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Cited by 113Protein Degradation and InhibitorsUbiquitin and proteasome pathwaysAdvanced Fluorescence Microscopy Techniques

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

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Daniel, Katrin, Icha, Jaroslav, Horenburg, Cindy, Müller, Doris, Norden, Caren, Mansfeld, Jörg·2018·Nature Communications
DOI 10.1038/s41467-018-05855-5PMC PMC6098157

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Abstract

The conditional and reversible depletion of proteins by auxin-mediated degradation is a powerful tool to investigate protein functions in cells and whole organisms. However, its wider applications require fusing the auxin-inducible degron (AID) to individual target proteins. Thus, establishing the auxin system for multiple proteins can be challenging. Another approach for directed protein degradation are anti-GFP nanobodies, which can be applied to GFP stock collections that are readily available in different experimental models. Here, we combine the advantages of auxin and nanobody-based degradation technologies creating an AID-nanobody to degrade GFP-tagged proteins at different cellular structures in a conditional and reversible manner in human cells. We demonstrate efficient and reversible inactivation of the anaphase promoting complex/cyclosome (APC/C) and thus provide new means to study the functions of this essential ubiquitin E3 ligase. Further, we establish auxin degradation in a vertebrate model organism by employing AID-nanobodies in zebrafish.

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Fig. 1 Auxin-dependent degradation of GFP-tagged proteins by an anti-GFP nanobody. Schematic illustration of AID-nanobody degradation. GFP-tagged proteins are recognized by an AID-nanobody fusion. Addition of auxin triggers recruitment of the SCFTIR1 ubiquitin E3 ligase. Ubiquitylation of the GFP fusion protein occurs at lysine residues present in GFP and/or the target protein leading to proteasomal degradation. AID, auxin-inducible degron; Cul1, Cullin-1; Rbx1, RING-box protein 1; Skp1, S-phase kinase-associated protein 1; TIR1, transport inhibitor response 1; vhh-GFP4, anti-GFP nanobody
Fig. 1 Auxin-dependent degradation of GFP-tagged proteins by an anti-GFP nanobody. Schematic illustration of AID-nanobody degradation. GFP-tagged proteins are recognized by an AID-nanobody fusion. Addition of auxin triggers recruitment of the SCFTIR1 ubiquitin E3 ligase. Ubiquitylation of the GFP fusion protein occurs at lysine residues present in GFP and/or the target protein leading to proteasomal degradation. AID, auxin-inducible degron; Cul1, Cullin-1; Rbx1, RING-box protein 1; Skp1, S-phase kinase-associated protein 1; TIR1, transport inhibitor response 1; vhh-GFP4, anti-GFP nanobodyCC BY · PubMed Central
Fig. 2 Kinetics of Venus degradation by mAID-nanobodies. a Time-lapse images and degradation kinetics of Venus targeted by the mAID-nanobody or b of Venus-AID after the addition of 0.5 mM auxin. Scale bar: 10 μm. Quantifications show the mean ± s.d. (n = 5, -auxin, +auxin +MG132; n = 10, +auxin). c Western blot analyses of cells treated as in a and b for the indicated time with 0.5 mM auxin or auxin together with 10 μM proteasome inhibitor MG132. d Titration of tetracycline-inducible expression of mAID-nanobodies and the effect on the degradation kinetics of Venus in the presence of 0.5 mM auxin. Note, the 100 pg curve is masked by the 20 and 0 pg curves. Quantifications show non-linear fit (one-phase decay) of (n = 6, 0 tetracyline ± auxin; n = 3, remaining concentrations). Note, images of –auxin, +auxin and 10 μM MG132-treated cells, degradation of Venus by deGradFP, and Western blot detection of mAID-nanobody expression during the titration are shown in Supplementary Figure 2
Fig. 2 Kinetics of Venus degradation by mAID-nanobodies. a Time-lapse images and degradation kinetics of Venus targeted by the mAID-nanobody or b of Venus-AID after the addition of 0.5 mM auxin. Scale bar: 10 μm. Quantifications show the mean ± s.d. (n = 5, -auxin, +auxin +MG132; n = 10, +auxin). c Western blot analyses of cells treated as in a and b for the indicated time with 0.5 mM auxin or auxin together with 10 μM proteasome inhibitor MG132. d Titration of tetracycline-inducible expression of mAID-nanobodies and the effect on the degradation kinetics of Venus in the presence of 0.5 mM auxin. Note, the 100 pg curve is masked by the 20 and 0 pg curves. Quantifications show non-linear fit (one-phase decay) of (n = 6, 0 tetracyline ± auxin; n = 3, remaining concentrations). Note, images of –auxin, +auxin and 10 μM MG132-treated cells, degradation of Venus by deGradFP, and Western blot detection of mAID-nanobody expression during the titration are shown in Supplementary Figure 2CC BY · PubMed Central
Fig. 3 Conditional and reversible depletion of GFP-fusion proteins localized to distinct cellular compartments and structures by mAID-nanobodies. a Time-lapse images showing the degradation and recovery of (a) nuclear lamina-associated Venus-LMNA and b nuclear and cytoplasmic CCND1-Venus in Hela cell lines expressing tetracycline-inducible mAID-nanobody or c the centromere-associated Inner centromere protein (INCENP-GFP) after transient transfection of mAID-nanobody and nuclear mCherry expressed from the same mRNA (mCherry + mAID-VHH, see Methods). a–c Degradation and recovery were induced by the addition or removal of 0.5 mM auxin for the indicated time. Scale bar: 10 μm. d Single cell degradation kinetics of Venus-LMNA, CCND1-Venus and INCENP-GFP from the experiments shown in a–c. Open blue circles indicate degradation INCENP-GFP cells that reach a plateau at ~50% of the initial fluorescence. Quantifications show the mean ± s.d. Venus-LMNA and CCND1-Venus (n = 40, –auxin; n = 40, +auxin). INCENP-GFP (n = 14, –auxin; n = 29, +auxin (plateau); n = 40, +auxin)
Fig. 3 Conditional and reversible depletion of GFP-fusion proteins localized to distinct cellular compartments and structures by mAID-nanobodies. a Time-lapse images showing the degradation and recovery of (a) nuclear lamina-associated Venus-LMNA and b nuclear and cytoplasmic CCND1-Venus in Hela cell lines expressing tetracycline-inducible mAID-nanobody or c the centromere-associated Inner centromere protein (INCENP-GFP) after transient transfection of mAID-nanobody and nuclear mCherry expressed from the same mRNA (mCherry + mAID-VHH, see Methods). a–c Degradation and recovery were induced by the addition or removal of 0.5 mM auxin for the indicated time. Scale bar: 10 μm. d Single cell degradation kinetics of Venus-LMNA, CCND1-Venus and INCENP-GFP from the experiments shown in a–c. Open blue circles indicate degradation INCENP-GFP cells that reach a plateau at ~50% of the initial fluorescence. Quantifications show the mean ± s.d. Venus-LMNA and CCND1-Venus (n = 40, –auxin; n = 40, +auxin). INCENP-GFP (n = 14, –auxin; n = 29, +auxin (plateau); n = 40, +auxin)CC BY · PubMed Central
Fig. 4 mAID-nanobodies allow the rapid but reversible inactivation of the APC/C. a Degradation of endogenously tagged Venus-ANAPC4 after the addition of 0.5 mM auxin. Scale bar: 10 μm. ANAPC4, APC/C subunit 4. See also Supplementary Figure 3 for characterization of the ANAPC4 knock-in. b Quantification of Venus-ANAPC4 fluorescence from time-lapse data in a showing the kinetics of Venus-ANAPC4 degradation in the nucleus and cytoplasm represented as the mean ± s.d. (n = 6, all conditions). c Time-lapse images and quantification of Venus-ANAPC4 cells arresting in mitosis in the presence of 0.5 mM auxin. Data represent the mean ± s.d. (n = 12, −auxin and +auxin). Scale bar: 10 μm. d Western blot analysis of cells treated with 0.5 mM auxin for the indicated time. Accumulation of cyclins CCNA2 and CCNB1 are indicative of a mitotic arrest. TIR1 serves as a loading control. e Time-lapse images of Venus-ANAPC4 cells treated for 3 h with 0.5 mM auxin followed by auxin removal and 26 h of recovery. Note, Venus-ANAPC4 recovery after 7.3 h is sufficient to allow mitotic progression. Scale bar: 10 μm. f Quantification of Venus-ANAPC4 recovery from cells treated as in e represented as mean ± s.d. (n = 18, −auxin; n = 39, +auxin). Scale bar: 10 μm. g Western blot analysis of cells treated as in e, TIR1 serves as a loading control
Fig. 4 mAID-nanobodies allow the rapid but reversible inactivation of the APC/C. a Degradation of endogenously tagged Venus-ANAPC4 after the addition of 0.5 mM auxin. Scale bar: 10 μm. ANAPC4, APC/C subunit 4. See also Supplementary Figure 3 for characterization of the ANAPC4 knock-in. b Quantification of Venus-ANAPC4 fluorescence from time-lapse data in a showing the kinetics of Venus-ANAPC4 degradation in the nucleus and cytoplasm represented as the mean ± s.d. (n = 6, all conditions). c Time-lapse images and quantification of Venus-ANAPC4 cells arresting in mitosis in the presence of 0.5 mM auxin. Data represent the mean ± s.d. (n = 12, −auxin and +auxin). Scale bar: 10 μm. d Western blot analysis of cells treated with 0.5 mM auxin for the indicated time. Accumulation of cyclins CCNA2 and CCNB1 are indicative of a mitotic arrest. TIR1 serves as a loading control. e Time-lapse images of Venus-ANAPC4 cells treated for 3 h with 0.5 mM auxin followed by auxin removal and 26 h of recovery. Note, Venus-ANAPC4 recovery after 7.3 h is sufficient to allow mitotic progression. Scale bar: 10 μm. f Quantification of Venus-ANAPC4 recovery from cells treated as in e represented as mean ± s.d. (n = 18, −auxin; n = 39, +auxin). Scale bar: 10 μm. g Western blot analysis of cells treated as in e, TIR1 serves as a loading controlCC BY · PubMed Central
Fig. 5 Degradation of endogenous ANAPC4 by mAID-nanobodies in comparison with AID and deGradFP systems. a Time-lapse images showing the degradation of endogenously tagged Venus-ANAPC4 and mAID-Venus-ANAPC4, respectively, in response to the addition of 0.5 mM auxin for mAID-nanobodies and AID-Venus-ANAPC4 or 1 μg/ml tetracycline to induce deGradFP expression. Cells that efficiently degraded Venus-ANAPC4 during 24 h of deGradFP expression are high-lighted by a dotted line. b Degradation kinetics of the time-lapse data shown in a represented as the mean ± s.d. for mAID-vhhGFP (n = 9, −auxin; n = 16, +auxin), mAID-Venus-ANAPC4 (n = 12, −auxin; n = 20, +auxin), and deGradFP (n = 8, −tetracycline; n = 11 +tetracycline). c Western blot analysis of cells treated as in a for the indicated time. Increased levels of CCNB1 are indicative of a mitotic arrest and thus functional inactivation of the APC/C. See also Supplementary Figure 4 for characterization of the mAID-Venus-ANAPC4 knock-in, the effect of prolonged expression of deGradFP on Venus-ANAPC4 and CCNB1 levels, and Venus-ANAPC4 fluorescence in response to expressing deGradFP and deGradFP∆Fbox
Fig. 5 Degradation of endogenous ANAPC4 by mAID-nanobodies in comparison with AID and deGradFP systems. a Time-lapse images showing the degradation of endogenously tagged Venus-ANAPC4 and mAID-Venus-ANAPC4, respectively, in response to the addition of 0.5 mM auxin for mAID-nanobodies and AID-Venus-ANAPC4 or 1 μg/ml tetracycline to induce deGradFP expression. Cells that efficiently degraded Venus-ANAPC4 during 24 h of deGradFP expression are high-lighted by a dotted line. b Degradation kinetics of the time-lapse data shown in a represented as the mean ± s.d. for mAID-vhhGFP (n = 9, −auxin; n = 16, +auxin), mAID-Venus-ANAPC4 (n = 12, −auxin; n = 20, +auxin), and deGradFP (n = 8, −tetracycline; n = 11 +tetracycline). c Western blot analysis of cells treated as in a for the indicated time. Increased levels of CCNB1 are indicative of a mitotic arrest and thus functional inactivation of the APC/C. See also Supplementary Figure 4 for characterization of the mAID-Venus-ANAPC4 knock-in, the effect of prolonged expression of deGradFP on Venus-ANAPC4 and CCNB1 levels, and Venus-ANAPC4 fluorescence in response to expressing deGradFP and deGradFP∆FboxCC BY · PubMed Central
Fig. 6 Auxin-dependent degradation of GFP-tagged proteins by mAID-nanobodies in zebrafish. a Eye of a 24 hpf zebrafish embryo in Tg(bactin:GFP-PCNA) mosaically injected with RNAs of TIR1, mAID-nanobody and H2B-RFP RNA to visualize affected cells. The dashed box shows the area displayed in b. RNE, retinal neuroepithelium. Scale bar: 20 μm. b GFP-PCNA degradation in the presence of IAA. Dashed line: Nuclei of cells expressing the degradation system. Scale bar: 10 μm. c Comparison of GFP-PCNA degradation at 22 hpf in response to IAA and NAA treatment. 1 represents no degradation and 0 represents complete degradation. Bars represent median, −auxin control (n = 11, 110 cells), IAA (n = 11, 108 cells), NAA (n = 11, 108 cells). Significance according to a two-tailed Mann–Whitney test. d Time-lapse imaging of GFP-PCNA degradation in response to NAA in cells without (H2B-RFP negative, left panel) and with degradation system (H2B-RFP positive, right panel). Scale bar: 5 μm. e Degradation of GFP-fusion proteins localized to different cellular structures. Nuclear membrane-localized Lamina-associated polypeptide 2 Tg(bactin:GFP-LAP2b), filamentous actin binding Utrophin fragment Tg(bactin:GFP-Utrophin) and actomyosin-associated non-muscle myosin II light chain Tg(bactin:Myl12.1-GFP). Cells containing the degradation system were visualized by cytoplasmic H2B-RFP or cytoplasmic mKate2 RNA co-injection. Scale bar: 10 μm. f Comparison of GFP-LAP2b degradation in response to IAA and NAA treatment as in c. Bars represent median, −auxin control (n = 11, 110 cells), IAA (n = 5, 50 cells), NAA (n = 11, 111 cells). Significance according to a two-tailed Mann–Whitney test. g Depletion of GFP from mitochondria, Tg(Eef1a1:mls-GFP). Cells containing the degradation system show cytoplasmic mKate2 signal. GFP intensity profile along dashed line is plotted, magenta corresponds to the degradation area. Scale bar: 10 μm. All data shown in this figure are representative examples from at least two independent biological experiments
Fig. 6 Auxin-dependent degradation of GFP-tagged proteins by mAID-nanobodies in zebrafish. a Eye of a 24 hpf zebrafish embryo in Tg(bactin:GFP-PCNA) mosaically injected with RNAs of TIR1, mAID-nanobody and H2B-RFP RNA to visualize affected cells. The dashed box shows the area displayed in b. RNE, retinal neuroepithelium. Scale bar: 20 μm. b GFP-PCNA degradation in the presence of IAA. Dashed line: Nuclei of cells expressing the degradation system. Scale bar: 10 μm. c Comparison of GFP-PCNA degradation at 22 hpf in response to IAA and NAA treatment. 1 represents no degradation and 0 represents complete degradation. Bars represent median, −auxin control (n = 11, 110 cells), IAA (n = 11, 108 cells), NAA (n = 11, 108 cells). Significance according to a two-tailed Mann–Whitney test. d Time-lapse imaging of GFP-PCNA degradation in response to NAA in cells without (H2B-RFP negative, left panel) and with degradation system (H2B-RFP positive, right panel). Scale bar: 5 μm. e Degradation of GFP-fusion proteins localized to different cellular structures. Nuclear membrane-localized Lamina-associated polypeptide 2 Tg(bactin:GFP-LAP2b), filamentous actin binding Utrophin fragment Tg(bactin:GFP-Utrophin) and actomyosin-associated non-muscle myosin II light chain Tg(bactin:Myl12.1-GFP). Cells containing the degradation system were visualized by cytoplasmic H2B-RFP or cytoplasmic mKate2 RNA co-injection. Scale bar: 10 μm. f Comparison of GFP-LAP2b degradation in response to IAA and NAA treatment as in c. Bars represent median, −auxin control (n = 11, 110 cells), IAA (n = 5, 50 cells), NAA (n = 11, 111 cells). Significance according to a two-tailed Mann–Whitney test. g Depletion of GFP from mitochondria, Tg(Eef1a1:mls-GFP). Cells containing the degradation system show cytoplasmic mKate2 signal. GFP intensity profile along dashed line is plotted, magenta corresponds to the degradation area. Scale bar: 10 μm. All data shown in this figure are representative examples from at least two independent biological experimentsCC BY · PubMed Central

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Metadata

Authors
Daniel, K., Icha, J., Horenburg, C., Müller, D., Norden, C., & Mansfeld, J.
Affiliations
  • Biotechnologisches Zentrum (DE)
  • University of Turku (FI)
  • Max Planck Institute of Molecular Cell Biology and Genetics (DE)
Corresponding author
  • Norden, Caren
  • Mansfeld, Jörg
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-08-17
Volume
9
Issue
1
Pages
3297-3297
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-05855-5
PMC
PMC6098157
OpenAlex
W2951829327
Cited by
113
References
45
Citations by year
2026: 9 · 2025: 22 · 2024: 13 · 2023: 9 · 2022: 7 · 2021: 12 · 2020: 23 · 2019: 14 · 2018: 4
FWCI
4.71
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • UTUPub (University of Turku) (submittedVersion, other-oa)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (MA 5831/1-1)
  • EC | European Research Council (680042)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
Keywords
auxin-inducible degron, nanobodies, anti-GFP nanobody, anaphase-promoting complex, E3 ubiquitin ligase, zebrafish, human cells
MeSH terms
Animals, Cell Compartmentation, HeLa Cells, Humans, Indoleacetic Acids, Kinetics, Lysine, Recombinant Fusion Proteins, Zebrafish, Green Fluorescent Proteins, Proteolysis, Single-Domain Antibodies, Anaphase-Promoting Complex-Cyclosome

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