A robust method for particulate detection of a genetic tag for 3D electron microscopy

  1. James Rae
  2. Charles Ferguson
  3. Nicholas Ariotti
  4. Richard I Webb
  5. Han-Hao Cheng
  6. James L Mead
  7. James D Riches
  8. Dominic JB Hunter
  9. Nick Martel
  10. Joanne Baltos
  11. Arthur Christopoulos
  12. Nicole Sarah Bryce
  13. Maria Lastra Cagigas
  14. Sachini Fonseka
  15. Marcel Ethan Sayre
  16. Edna C Hardeman
  17. Peter W Gunning
  18. Yann Gambin
  19. Thomas E Hall
  20. Robert G Parton  Is a corresponding author
  1. University of Queensland, Australia
  2. University of New South Wales, Australia
  3. Queensland University of Technology, Australia
  4. Monash University, Australia

Abstract

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

Data availability

All data generated or analysed during this study are included in the manuscript.

Article and author information

Author details

  1. James Rae

    Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
  2. Charles Ferguson

    Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
  3. Nicholas Ariotti

    Electron Microscope Unit, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  4. Richard I Webb

    Centre for Microscopy and Microanaysis, University of Queensland, St.Lucia, Australia
    Competing interests
    The authors declare that no competing interests exist.
  5. Han-Hao Cheng

    Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
  6. James L Mead

    Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
  7. James D Riches

    Science and Engineering Faculty, School of Biology & Environmental Science, Queensland University of Technology, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8494-4743
  8. Dominic JB Hunter

    The Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1826-6902
  9. Nick Martel

    Cell Biology and Molecular Medicine, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
  10. Joanne Baltos

    Drug Discovery Biology, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  11. Arthur Christopoulos

    Drug Discovery Biology, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  12. Nicole Sarah Bryce

    School of Medical Sciences, University of New South Wales, Kensington, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9799-7393
  13. Maria Lastra Cagigas

    School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
  14. Sachini Fonseka

    Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
  15. Marcel Ethan Sayre

    Vision Group, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
  16. Edna C Hardeman

    School of Medical Sciences, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1649-7712
  17. Peter W Gunning

    School of Medical Sciences, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0833-3128
  18. Yann Gambin

    EMBL Australia Node in Single Molecule Sciences, University of New South Wales, sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7378-8976
  19. Thomas E Hall

    Cell Biology and Molecular Medicine, University of Queensland, Brisbane, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7718-7614
  20. Robert G Parton

    Vision Group, University of Queensland, Brisbane, Australia
    For correspondence
    r.parton@imb.uq.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7494-5248

Funding

National Health and Medical Research Council (APP1140064)

  • Robert G Parton

The Kid's Cancer Project

  • Edna C Hardeman
  • Peter W Gunning

International Cotutelle Macquarie University Research Excellence Scholarship (iMQRES 2019060)

  • Marcel Ethan Sayre

National Health and Medical Research Council (APP1150083)

  • Arthur Christopoulos
  • Robert G Parton

National Health and Medical Research Council (APP1156489)

  • Robert G Parton

National Health and Medical Research Council (APP1185021)

  • Nicholas Ariotti

Australian Research Council centre of excellence in Convergent Bio-Nano Science and Technology (CE140100036)

  • Robert G Parton

Australian Department of Industry, Innovation and Science Cooperative Research Centre Project (CRC-P)

  • Edna C Hardeman
  • Peter W Gunning

Australian Research Council (DP160101623)

  • Edna C Hardeman
  • Peter W Gunning

National Health and Medical Research Council (APP1100202)

  • Edna C Hardeman
  • Peter W Gunning

National Health and Medical Research Council (APP1079866)

  • Edna C Hardeman
  • Peter W Gunning

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Adam Frost, University of California, San Francisco, United States

Publication history

  1. Received: November 5, 2020
  2. Accepted: April 26, 2021
  3. Accepted Manuscript published: April 27, 2021 (version 1)
  4. Version of Record published: May 7, 2021 (version 2)

Copyright

© 2021, Rae et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. James Rae
  2. Charles Ferguson
  3. Nicholas Ariotti
  4. Richard I Webb
  5. Han-Hao Cheng
  6. James L Mead
  7. James D Riches
  8. Dominic JB Hunter
  9. Nick Martel
  10. Joanne Baltos
  11. Arthur Christopoulos
  12. Nicole Sarah Bryce
  13. Maria Lastra Cagigas
  14. Sachini Fonseka
  15. Marcel Ethan Sayre
  16. Edna C Hardeman
  17. Peter W Gunning
  18. Yann Gambin
  19. Thomas E Hall
  20. Robert G Parton
(2021)
A robust method for particulate detection of a genetic tag for 3D electron microscopy
eLife 10:e64630.
https://doi.org/10.7554/eLife.64630

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