strong sqh alleles (Young et al., 1993), indicating that the sqhSqh::GFP protein was successfully degraded in the amnioserosa (Fig. 5B). Therefore, mutant phenotypes can be phenocopied using DARPin based reagents.


Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry.

Brauchle M, Hansen S, Caussinus E, Lenard A, Ochoa-Espinosa A, Scholz O, Sprecher SG, Plückthun A, Affolter M - Biol Open (2014)

Tissue specific expression of a Slmb-anti-GFP-DARPin fusion in Drosophila phenocopies a non-muscle myosin II mutant phenotype.(A) Shown is an embryo with the following genotype: sqhAX3; sqhSqh::GFP. The arrow points to the normal dorsal closure. (B) Shown is an embryo with the following genotype: sqhAX3/Y; sqhSqh::GFP/Gal4; NSlmb-3G86.32/+. The dotted line outlines the “dorsal open” phenotype exposing the amnioserosa. Scale bars are 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265764&req=5

f05: Tissue specific expression of a Slmb-anti-GFP-DARPin fusion in Drosophila phenocopies a non-muscle myosin II mutant phenotype.(A) Shown is an embryo with the following genotype: sqhAX3; sqhSqh::GFP. The arrow points to the normal dorsal closure. (B) Shown is an embryo with the following genotype: sqhAX3/Y; sqhSqh::GFP/Gal4; NSlmb-3G86.32/+. The dotted line outlines the “dorsal open” phenotype exposing the amnioserosa. Scale bars are 20 µm.
Mentions: Next, we attempted to degrade an endogenously GFP-tagged protein in Drosophila in order to assess whether we were able to phenocopy the mutant phenotype of the gene of interest. For this purpose, we used a fly line expressing Spaghetti squash (Sqh) (a subunit of the non-muscle myosin II complex) as a GFP fusion protein under the control of its own promoter, hereafter called sqhSqh::GFP, thereby rescuing the sqh mutant (Fig. 5A) (Royou et al., 2004; also see Caussinus et al., 2012). In such embryos, we expressed the F-Box anti-GFP DARPin fusion protein, Slmb-3G86.32, using an amnioserosa-specific GAL4 driver. These flies exhibited a clear phenotype where the lateral epidermis could not close at the dorsal side, similar to what is observed in strong sqh alleles (Young et al., 1993), indicating that the sqhSqh::GFP protein was successfully degraded in the amnioserosa (Fig. 5B). Therefore, mutant phenotypes can be phenocopied using DARPin based reagents.

Bottom Line: For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM.Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations.Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland Department of Zoology, University of Fribourg, Chemi du Musée 10, 1700 Fribourg, Switzerland.

No MeSH data available.