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Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells

Nature Methods volume 17, pages 937–946 (2020)Cite this article

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An Author Correction to this article was published on 16 October 2020

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Abstract

Genetically encoded tags for single-molecule imaging in electron microscopy (EM) are long-awaited. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated tags. Next, we exploited this mechanism to develop approaches for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies and mitochondrial matrices. We further implemented cysteine-rich tag–antibody fusion proteins as new immuno-EM probes. Thus, our approaches should allow biologists to address a wide range of biological questions at the single-molecule level in cellular ultrastructural contexts.

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Fig. 1: Schematics showing the principles of genetically encoded tags for EM.
Fig. 2: The implementation of ANSM-based synthesis of AuNPs on cysteine-rich tags.
Fig. 3: Develop ANSM-based AuNP synthesis protocols with E. coli cells.
Fig. 4: Single-molecule visualization of MTn fusion proteins in S. pombe cells.
Fig. 5: MTn tags specific localized to the lumen of the ER, ER membrane and the mitochondrial matrix in HeLa cells.
Fig. 6: EM visualization of GFP in cells with GBP-MTn probes.

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Data availability

All the plasmids shown in Supplementary Table 2 have been deposited in Addgene with the indicated accession codes. All raw data images used in all figures presented in the paper are available upon request. Source data are provided with this paper.

Change history

  • 16 October 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank X.-D. Wang, for his vision and long-term support for this high-risk exploring project. W.H. thanks R. He for constantly standing by him during this time-consuming adventure and D. DeRosier for helpful discussions. We are grateful to X.-C. Wang, D.-F. Zhao, X.-G. Lei, N. Huang, Y. Cai, W. Hunziker, Y.-M. Yuan, K. Ye, F.-C. Wang, G.-S. Ou, G.-H. Liu, P.-Y. Xu., F. Shao and Y. Rao for their help and discussions. We thank D.R. Winge for providing mouse MT-1 plasmid. We are grateful to J.H. Snyder for his professional editing service. L.-L.D. was supported by grant from a MOST (973 Program no. 2014CB849901). W.H. was supported by grants from the MOST (973 Programs nos. 2011CB812502 and 2014CB849902) and by funding support from the Beijing Municipal Government.

Author information

Author notes

  1. These authors contributed equally: Zhaodi Jiang, Xiumei Jin, Yuhua Li.

Authors and Affiliations

  1. PTN Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China

    Zhaodi Jiang

  2. National Institute of Biological Sciences, Beijing, China

    Zhaodi Jiang, Xiumei Jin, Yuhua Li, Sitong Liu, Xiao-Man Liu, Ying-Ying Wang, Pei Zhao, Xinbin Cai, Ying Liu, Yaqi Tang, Xiaobin Sun, Yan Liu, Yanyong Hu, Ming Li, Gaihong Cai, Xiangbing Qi, She Chen, Li-Lin Du & Wanzhong He

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Contributions

W.H. conceived the project, designed the experiments, supervised the research, initially discovered and conceptualized the ANSM-based AuNP synthesis, analyzed the data and wrote the paper. X.J. performed the experiments for developing the ANSM/AuNP synthesis method, optimizing the AuNP synthesizing conditions for both isolated tags and tags expressed in E. coli cells, and also the early efforts on developing AuNP synthesis in S. pombe cells using the ‘cold MeOH’ approach. Z.J. implemented and optimized the protocols for eukaryotic cells and the related EM work, and also performed some the pre-embedding staining work. Y. Li helped to design the tags, established HeLa cell lines, performed the molecular biology and immunogold labeling work, prepared GFP-tag-KDEL HeLa cell lines and conducted the in vitro protein experiments. S.L. conducted the initial development of a freeze-substitution protocol for synthesizing gold particles in S. pombe cells and helped to design the cartoon figures. P.Z., X.C., Yan Liu and Y. Li performed the molecular biology work with the E. coli system. L.-L.D. established and supervised the S. pombe cells and GBP-related collaborations. X.-M.L. and Y.-Y.W. created the S. pombe strains (Ost4, Nup124, Sad1) and took the images. Ying Liu, Yan Liu, X.S., Y.T., Y.H. and M.L. performed a series of EM-related work. X.Q. for supported part of the chemistry-related work. S.C. and G.C. for the MALDI–TOF analysis. All authors participated in discussions and data interpretation.

Corresponding author

Correspondence to Wanzhong He.

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Competing interests

The National Institute of Biological Sciences, Beijing, filed a PCT patent (WO2019024707A1) (inventors W.H. and X.J.) covering part of the information contained in this article.

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Au(I)-thiolate precipitates formed in the mixtures of HAuCl4 and RSH at classic BSM conditions.

a, Cloudy precipitates formed in the mixture of 2-ME and HAuCl4 at RS−/Au(III) < 2:1 conditions, but no detectable precipitates formed in the mixture at RS−/Au(III) > 2:1 conditions (top row); AuNPs formed (as indicated by the brown colors) in these mixtures at RS−/Au(III) < 2:1 conditions when reduced with NaBH4, while no AuNPs formed (colorless) in those mixtures at RS−/Au(III) > 2:1 conditions (bottom row). b, In D-P and HAuCl4 mixtures: at the state of RS−/Au(III) = 1:1, the solution turned to light brown due to the formation of the unstable (D-P)Au(I) species, but no detectable colors at other conditions (top row); When reduced by NaBH4, the solutions of those RS−/Au(III) < 2:1 conditions turned to brown color, only those RS−/Au(III) > 2:1 cases were still colorless (bottom row). c, Five pairs of experiments revealing the effect of 30 mM D-P for dissolving the cloudy precipitates formed in the mixtures of 0.5 mM HAuCl4 and various concentration of 2-ME. Cloudy precipitates formed in the 0.5 mM HAuCl4 and 10 mM, 20 mM, 40 mM and 80 mM of 2-ME mixtures (the left ones); when added extra 30 mM of D-P into the same mixtures the precipitates were dissolved completely (the right ones). Representative images for (a-c) were selected from n = 3 independent experiments with similar results.

Extended Data Fig. 2 Gold chelating orders of 6 amino acids, 2-mercaptoethanol (2-ME), and D-penicillamine (D-P).

a, Tests of AuNPs synthesis with the six mixtures of 0.2 mM 6 typical amino acids and 0.5 mM HAuCl4, reduced by 1 mM NaBH4, the solutions all changed to purple or dark brown color, indicating the formation of AuNPs. b, Tests of AuNPs synthesis with the additional 140 mM 2-ME to the six mixtures of the 0.2 mM amino acids and 0.5 mM HAuCl4, reduced by 1 mM NaBH4; Only the sample with cysteine changed color to brown (an indication of AuNPs formation), other specimens remained colorless (no AuNPs formed). c, Tests of AuNPs synthesis with the additional 30 mM D-P to the six mixtures of the 0.2 mM amino acids and 0.5 mM HAuCl4, reduced by 1 mM NaBH4; No AuNPs formed in all the six samples. These results implied a gold chelating order (from weak to strong): -COOH, -NH2 group of amino acid< thiol group of 2-ME < thiol group of cysteine < thiol group of D-P. The results in b and c also demonstrated the capabilities of 2-ME and D-P for auto-nucleation suppression. Representative images for (a-c) were selected from n = 4 independent experiments with similar results.

Extended Data Fig. 3 Identification of the species of gold-thiolate in the mixtures of HAuCl4 and RSH by MALDI-TOF analysis.

a, Two types of gold thiolate compounds identified in the 1:1 Au(III)/(D-P) mixture at RS−/Au(III) < 2:1 condition: Au(I)n(D-P)n+1, (n = 2,3,4) in zigzag chain forms (labeled as black), and the Na(1-2)Au2(D-P)4Cl2O compounds (labeled as red). b, Only the soluble Na(1-2)Au2(D-P)4Cl2O formed in the Au/D-P mixture at RS−/Au(III) > 2:1 condition. c, In the mixture of 0.5 mM HAuCl4 + 60 mM 2-ME (RS−/Au(III) < 2:1 condition), the major Au(I) thiolate species were zigzag chain-like 1:1 Au(I)/(2-ME) compounds (in black), mixed with a little amount of Na2Au(III)(2-ME)4 (in green) and 1:2 Au(I)/(2-ME) compounds (in red). d, In the mixture of 0.5 mM HAuCl4 + 160 mM 2-ME (RS−/Au(III) > 2:1 condition), the amount of zigzag chain-like 1:1 Au(I)/(2-ME) compounds (in back) were largely reduced, and Na(0-2)Au(III)(2-ME)4 (in green) became the major species, and also found some 1:2 Au(I)/(2-ME) compounds (in red).e, Additional 20 mM D-P to the 0.5 mM HAuCl4 + 60 mM 2-ME mixture, only the Au(III) compounds (in green) and a little amount of NaAu2(D-P)4Cl2O (red) found in the mixture. f, In the 0.1 mM HAuCl4 and 150 mM 2-ME mixture, only three 1:2 Au(I)/(2-ME) compounds (red) formed at such RS−/Au(III) > 2:1 condition. Representative images for (a-f) were selected from n = 2 independent experiments with similar results.

Extended Data Fig. 4 Single-molecule counting level imaging of AuNPs direct-synthesized on individual tag fusion proteins.

a, c, e, g, EM images of negative stained MBP-2MT, MBP-MTn, MBP-MTa and MBP-2AFP, showing single molecules (~7 nm in sizes) marked with 8 nm x 8 nm red boxes. b, d, f, h, EM images of MBP-2MT, MBP-MTn, MBP-MTa, and MBP-2AFP molecules underwent ANSM-based AuNP synthesis, followed by 2 % UA negative staining; the 8 nm x 8 nm red boxes marked out these proteins forming AuNPs on the tags in 1:1 ratio. The 8 nm x 8 nm white boxes marked out those proteins without visible AuNPs, which might be caused by insufficient exposure to gold thiolate sources due to protein aggregations (for example, the purified MBP-MTa seems unstable and tends to form aggregates). To avoid multiple AuNPs aggregations with the standard 2-ME/D-P AuNP synthesis protocol (Fig. 2), here we a TCEP/D-P AuNP synthesis protocol used for (b, d, f, h) (Methods). Representative images for (a-h) were selected from n = 4 independent experiments with similar results.

Extended Data Fig. 5 Specific distributions of AuNPs in the E. Coli cells expressing MBP-MTn-FliG.

a, Hardly seen any AuNPs in the wild-type BL21(DE3) cells; b, Relatively uniform distribution of AuNPs in a cross-section of a E. coli cell expressing MBP-MTn-FliG. c, AuNPs accumulated at the pole of an E. coli cell that expressed MBP-MTn-FliG, only a few AuNPs found in the nucleoid region, but no AuNPs found in the periplasmic spaces (pointed by the black arrows). Specimen prepared with ‘-60˚C MeOH’ fixation followed with 2-ME/D-P AuNP synthesis. Representative images for (a-c) were selected from n = 10 independent experiments with similar results.

Extended Data Fig. 6 A comparison of the performance of MTn tag labeling, Tokuyasu technique, and conventional immuno-EM method.

a, b, Expression patterns of the ER-located membrane proteins, Ost4-GFP and Ost4-GFP-MTn, in S. pombe observed by live-cell fluorescence imaging. c, Immunogold staining of Ost4-GFP in yeast cell using anti-GFP (goat) antibody (Cat. No.600-101-215, Lot.35059, Rockland) and protein A-10nm gold particles (Cat.No.25285, Code:110.111, EMS) on a cryosection prepared with Tokuyasu technique. d, Direct synthesis of AuNPs on Ost4-GFP-MTn in a yeast cell using Scheme 2b (PIPES) for ANSM AuNP synthesis. e, Immunogold staining of Ost4-GFP using anti-GFP (Rabbit) antibody (Cat. No. ab6556, Lot.GR3252667-1, Abcam) and anti-rabbit IgG antibody-10nm gold particles (Cat.No.G7402-.4 ml, Lot.SLBT0649, Sigma) on LR white embedded plastic section of a yeast cell (cell prepared with HPF, FSF with 0.1% UA + 5% H2O in acetone). f, Immunogold staining of Ost4-GFP-MTn in yeast cell (the same experimental setting as used for e, except embedded in HM20 resin). Notably, the labeling density with ANSM AuNPs synthesis on Ost4-GFP-MTn (d) is ~10-20x higher than those with immunogold staining (c, e, f). Representative images were selected from n = 3 (a, b, c, e, f), 5 (d) independent experiments with similar results.

Extended Data Fig. 7 An overview image showing the specific distribution of GFP-MTn-KDEL molecules in a HeLa cell treated with Scheme 3c (HPF/FSF).

An EM image of a 90 nm thick section of a GFP-MTn-KDEL expressing cell showing an overview of the ultrastructural preservation as well as the highly specific localization of the AuNPs synthesized on the fusion protein molecules accumulated in ER lumens, almost no AuNPs in mitochondria (M), multivesicular bodies (MVB), cytosol and nucleus. A high magnification image of the boxed region showing more details (Supplementary Fig. 17H(a)). Representative image was selected from n = 3 independent experiments with similar results.

Extended Data Fig. 8 Specific distributions of AuNPs synthesized on Ost4-GFP-MTn in HeLa cells treated with Scheme 3c (HPF/FSF).

a, b, AuNPs specifically localized on the outer surface of irregular-shaped ER networks in images of Ost4-GFP-MTn expressing cells treated with Scheme 3c; Only see a few AuNPs on outer surfaces of mitochondria (those AuNPs were likely attached to ER membranes adjacent to the surfaces of mitochondria (M), see Supplementary Video 2 & 3). Representative images for (a-b) were selected from n = 2 independent experiments with similar results.

Extended Data Fig. 9 Specific distributions of AuNPs synthesized on Mito-acGFP-MTn in HeLa cells treated with Scheme 3c (HPF/FSF).

An EM image of 90 nm thick sections of HeLa cells expressing Mito-acGFP-MTn showing the highly-specific distribution of AuNPs in mitochondrial matrices as expected, and the well-preserved ultrastructures of mitochondria (M). Representative images were selected from n = 2 independent experiments with similar results.

Extended Data Fig. 10 Subcellular distribution patterns of AuNPs synthesized on cysteine-rich tags in cells.

The chart is showing the statistical distribution patterns of the average AuNPs densities (AuNP counts per 0.25 µm2 obtained from 90nm-thick sections) in various subcellular organelles in S. pombe cells or HeLa cells expressing cysteine-rich tags (MTn or AFP). The averaged AuNPs densities in endoplasmic reticulum (ER), cytosol, nuclei (Nu), lysosomes (Lyso), multivesicular bodies (MVB) and mitochondria (Mito) were obtained from 19-26 EM images of 90n-thick sections of cells (Supplementary Note 15). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparisons using GraphPad Prism 8.4.2 software. Quantitative data were expressed as box-and-whisker plots (center line, average; limits, 75% and 25%; whiskers, maximum and minimum). The specimens in the left column (a, c, e, g) were prepared with Scheme 2b or 2d (using a strategy of oxidization and G A fixation). The specimens in the right column (b, d, f, h) were prepared with Scheme I (-60 °C MeOH) or Scheme 3c (HPF/FSF) (using a rapid freezing strategy without using oxidization and GA fixation) (Methods). The averaged densities of AuNPs in different organelles demonstrated that the tags had significantly specific localizations to the targeted organelles, while the background noises were quite low (< 2-5%). All the P values as shown in (a-h) are: P < 0.0001 (95% CI). The numbers of EM images used for (a-h) are: (a) n = 20; (b) n = 19; (c) n = 23; (d) n = 22; (e) n = 19; (f) n = 22; (g) n = 22; (f) n = 26. Size (n) for each organelle is labeled on the figure.

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Supplementary information

Supplementary Information

Supplementary Tables 1–3, Notes 1–16 and Figs. 1–21.

Reporting Summary

Supplementary Video 1

Tomogram of a S. pombe cell expressing Ost4-GFP-MTn. A tomogram of a 150-nm-thick section from an Ost4-GFP-MTn expressing S. pombe cell treated with Scheme 2b (PIPES) showing the AuNPs attached to membranes of NE or ER, which obviously untangled the ambiguous features in the projection image (Supplementary Fig. 18c). This video corresponds to a volume of 2,287 × 2,287 × 93 nm3. Dual-axes tilt series (±70°, interval 1°) reconstruction with IMOD software (bio3d.colorado.edu/imod).

Supplementary Video 2

A Tomogram of a HeLa cell expressing Ost4-GFP-MTn. A tomogram of a 130-nm-thick section from an Ost4-GFP-MTn expressing HeLa cell treated with Scheme 3c (HPF/FSF) showing the AuNPs attached to the cytosolic surfaces of the ER membrane, which also demonstrated that those AuNPs attached to the mitochondrial surface in the projection image seemed still on the ER membrane. This video corresponds to a volume of 1,832 × 1,498 × 110 nm3. Dual-axes tilt series (±70°, interval 1°) reconstruction with IMOD software (bio3d.colorado.edu/imod).

Supplementary Video 3

A Tomogram of a HeLa cell expressing Ost4-GFP-MTn. A tomogram of a 130-nm-thick section from an Ost4-GFP-MTn expressing HeLa cells treated with Scheme 3c (HPF/FSF) showing the AuNPs attached to the cytosolic surfaces of the ER membrane, which also demonstrated that those AuNPs attached to the mitochondrial surface in the projection image seemed still on the ER membrane. This video corresponds to a volume of 1,238 × 1,175 × 117.5 nm3. Dual-axes tilt series (±70°, interval 1°) reconstruction with IMOD software (bio3d.colorado.edu/imod).

Supplementary Protocol 1

Supplementary Protocol 1

Supplementary Protocol 2

Supplementary Protocol 2

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Source Data Supplementary Fig. 17

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Jiang, Z., Jin, X., Li, Y. et al. Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells. Nat Methods 17, 937–946 (2020). https://doi.org/10.1038/s41592-020-0911-z

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