A robust method for particulate detection of a genetic tag for 3D electron microscopy
- University of Queensland, Australia
- University of New South Wales, Australia
- Queensland University of Technology, Australia
- 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
Nicole Sarah Bryce
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.
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.
Metrics
-
- 3,287
- views
-
- 461
- downloads
-
- 21
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A robust method for particulate detection of a genetic tag for 3D electron microscopy
eLife 10:e64630.
https://doi.org/10.7554/eLife.64630
Further reading
-
- Cell Biology
- Genetics and Genomics
Single-nucleus RNA sequencing (snRNA-seq), an alternative to single-cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying mouse adipose tissue remodeling during obesity. By integrating bulk nuclear RNA-seq from adipocyte nuclei of different sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. These subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot. Specifically, we identify a key molecular feature of dysfunctional hypertrophic adipocytes, a global shutdown in gene expression, along with elevated stress and inflammatory responses. Furthermore, our differential gene expression analysis reveals distinct contributions of adipocyte subpopulations to the overall pathophysiology of adipose tissue. Our study establishes a robust snRNA-seq method, providing novel insights into the biological processes involved in adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.
-
- Cell Biology
A combination of intermittent fasting and administering Wnt3a proteins to a bone injury can rejuvenate bone repair in aged mice.
