Article Figures & Tables
Figures
- Fig. 1. Specificity, oligomeric state and affinity of anti-GFP and anti-mCherry DARPins.
(A) ELISA experiments show a high specificity of selected DARPins towards their cognate target and closely related proteins. Off7 is a control DARPin binding to maltose binding protein (MBP), E 3_5 is an unselected DARPin. All bars represent mean values of duplicates, error bars represent standard deviations. (B) Analysis of the oligomeric state by SEC shows that most DARPins are predominantly monomeric. Due to low extinction coefficients at 280 nm for the chromatograms of 2m22 and 2m74 the absorption at 230 nm is shown. Arrows indicate the elution volumes of the molecular weight standard with the respective MWs. (C) Example of FA assays of different DARPins binding to sfGFP. The solid line indicates a fit to a 1:1 binding model. Extracted KDs for all DARPins can be found in Table 1. (D) Example of a kinetic titration SPR experiment of 3G124 binding to GFP. The concentrations of the five DARPin injections are indicated in the graph. Fit to a global 1:1 kinetic titration binding model is indicated in red. Extracted association and dissociation rates and KDs for several DARPins are summarized in Table 1; additional sensograms are shown in supplementary material Fig. S4.
- Fig. 2. Binding of anti-GFP-DARPin-Ruby2 fusions to GFP in HeLa cells.
Shown are HeLa cells transiently overexpressing a DARPin-mRuby2 fusion protein (A–G) (H is an mRuby2-nanobody fusion) together with a GFP version tethered to the plasma membrane (GFP-CVIM, A′–H′). Overlap of fluorescent signal indicates binding of the respective anti-GFP-DARPin-mRuby2 fusion to GFP-CVIM, indicated by the yellow fluorescent signal at the plasma membrane. (A–A″) As expected, the anti-mCherry DARPin 2m22-mRuby2 fusion protein, which does not recognize GFP, is not recruited to the plasma membrane. (B–B″) 3G86.32-mRuby2, (C–C″) 3G168-mRuby2, (D–D″) 3G124-mRuby2 as well as (E–E″) 3G86.1-mRuby2 fusion proteins localize to the plasma membrane where they interact with GFP-CVIM. On the other hand, low affinity (F–F″) 3G61-mRuby2 and (G–G″) 3G146-mRuby2 fusion proteins localize to the cytoplasm and nucleus, indicating that they cannot interact sufficiently with GFP-CVIM anchored in the plasma membrane. (H–H″) Positive control mRuby2-VHH-GFP4 fusion protein localizes to the plasma membrane. Unprimed letters, mRuby2 channel; primed letters, GFP channel; double primed letters, overlay. Scale bars are 20 µm.
- Fig. 3. Binding of anti-mCherry-Darpin-GFP fusions to mCherry in HeLa cells.
Shown are HeLa cells transiently overexpressing GFP (A) or anti-mCherry-GFP fusion proteins (B–E) together with a mCherry version tethered to the plasma membrane (mCherry-CVIM, A′–E′). Overlap of fluorescent signal indicates binding of the respective anti-mCherry-DARPin-GFP fusion to mCherry-CVIM, indicated by the yellow fluorescent signal at the plasma membrane. (A–A″) As expected, the untethered GFP control does not change its subcellular localization upon co-expression with mCherry-CVIM and remains cytoplasmic and nuclear. (B–B″) 2m22-GFP re-localizes to the plasma membrane where it binds to mCherry-CVIM. (C–C″) The 3m160-GFP fusion protein results in some fluorescent signal at the plasma membrane indicating the weaker affinity to mCherry-CVIM. (D–D″) 2m151-GFP and (E–E″) 2m74-GFP fusion proteins are not significantly recruited to the plasma because of their only micromolar affinity. Unprimed letters, GFP channel; primed letters, mCherry channel; double primed letters, overlay. Scale bars are 20 µm.
- Fig. 4. Expression of a Slmb-anti-GFP-DARPin fusion in Drosophila embryos degrades eYFP.
(A) Shown is an embryo with the following genotype: H2A-eYFP; en-GAL4, UAS-mCherryNLS; UAS-Slmb-3G86.32. Nuclei that express the Slmb-anti-GFP-DARPin in the engrailed pattern are also expressing unfused mCherry. It can be seen that red cells lost the eYFP signal. (B–B″) Close-up of another embryo showing the channel-specific signal of eYFP (B), mCherry (B′) and the overlay (B″). Note again that cells expressing mCherry have strongly reduced H2A-eYFP signal, indicating efficient eYFP degradation due to the expression of a Slmb-anti-GFP-Darpin. Scale bars are 20 µm.
- Fig. 5. 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.
- Fig. 6. anti-GFP-DARPin fusion proteins can relocalize fluorescent fusion proteins in D. rerio embryos.
(A,B) 3G86.2-mRuby2 binds GFP in living zebrafish embryos. (A) Control embryo showing the localization of 3G86.32-mRuby2 in two adjacent skin cells. (B–B″) In a zebrafish embryo co-expressing membrane-bound GFP-CVIM (B), 3G86.32-mRuby2 now localizes to the plasma membrane (B′) and shows a virtually complete co-localization with GFP-CVIM (B″). (C,D) membrane-anchored 3G86.32-mRuby2-CVIM recruits GFP-rab5c in living zebrafish embryos. (C) Control embryo showing the localization of GFP-rab5c in two adjacent skin cells. (D–D″) In a zebrafish embryo co-expressing membrane-bound 3G86.32-mRuby2-CVIM (D′), GFP-Rab5C also localizes to the plasma membrane (D) and shows a virtually complete co-localization with 3G86.32-mRuby2-CVIM (D″). Scale bars are 20 µm.
Tables
- Table 1. Affinities and oligomeric states of GFP and mCherry-binding DARPins
Keywords
Article Navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Other journals from The Company of Biologists
Cover article - Apolipoprotein L9 interacts with LC3/GABARAP and is a microtubule-associated protein with a widespread subcellular distribution
Arvind A. Thekkinghat, Kamlesh K. Yadav and Pundi N. Rangarajan demonstrate the versatile properties of apolipoprotein L9, a lipid-binding protein, and its influence on a variety of activities taking place inside an animal cell.
James J. Dowling and his colleagues conducted the first large-scale screening efforts for NEBLUN-related nemaline myopathy to address the urgent need for effective treatment for patients affected by the disease.
Have you seen our interviews with the early-career first authors of our papers? Recently, we caught up with first authors Arvind Thekkinghat, Shannon Taylor and Daisuke Takao.
Biology Open has strong credentials and publishing with us is easy and fast. BiO aims to provide rapid publication for scientifically sound observations and valid conclusions in developmental, cell, experimental and translational biology. Submit your paper here – you’ll be in good company!
Recent developmental biology highlights in BiO – Anna Nele Herdina and her colleagues define the spectrum of phenotypic abnormalities linked with Col4a2 disruption, and demonstrate the opportunities the Deciphering the Mechanisms of Developmental Disorders (DMDD) program offers for exploring rare human diseases.
Transfer to Biology Open
Did you know: if your submission to one of our sister journals, Development, Journal of Cell Science, Journal of Experimental Biology or Disease Models & Mechanisms, is unsuccessful, you can transfer your paper and any reviews directly to Biology Open. The majority of papers transferred with reviews are accepted for publication. Read here to find out more.
BiO partners with Publons.
Biology Open is pleased to announce a new partnership with Publons. This allows reviewers to easily track and verify every review by choosing to add the review to their Publons profile when completing the review submission form. Publons makes it simple for reviewers to showcase their peer review contributions in a format that can be included in job and funding applications (without breaking reviewer anonymity). Read the official announcement here!
preLights - preLights reaches 500 posts!
The success of preLights is down to the hard work of our preLighters. Head over to the preLights website to find out more about our most active early-career researchers.