Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

Daniel, Katrin; Icha, Jaroslav; Horenburg, Cindy; Müller, Doris; Norden, Caren; Mansfeld, Jörg

doi:10.1038/s41467-018-05855-5

Cited by 96 publications

(129 citation statements)

References 44 publications

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“…Degradation of GFP-tagged proteins via the expression of a GFP nanobody fused to a F-box is a powerful tool to investigate protein function. While the deGradFP approach has originally been reported to be effective in Drosophila, similar methods have in the meantime been reported in C. elegans (Wang et al, 2017), zebrafish (Daniel et al, 2018;Shin et al, 2015;Yamaguchi et al, 2019) and in plants (Baudisch, Pfort, Sorge, & Conrad, 2018).…”

Section: Degradfpmentioning

confidence: 90%

“…Upon binding to the GFP-tagged POI, a GFP nanobody fused to an F-box domain can target the POI for degradation (Caussinus, Kanca, & Affolter, 2011) (Figure 1c). Several similar approaches have been recently developed to achieve protein degradation in different model organisms (Daniel et al, 2018;Wang et al, 2017;Yamaguchi, Colak-Champollion, & Knaut, 2019).…”

Section: Protein Degradationmentioning

confidence: 99%

“…However, given the relatively large size, GFP may not be optimal for tagging certain proteins. Therefore, the use of recently reported intrabodies against small tags, such as SunTag (19aa) Degraded in absence of the antigen (GFP) (Tang et al, 2016) Lysine-less VHH4 (GFP, YFP, Venus) VHH4 in which lysines have been substituted for Arginines (Daniel et al, 2018) mCherry VHH (Fridy et al, 2014) MoonTag (gp41, 15aa linear epitope) (Boersma et al, 2019) BC2 VHH (BC2 tag, 12aa) Not tested as intrabody. (Braun et al, 2016) NbALFA (ALFAtag, 15aa) (Götzke et al, 2019) Darpin 3G86.32 Anti-GFP DARPin (GFP) (Brauchle et al, 2014) 2 m22 Anti-mCherry DARPin (mCherry) (Brauchle et al, 2014) E11 and G01 Anti-TFP DARPins (TFP) (Vigano et al, 2018) scFv HA frankenbody (HA, 9aa linear epitope) (Zhao et al, 2018) Suntag (GCN4 v4, 19aa linear epitope) Aggregation at high levels unless fused to sfGFP-GB1 scaffold (Tanenbaum, Gilbert, Qi, Weissman, & Vale, 2014) BGP7 ScFv (BGP7, 7aa linear epitope) (Lim, Ichinose, Shinoda, & Ueda, 2007;Wongso, Dong, Ueda, & Kitaguchi, 2017) (15aa), promises to be a great addition to the protein binder toolbox (Boersma et al, 2019;Gotzke et al, 2019;Tanenbaum et al, 2014;Zhao et al, 2018).…”

Section: Protein Binders Against Commonly Used Tagsmentioning

confidence: 99%

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Reflections on the use of protein binders to study protein function in developmental biology

Aguilar

Viganò

Affolter

et al. 2019

WIREs Developmental Biology

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Studies in the field of developmental biology aim to unravel how a fertilized egg develops into an adult organism and how proteins and other macromolecules work together during this process. With regard to protein function, most of the developmental studies have used genetic and RNA interference approaches, combined with biochemical analyses, to reach this goal. However, there always remains much room for interpretation on how a given protein functions, because proteins work together with many other molecules in complex regulatory networks and it is not easy to reveal the function of one given protein without affecting the networks. Likewise, it has remained difficult to experimentally challenge and/or validate the proposed concepts derived from mutant analyses without tools that directly manipulate protein function in a predictable manner. Recently, synthetic tools based on protein binders such as scFvs, nanobodies, DARPins, and others have been applied in developmental biology to directly manipulate target proteins in a predicted manner. Although such tools would have a great impact in filling the gap of knowledge between mutant phenotypes and protein functions, careful investigations are required when applying functionalized protein binders to fundamental questions in developmental biology. In this review, we first summarize how protein binders have been used in the field, and then reflect on possible guidelines for applying such tools to study protein functions in developmental biology.This article is categorized under:Technologies > Analysis of ProteinsEstablishment of Spatial and Temporal Patterns > GradientsInvertebrate Organogenesis > Flies

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Section: Degradfpmentioning

confidence: 90%

Section: Protein Degradationmentioning

confidence: 99%

Section: Protein Binders Against Commonly Used Tagsmentioning

confidence: 99%

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Reflections on the use of protein binders to study protein function in developmental biology

Aguilar

Viganò

Affolter

et al. 2019

WIREs Developmental Biology

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“…Since these nanobodies can be readily expressed in cells, it is possible to use them as "dongles": to extend the functionality of GFP by attaching a new protein domain to the GFP-tagged protein-of-interest via fusion with the nanobody. This approach has been exploited to degrade proteins-ofinterest (Caussinus et al, 2011;Kanner et al, 2017;Daniel et al, 2018;Yamaguchi et al, 2019), to introduce additional tags (Rothbauer et al, 2008;Ariotti et al, 2015;Derivery et al, 2017;Zhao et al, 2018), or to constitutively relocalize GFP-tagged proteins (Schornack et al, 2009;Derivery et al, 2015). Recently a suite of functionalized nanobodies to GFP or RFP were generated enabling recoloring, inactivation, ectopic recruitment, and calcium sensing (Prole and Taylor, 2019).…”

Section: Introductionmentioning

confidence: 99%

Unintended inhibition of protein function using GFP nanobodies in human cells

Küey

Larocque

Clarke

et al. 2019

Preprint

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Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed "dongles", that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further. clathrin-mediated endocytosis | dynamin | nanobody | GFP-binding protein | knocksideways Correspondence: s.j.royle@warwick.ac.uk Küey et al. | bioRχiv | May 30, 2019 | 1-11

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“…The auxin-inducible degradation system allows for rapid and conditional 74 degradation of auxin-inducible degron (AID)-tagged proteins in C. elegans as well as in 75 other commonly used model systems including yeast (Nishimura et al 2009), Drosophila 76 (Trost et al 2016), zebrafish (Daniel et al 2018), cultured mammalian cells (Nishimura et 77 7 The pJW1747 repair template was purified using the Invitrogen PureLink HQ Mini 127 Plasmid DNA Purification Kit (K210001). The optional wash step in the protocol using a 128 4 M guanidine-HCl + 40% isopropanol solution is highly recommended, as excluding it 129 dramatically reduced injection efficiency in our hands.…”

mentioning

confidence: 99%

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Martinez

Kinney

Medwig-Kinney

et al. 2019

Preprint

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As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system, allows for spatial and temporal control of protein degradation, functioning through the activity of a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID method, utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 minutes of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 interacts with C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the Ftz-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work provides a conceptual improvement to the AID system for dissecting gene function at the single-cell level during C. elegans development.

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Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

Cited by 96 publications

References 44 publications

Reflections on the use of protein binders to study protein function in developmental biology

Reflections on the use of protein binders to study protein function in developmental biology

Unintended inhibition of protein function using GFP nanobodies in human cells

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

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