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

James Rae; Charles Ferguson; Nicholas Ariotti; Richard I. Webb; Han Hao Cheng; James L. Mead; James D. Riches; Dominic J.B. Hunter; Nick Martel; Joanne Baltos; Arthur Christopoulos; Nicole S. Bryce; Maria Lastra Cagigas; Sachini Fonseka; Marcel E. Sayre; Edna C. Hardeman; Peter W. Gunning; Yann Gambin; Thomas E. Hall; Robert G. Parton

doi:10.7554/ELIFE.64630

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

James Rae, Charles Ferguson, Nicholas Ariotti, Richard I. Webb, Han Hao Cheng, James L. Mead, James D. Riches, Dominic J.B. Hunter, Nick Martel, Joanne Baltos, Arthur Christopoulos, Nicole S. Bryce, Maria Lastra Cagigas, Sachini Fonseka, Marcel E. Sayre, Edna C. Hardeman, Peter W. Gunning, Yann Gambin, Thomas E. Hall, Robert G. Parton

Drug Discovery Biology

Research output: Contribution to journal › Article › Research › peer-review

16 Citations (Scopus)

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 (EM). 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 EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, 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.

Original language	English
Article number	e64630
Number of pages	17
Journal	eLife
Volume	10
DOIs	https://doi.org/10.7554/ELIFE.64630
Publication status	Published - 27 Apr 2021

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10.7554/ELIFE.64630Licence: CC BY

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Rae, J., Ferguson, C., Ariotti, N., Webb, R. I., Cheng, H. H., Mead, J. L., Riches, J. D., Hunter, D. J. B., Martel, N., Baltos, J., Christopoulos, A., Bryce, N. S., Cagigas, M. L., Fonseka, S., Sayre, M. E., Hardeman, E. C., Gunning, P. W., Gambin, Y., Hall, T. E., & Parton, R. G. (2021). A robust method for particulate detection of a genetic tag for 3D electron microscopy. eLife, 10, Article e64630. https://doi.org/10.7554/ELIFE.64630

@article{e235a0e44d914ff1a8598ead82d17652,

title = "A robust method for particulate detection of a genetic tag for 3D electron microscopy",

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 (EM). 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 EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, 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.",

author = "James Rae and Charles Ferguson and Nicholas Ariotti and Webb, {Richard I.} and Cheng, {Han Hao} and Mead, {James L.} and Riches, {James D.} and Hunter, {Dominic J.B.} and Nick Martel and Joanne Baltos and Arthur Christopoulos and Bryce, {Nicole S.} and Cagigas, {Maria Lastra} and Sachini Fonseka and Sayre, {Marcel E.} and Hardeman, {Edna C.} and Gunning, {Peter W.} and Yann Gambin and Hall, {Thomas E.} and Parton, {Robert G.}",

note = "Funding Information: The authors acknowledge the help of staff and use of facilities in the Microscopy Australia NCRIS Facility at the Centre for Microscopy and Microanalysis at The University of Queensland. The authors acknowledge the use of the Cryo Electron Microscopy Facility through the Victor Chang Innovation Centre, funded by the NSW government, the Electron Microscope Unit within the Mark Wainwright Analytical Centre at UNSW Sydney and Microscopy Australia. This work was supported by the National Health and Medical Research Council of Australia (grants APP1140064 and APP1150083 and fellowship APP1156489 to RGP; APP1185021 to NA). RGP is supported by the Australian Research Council (ARC) Centre of Excellence in Convergent Bio-Nano Science and Technology. This work was also supported by an Australian Department of Industry, Innovation and Science Cooperative Research Centre Project (CRC-P) grant to PWG and ECH, and grants from the Australian Research Council (ARC grant DP160101623), the Australian National Health and Medical Research Council (NHMRC grant APP1100202, APP1079866), and The Kid{\textquoteright}s Cancer Project to PWG and ECH. MES is funded by an International Cotutelle Macquarie University Research Excellence Scholarship (iMQRES 2019060). Funding Information: The authors acknowledge the help of staff and use of facilities in the Microscopy Australia NCRIS Facility at the Centre for Microscopy and Microanalysis at The University of Queensland. The authors acknowledge the use of the Cryo Electron Microscopy Facility through the Victor Chang Innovation Centre, funded by the NSW government, the Electron Microscope Unit within the Mark Wainwright Analytical Centre at UNSW Sydney and Microscopy Australia. This work was supported by the National Health and Medical Research Council of Australia (grants APP1140064 and APP1150083 and fellowship APP1156489 to RGP; APP1185021 to NA). RGP is supported by the Australian Research Council (ARC) Centre of Excellence in Convergent Bio-Nano Science and Technology. This work was also supported by an Australian Department of Industry, Innovation and Science Cooperative Research Centre Project (CRC-P) grant to PWG and ECH, and grants from the Australian Research Council (ARC grant DP160101623), the Australian National Health and Medical Research Council (NHMRC grant APP1100202, APP1079866), and The Kid?s Cancer Project to PWG and ECH. MES is funded by an International Cotutelle Macquarie University Research Excellence Scholarship (iMQRES 2019060). Publisher Copyright: {\textcopyright} Rae et al. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = apr,

day = "27",

doi = "10.7554/ELIFE.64630",

language = "English",

volume = "10",

journal = "eLife",

issn = "2050-084X",

publisher = "eLife Sciences Organisation, Ltd.",

}

Rae, J, Ferguson, C, Ariotti, N, Webb, RI, Cheng, HH, Mead, JL, Riches, JD, Hunter, DJB, Martel, N, Baltos, J , Christopoulos, A, Bryce, NS, Cagigas, ML, Fonseka, S, Sayre, ME, Hardeman, EC, Gunning, PW, Gambin, Y, Hall, TE & Parton, RG 2021, 'A robust method for particulate detection of a genetic tag for 3D electron microscopy', eLife, vol. 10, e64630. https://doi.org/10.7554/ELIFE.64630

TY - JOUR

T1 - A robust method for particulate detection of a genetic tag for 3D electron microscopy

AU - Rae, James

AU - Ferguson, Charles

AU - Ariotti, Nicholas

AU - Webb, Richard I.

AU - Cheng, Han Hao

AU - Mead, James L.

AU - Riches, James D.

AU - Hunter, Dominic J.B.

AU - Martel, Nick

AU - Baltos, Joanne

AU - Christopoulos, Arthur

AU - Bryce, Nicole S.

AU - Cagigas, Maria Lastra

AU - Fonseka, Sachini

AU - Sayre, Marcel E.

AU - Hardeman, Edna C.

AU - Gunning, Peter W.

AU - Gambin, Yann

AU - Hall, Thomas E.

AU - Parton, Robert G.

N1 - Funding Information: The authors acknowledge the help of staff and use of facilities in the Microscopy Australia NCRIS Facility at the Centre for Microscopy and Microanalysis at The University of Queensland. The authors acknowledge the use of the Cryo Electron Microscopy Facility through the Victor Chang Innovation Centre, funded by the NSW government, the Electron Microscope Unit within the Mark Wainwright Analytical Centre at UNSW Sydney and Microscopy Australia. This work was supported by the National Health and Medical Research Council of Australia (grants APP1140064 and APP1150083 and fellowship APP1156489 to RGP; APP1185021 to NA). RGP is supported by the Australian Research Council (ARC) Centre of Excellence in Convergent Bio-Nano Science and Technology. This work was also supported by an Australian Department of Industry, Innovation and Science Cooperative Research Centre Project (CRC-P) grant to PWG and ECH, and grants from the Australian Research Council (ARC grant DP160101623), the Australian National Health and Medical Research Council (NHMRC grant APP1100202, APP1079866), and The Kid’s Cancer Project to PWG and ECH. MES is funded by an International Cotutelle Macquarie University Research Excellence Scholarship (iMQRES 2019060). Funding Information: The authors acknowledge the help of staff and use of facilities in the Microscopy Australia NCRIS Facility at the Centre for Microscopy and Microanalysis at The University of Queensland. The authors acknowledge the use of the Cryo Electron Microscopy Facility through the Victor Chang Innovation Centre, funded by the NSW government, the Electron Microscope Unit within the Mark Wainwright Analytical Centre at UNSW Sydney and Microscopy Australia. This work was supported by the National Health and Medical Research Council of Australia (grants APP1140064 and APP1150083 and fellowship APP1156489 to RGP; APP1185021 to NA). RGP is supported by the Australian Research Council (ARC) Centre of Excellence in Convergent Bio-Nano Science and Technology. This work was also supported by an Australian Department of Industry, Innovation and Science Cooperative Research Centre Project (CRC-P) grant to PWG and ECH, and grants from the Australian Research Council (ARC grant DP160101623), the Australian National Health and Medical Research Council (NHMRC grant APP1100202, APP1079866), and The Kid?s Cancer Project to PWG and ECH. MES is funded by an International Cotutelle Macquarie University Research Excellence Scholarship (iMQRES 2019060). Publisher Copyright: © Rae et al. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/4/27

Y1 - 2021/4/27

N2 - 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 (EM). 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 EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, 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.

AB - 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 (EM). 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 EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, 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.

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U2 - 10.7554/ELIFE.64630

DO - 10.7554/ELIFE.64630

M3 - Article

C2 - 33904409

AN - SCOPUS:85105904091

SN - 2050-084X

VL - 10

JO - eLife

JF - eLife

M1 - e64630

ER -

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

Abstract

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Translating membrane proteins into therapeutics; from bedside to bench

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology

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

Abstract

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Translating membrane proteins into therapeutics; from bedside to bench

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology

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