In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish
- Institute for Molecular Bioscience, The University of Queensland, Australia
- QIMR Berghofer Medical Research Institute, Australia
- Centre for Microscopy and Microanalysis, The University of Queensland, Australia
Figures
Figure 1 with 2 supplements
Testing and optimising biotin ligases: BASU, TurboID, and miniTurbo.
(A) A schematic overview of the workflow. The BASU/TurboID/MiniTurbo was transiently expressed in zebrafish embryos by RNA injection. Chorion-removed fish embryos with green fluorescence were selected for incubation in biotin supplemented embryo media for 18 hr. After biotin incubation, embryos were analysed by western blotting and immunofluorescence. (B) The streptavidin-HRP blot showing biotinylated proteins in two dpf zebrafish embryos expressing eGFP-tagged BASU, TurboID, and miniTurbo. Fish embryos were incubated in biotin concentrations of 50 or 500 μM biotin for 18 hr before embryo solubilisation and Western blot analysis. Actin immunoblot (IB:Actin) serves as a loading control; the anti-Myc immunoblot (IB:Myc) reflects the protein level of each biotin ligases; each sample is a pool of 30 embryos. (C) Representative images of NeutrAvidin staining of biotinylated proteins in 2 dpf zebrafish embryo transiently expressing TurboID-eGFP. Fish muscle and yolk were outlined with dashed lines. White arrows indicate muscle fibres expressing TurboID-eGFP. n = 6. Scale bar denotes 100 µm (D and E) Dependency of TurboID activity on biotin concentration and incubation time. Zebrafish embryos transiently expressing TurboID-eGFP were incubated with embryo media containing 0 to 750 μM biotin for 18 hr (D) or incubated with 500 μM biotin for 0 to 18 hr (E) before protein extraction and immunoblot analysis with streptavidin-HRP, anti-Actin and anti-Myc antibodies; each sample is a pool of 30 embryos. For immunoblots showing the biotinylation of BioID and BioID2 in zebrafish embryos see Figure 1—figure supplement 1. For biotin tolerance of zebrafish embryos see Figure 1—figure supplement 2. For original western blot images see Figure 1—source data 1.
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Figure 1—source data 1
Raw images of blots.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig1-data1-v2.pdf
Figure 1—figure supplement 1
Testing BioID and BioID2 in transgenic zebrafish.
(A) Streptavidin-HRP blot showing biotinylated proteins in BioID (BirA*) transgenic zebrafish embryos. Zebrafish embryos were incubated with 50 µM biotin for 18 hr prior to protein extraction and western blotting. (B, C and D) Streptavidin-HRP blots showing biotinylated proteins in zebrafish embryos with (B) increased biotin concentration, (C) increased biotin incubation time and (D) addition of DMSO. (E and F) Streptavidin-HRP blots showing biotinylated proteins in BioID2 transgenic zebrafish embryos under (E) standard incubation temperature (28°C) and (F) elevated temperature (32.5°C) during biotin incubation. Each sample is a pool of 30 embryos at 3 dpf. The two prominent protein bands present in all samples were endogenous biotinylated proteins.
Figure 1—figure supplement 2
Determining biotin toxicity and tolerance in zebrafish embryos.
(A) The fish embryos were incubated with biotin solution at indicated concentration from 1 to 5 dpf. Fifty WT embryos were used for each treatment group. Dead embryos were removed and counted each day. The statistics were performed using Log-rank (Mantel-Cox) test; ns denotes not significant. (B) representative images showing the fish embryo morphology at 3 dpf with or without biotin (600 µM) incubation.
Figure 2
Spatial resolution of TurboID-catalysed biotinylation in zebrafish embryos.
(A) Schematic representation of eGFP-, NLS-, CaaX-, CD44b-, and Cavin4b-tagged TurboID constructs for mRNA injection in zebrafish embryos. TurboID-eGFP was used as a positive control. (B) Representative images showing the distribution of biotinylated proteins in two dpf zebrafish embryos transiently expressing different TurboID constructs. Negative control fish were injected with eGFP only. Fish embryos were fixed and permeabilised before NeutrAvidin-DyLight staining for biotin and DAPI staining to indicate nuclei. Regions within the white box were magnified and shown in the gray scale for NeutrAvidin and DAPI staining in the right panel; n = 3. Scale bar represents 50 µm. (C) Streptavidin-HRP blots showing the ‘protein barcode’ produced by biotinylated proteins in fish embryo transiently expressing different TurboID constructs. Actin immunoblot served as a loading control. Each sample is a pool of 30 embryos. (D) SYPRO Ruby protein stain showing proteins isolated by streptavidin-pulldown. Approximately three hundred embryos transiently expressing each TurboID constructs were subjected to streptavidin-pulldown after biotin incubation and embryo lysis. For original western blot/gel images see Figure 2—source data 1.
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Figure 2—source data 1
Raw images of blots.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig2-data1-v2.pdf
Figure 3
In vivo binding of GFP-nanobody, GBP and dGBP, in stable transgenic zebrafish embryos.
(A and B) Representative images showing the colocalisation between Cavin1a-Clover and mRuby2-GBP/dGBP in live zebrafish embryos. Cavin1a-Clover zebrafish embryos transiently expressing mRuby2-tagged GBP (A) or dGBP (B). Injected embryos were imaged at three dpf. The approximate amount of injected RNA was indicated in the mRuby2 images. Line scan (indicated by the blue line) shows the fluorescent intensity of Clover and mRuby2 across the sarcolemma of mRuby2-positive muscle cells. Scale bar denotes 10 µm in both (A) and (B).
Figure 4 with 1 supplement
GFP-directed in vivo biotin labelling.
(A) Representative images of TurboID-dGBP catalysing GFP-dependent biotinylation in transgenic zebrafish embryos at 3 dpf. The TurboID-dGBP line was crossed with different GFP-tagged zebrafish lines: Cavin1a-Clover (plasma membrane), Cavin4a-Clover (sarcolemma and T-tubules), kdrl:eGFP (vasculature), and MotoN:eGFP (motor neurons). After biotin incubation, embryos were fixed, permeabilised, and stained with NeutrAvidin to visualise the biotinylated protein. mKate2 is a fluorescent indicator for expression of TurboID-dGBP. Controls were carried out by using siblings from the same clutch without GFP expression (TurboID only) and siblings without TurboID expression (Cavin1a-Clover only), as well as omitting biotin incubation. The scale bar denotes 40 µm; n = 3. (B) Western blot analysis showing the biotinylated proteins in 3 dpf zebrafish embryos from TurboID-dGBP outcrossing with Cavin1a-Clover line. Each sample is a pool of 30 embryos. (C) Western blot analysis of fish lysates and streptavidin pulldown with embryos from TurboID-dGBP line outcrossing with Cavin1a-Clover line. Each pulldown sample is a pool of 200 embryos. For confocal images comparing the biotin labelling specificity in zebrafish embryos with different expression level of TurboID-dGBP see Figure 4—figure supplement 1. For table summarising proteins identified in control embryos expressing only TurboID-dGBP, see Supplementary file 3. For original western blot images see Figure 4—source data 1.
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Figure 4—source data 1
Raw images of blots.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig4-data1-v2.pdf
Figure 4—figure supplement 1
The specificity of TurboID-dGBP biotin labelling is independent of its expression level in zebrafish embryos.
(A) Representative images showing biotinylated proteins in zebrafish embryos from outcrossing Cavin1a-Clover with different TurboID-dGBP lines. The number of ‘+' denotes the expression level of TurboID as reflected by mKate2 fluorescent reporter. The scale bar represents 20 µm.
Figure 5
Proteomes identified by BLITZ system in Clover-tagged cavin zebrafish.
(A) A schematic overview of applying TurboID-dGBP fish to identify cavin-associated proteins. The TurboID-dGBP zebrafish was crossed with Clover-tagged cavin fish lines. The embryos carrying both transgenes were selected for subsequent biotin incubation and biotin affinity purification coupled MS sequencing. Identified proteins were refined by subtracting proteins identified in control embryos expressing only TurboID-dGBP. (B) Venn diagram showing the overlap of identified proteins in Cavin1a, Cavin4a, and Cavin4b samples. (C) Heatmap showing relative abundance of identified proteins based on normalised MS2Count in Cavin1a, Cavin4a, and Cavin4b proteomes. (D) Bar graph showing the distribution of proteins at subcellular level. The cellular component information was curated from Uniport database and literature. For table summarising all identified and enriched proteins, see Supplementary file 1 – Tables 1-3. For table annotating all identified and enriched protein with subcellular localisation and functions, see Supplementary file 1 – Table 4. For table showing all identified peptides in Cavin1a sample and sibling control sample see Supplementary file 2 – Tables 1-2. For protein identification report generated by ProteinPilot, see Figure 5—source datas 1–6.
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Figure 5—source data 1
Protein identification report for Cavin1a sample generated by ProteinPilot.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig5-data1-v2.xlsx
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Figure 5—source data 2
Protein identification report for Cavin1a control sample generated by ProteinPilot.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig5-data2-v2.xlsx
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Figure 5—source data 3
Protein identification report for Cavin4a sample generated by ProteinPilot.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig5-data3-v2.xlsx
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Figure 5—source data 4
Protein identification report for Cavin4a control sample generated by ProteinPilot.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig5-data4-v2.xlsx
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Figure 5—source data 5
Protein identification report for Cavin4b sample generated by ProteinPilot.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig5-data5-v2.xlsx
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Figure 5—source data 6
Protein identification report for Cavin4b control sample generated by ProteinPilot.
- https://cdn.elifesciences.org/articles/64631/elife-64631-fig5-data6-v2.xlsx
Figure 6
A schematic overview of the BLITZ system.
The TurboID-dGBP lines can be crossed with existing GFP-tagged lines. In the embryos carrying both transgenes, the binding between dGBP and GFP stabilise TurboID-dGBP, which leads to proximity biotinylation around the GFP-tagged POIs. The unbound TurboID-dGBP will be rapidly degraded by the ubiquitin proteasome system, which minimises non-specific labelling when dGBP-GFP binding saturates, as well as achieving tissue specificity by averting labelling in cells/tissues that do not express GFP. The biotin-labelled proteins can be isolated by biotin affinity purification and identified by MS analysis.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (E. coli- modified) | BirA* | Roux et al., 2012; DOI: 10.1083/jcb.201112098 | R118G mutant of WT BirA | |
| Gene (Bacillus subtilis - modified) | BASU | Ramanathan et al., 2018; DOI: 10.1038/NMETH.4601 | R124G, E323S, G325R mutation and N-terminus deletion of WT biotin ligase from B. subtilis | |
| Gene (Aquifex aeolicus - modified) | BioID2 | Kim et al., 2016; DOI: 10.1091/mbc.E15-12-0844 | R40G mutation of WT biotin ligase from A. aeolicus | |
| Gene (E. coli- modified) | miniTurbo | Branon et al., 2018; DOI: 10.1038/nbt.4201 | 13 point mutations and N-terminal deletion of WT BriA | |
| Gene (E. coli- modified) | TurboID | Branon et al., 2018; DOI: 10.1038/nbt.4201 | 15 point mutations of WT BirA | |
| Strain, strain background (Danio rerio) | TAB | University of Queensland (UQ) Biological Resources Aquatics | Wild-type (TAB), an AB/TU line generated in UQBR Aquatics (UQ Biological Resources) | |
| Strain, strain background (Danio rerio) | TurboID-dGBP | Generated in this paper | Tg(actb2:mKate2-P2A-TurboID-dGBP) | |
| Strain, strain background (Danio rerio) | Cavin1a-Clover | Generated in this paper | Tg(actc1b:Cavin1a-Clover) | |
| Strain, strain background (Danio rerio) | Cavin4a-Clover | Generated in this paper | Tg(actc1b:Cavin4a-Clover) | |
| Strain, strain background (Danio rerio) | Cavin4b-Clover | Generated in this paper | Tg(actc1b:Cavin4b-Clover) | |
| Strain, strain background (Danio rerio) | Kdrl:GFP | (Beis et al., 2005); DOI: 10.1242/dev.01970 | Tg(kdrl:EGFP) | |
| Strain, strain background (Danio rerio) | MotoN:GFP | (Punnamoottil et al., 2015); DOI: 10.1002/dvg.22852 | Tg(miR218:EGFP) | |
| Genetic reagent (Danio rerio) | actb2:mKate2-P2A-TurboID-dGBP | Generated in this paper | Addgene: 163857 | Construct for generating stable transgenic fish line; see Materials and methods for line generation |
| Genetic reagent (Danio rerio) | actc1b:Cavin1a-Clover | Generated in this paper | Addgene: 163852 | Construct for generating stable transgenic fish line; see Materials and methods for line generation |
| Genetic reagent (Danio rerio) | actc1b:Cavin4a-Clover | Generated in this paper | Addgene: 163853 | Construct for generating stable transgenic fish line; see Materials and methods for line generation |
| Genetic reagent (Danio rerio) | actc1b:Cavin4b-Clover | Generated in this paper | Addgene: 163854 | Construct for generating stable transgenic fish line; see Materials and methods for line generation |
| Genetic reagent (Danio rerio) | pT3TS-BASU-EGFP | Generated in this paper | Addgene: 163845 | Construct for in vitro RNA synthesis and RNA injection |
| Genetic reagent (Danio rerio) | pT3TS-TurboID-EGFP | Generated in this paper | Addgene: 163846 | Construct for in vitro RNA synthesis and RNA injection |
| Genetic reagent (Danio rerio) | pT3TS-miniTurbo-EGFP | Generated in this paper | Addgene: 163847 | Construct for in vitro RNA synthesis and RNA injection |
| Genetic reagent (Danio rerio) | pT3TS-TurboID-CaaX | Generated in this paper | Addgene: 163848 | Construct for in vitro RNA synthesis and RNA injection |
| Genetic reagent (Danio rerio) | pT3TS-nls-TurboID | Generated in this paper | Addgene: 163849 | Construct for in vitro RNA synthesis and RNA injection |
| Genetic reagent (Danio rerio) | pT3TS-CD44b-TurboID | Generated in this paper | Addgene: 163850 | Construct for in vitro RNA synthesis and RNA injection |
| Genetic reagent (Danio rerio) | pT3TS-Cavin4b-TurboID | Generated in this paper | Addgene: 163851 | Construct for in vitro RNA synthesis and RNA injection |
| Genetic reagent (Danio rerio) | actc1b:mRuby2-GBP | Generated in this paper | Addgene: 163856 | Construct for transient expression in zebrafish |
| Genetic reagent (Danio rerio) | actc1b:mRuby2-dGBP | Generated in this paper | Addgene: 163855 | Construct for transient expression in zebrafish |
| Antibody | Anti-Myc (Mouse monoclonal) | Cell Signaling Technology | 2276S | (1:2000) dilution with 5% skim milk in PBST |
| Antibody | Anti-Actin (Mouse monoclonal) | EMD Millipore | MAB1501 | (1:5000) dilution with 5% skim milk in PBST |
| Antibody | Anti-Cavin4b (Rabbit polyclonal) | Boster Biological Technology | DZ33949 | Customised antibody against zebrafish Cavin4b; (1:1000) dilution with 3% skim milk in PBST |
| Recombinant DNA reagent | p5E-actb2 | Kwan et al., 2007; DOI: 10.1002/dvdy.21343 | N/A | |
| Recombinant DNA reagent | p5E-actc1b | Ariotti et al., 2018b; DOI: 10.1242/dev.034561 | N/A | |
| Recombinant DNA reagent | pME-BASU-NS | generated in this paper | Addgene: 166565 | Gateway Entry clone contains BASU without a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | pME-TurboID-NS | generated in this paper | Addgene: 166566 | Gateway Entry clone contains TurboID without a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | pME-nls | Ariotti et al., 2018a; DOI: 10.1371/journal.pbio.2005473 | Addgene: 108882 | |
| Recombinant DNA reagent | pME-CD44b | Hall et al., 2020 DOI: 10.1038/s41467-020-17486-w | Addgene: 109576 | |
| Recombinant DNA reagent | pME-miniTurbo-NS | generated in this paper | Addgene: 166567 | Gateway Entry clone contains miniTurbo without a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | pME-Cavin1a | Hall et al., 2020; DOI: 10.1038/s41467-020-17486-w | Addgene: 126927 | |
| Recombinant DNA reagent | pME-Cavin4a | Hall et al., 2020; DOI: 10.1038/s41467-020-17486-w | Addgene: 109562 | |
| Recombinant DNA reagent | pME-Cavin4b | Hall et al., 2020; DOI: 10.1038/s41467-020-17486-w | Addgene: 109563 | |
| Recombinant DNA reagent | pME-mKate2-P2A-TurboID-NS | Generated in this paper | Addgene: 166568 | Gateway Entry clone contains mKate2-P2A-TurboID without a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | pME-mRuby2-NS | Generated in this paper | Addgene: 166569 | Gateway Entry clone contains mRuby2 without a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | p3E-TurboID | Generated in this paper | Addgene: 166570 | Gateway Entry clone contains TurboID with a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | p3E-Clover | Generated in this paper | Addgene: 126572 | Gateway Entry clone contains Clover with a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | p3E-EGFP | Generated in this paper | Addgene: 126573 | Gateway Entry clone contains EGFP with a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | p3E-csGBP (dGBP) | Ariotti et al., 2018a; DOI: 10.1371/journal.pbio.2005473 | Addgene: 108891 | Gateway Entry clone contains csGBP with a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | p3E-GBP | Ariotti et al., 2015a; DOI: 10.1016/j.devcel.2015.10.016 | Addgene: 67672 | Gateway Entry clone contains GBP with a stop codon; see Materials and methods for cloning and Addgene for vector map |
| Recombinant DNA reagent | p3E-CaaX (tH) | Hall et al., 2020; DOI: 10.1038/s41467-020-17486-w | Addgene: 109539 | |
| Recombinant DNA reagent | pT3TS-DEST | Generated in this paper | Addgene: 166571 | Gateway Destination vector contains T3 and T7 promoters for in vitro RNA synthesis; see Materials and methods for cloning and Addgene for vector map |
| Peptide, recombinant protein | Streptavidin-HRP | Abcam | Ab7403 | (1:5000) dilution with 5% BSA in PBST |
| Peptide, recombinant protein | Proteinase K | Invitrogen | 25530015 | |
| Peptide, recombinant protein | Pronase | Roche | 10165921001 | |
| Peptide, recombinant protein | Trypsin/Lys-C Mix, Mass Spec Grade | Promega | V5073 | |
| Commercial assay or kit | Pierce BCA protein assay kit | Thermo Scientific | 23225 | |
| Commercial assay or kit | Clarity Western ECL Substrate | Bio-Rad | 1705061 | |
| Commercial assay or kit | InstantBlue | Expedeon | ISB1L-1L | |
| Commercial assay or kit | SYPRO Ruby Protein Gel Stain | Invitrogen | S12000 | |
| Chemical compound, drug | Biotin | Sigma-Aldrich | B4639-1G | |
| Chemical compound, drug | Phenol Red | Sigma-Aldrich | P0290-100ML | |
| Chemical compound, drug | NeutrAvidin-DyLight 405 | Invitrogen | 22831 | |
| Chemical compound, drug | NeutrAvidin-DyLight 550 | Invitrogen | 84606 | |
| Chemical compound, drug | Sodium deoxycholate | Sigma-Aldrich | D6750-10G | |
| Chemical compound, drug | NP-40 | Sigma-Aldrich | 18896–50 ML | |
| Chemical compound, drug | EDTA | Astral Scientific | BIOEB0185-500G | |
| Chemical compound, drug | Complete Protease Inhibitor Cocktail | Sigma-Aldrich | 11836145001 | |
| Chemical compound, drug | Paraformaldehyde | Sigma-Aldrich | P6148-500G | |
| Chemical compound, drug | PBS tablets | Medicago | 09-8912-100 | |
| Chemical compound, drug | Triton-X100 | Sigma-Aldrich | T9284-500ML | |
| Chemical compound, drug | Tween 20 | Sigma-Aldrich | P1379-500ML | |
| Chemical compound, drug | DAPI | Sigma-Aldrich | D9542-5MG | |
| Chemical compound, drug | Bolt LDS sample buffer (4X) | Invitrogen | B0008 | |
| Chemical compound, drug | Dynabeads MyOne Streptavidin C1 | Invitrogen | 65001 | |
| Chemical compound, drug | Agarose, low gelling temperature | Sigma-Aldrich | A9414-100G | |
| Software, algorithm | ProteinPilot | SCIEX | Version 5.0.1 | |
| Software, algorithm | Analyst TF | SCIEX | Version 1.7 | |
| Software, algorithm | Excel | Microsoft | Version 16.45 | |
| Software, algorithm | Prism8 | GraphPad | Version 8.0.2 | |
| Software, algorithm | Fiji | ImageJ | Version 2.0.0-rc-69/1.52 p | |
| Software, algorithm | Illustrator | Adobe | Version 23.1.1 | |
| Other | PD-10 desalting column | GE Healthcare | 17-0851-01 | |
| Other | LoBind tube | Eppendorf | 022431048 | |
| Other | Blot 4–12% Bis-Tris Plus precast gels | Invitrogen | NW04120BOX |
Additional files
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Supplementary file 1
Tables showing all identified and enriched proteins in Cavin1a (Table 1), Cavin4a (Table 2), and Cavin4b (Table 3) samples as well as localisation and functions annotation (Table 4).
- https://cdn.elifesciences.org/articles/64631/elife-64631-supp1-v2.xlsx
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Supplementary file 2
Table showing all the peptides identified in Cavin1a and sibiling control samples.
- https://cdn.elifesciences.org/articles/64631/elife-64631-supp2-v2.xlsx
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Supplementary file 3
Table showing proteins identified in control zebrafish embryos expressing only TurboID-dGBP.
- https://cdn.elifesciences.org/articles/64631/elife-64631-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/64631/elife-64631-transrepform-v2.pdf
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In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish
eLife 10:e64631.
https://doi.org/10.7554/eLife.64631
