Cargando…
Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2
Determining the cellular localization of proteins of interest at nanometer resolution is necessary for elucidating their functions. Besides super-resolution fluorescence microscopy, conventional electron microscopy (EM) combined with immunolabeling or clonable EM tags provides a unique approach to c...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719812/ https://www.ncbi.nlm.nih.gov/pubmed/29238746 http://dx.doi.org/10.1007/s41048-017-0043-x |
_version_ | 1783284563833782272 |
---|---|
author | Shi, Yang Wang, Li Zhang, Jianguo Zhai, Yujia Sun, Fei |
author_facet | Shi, Yang Wang, Li Zhang, Jianguo Zhai, Yujia Sun, Fei |
author_sort | Shi, Yang |
collection | PubMed |
description | Determining the cellular localization of proteins of interest at nanometer resolution is necessary for elucidating their functions. Besides super-resolution fluorescence microscopy, conventional electron microscopy (EM) combined with immunolabeling or clonable EM tags provides a unique approach to correlate protein localization information and cellular ultrastructural information. However, there are still rare cases of such correlation in three-dimensional (3D) spaces. Here, we developed an approach by combining the focus ion beam scanning electron microscopy (FIB-SEM) and a promising clonable EM tag APEX2 (an enhanced ascorbate peroxidase 2) to determine the target protein localization within 3D cellular ultrastructural context. We further utilized this approach to study the 3D localization of mitochondrial dynamics-related proteins (MiD49/51, Mff, Fis1, and Mfn2) in the cells where the target proteins were overexpressed. We found that all the target proteins were located at the surface of the mitochondrial outer membrane accompanying with mitochondrial clusters. Mid49/51, Mff, and hFis1 spread widely around the mitochondrial surface while Mfn2 only exists at the contact sites. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s41048-017-0043-x) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5719812 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-57198122017-12-11 Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2 Shi, Yang Wang, Li Zhang, Jianguo Zhai, Yujia Sun, Fei Biophys Rep Method Determining the cellular localization of proteins of interest at nanometer resolution is necessary for elucidating their functions. Besides super-resolution fluorescence microscopy, conventional electron microscopy (EM) combined with immunolabeling or clonable EM tags provides a unique approach to correlate protein localization information and cellular ultrastructural information. However, there are still rare cases of such correlation in three-dimensional (3D) spaces. Here, we developed an approach by combining the focus ion beam scanning electron microscopy (FIB-SEM) and a promising clonable EM tag APEX2 (an enhanced ascorbate peroxidase 2) to determine the target protein localization within 3D cellular ultrastructural context. We further utilized this approach to study the 3D localization of mitochondrial dynamics-related proteins (MiD49/51, Mff, Fis1, and Mfn2) in the cells where the target proteins were overexpressed. We found that all the target proteins were located at the surface of the mitochondrial outer membrane accompanying with mitochondrial clusters. Mid49/51, Mff, and hFis1 spread widely around the mitochondrial surface while Mfn2 only exists at the contact sites. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s41048-017-0043-x) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2017-11-04 2017 /pmc/articles/PMC5719812/ /pubmed/29238746 http://dx.doi.org/10.1007/s41048-017-0043-x Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Method Shi, Yang Wang, Li Zhang, Jianguo Zhai, Yujia Sun, Fei Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2 |
title | Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2 |
title_full | Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2 |
title_fullStr | Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2 |
title_full_unstemmed | Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2 |
title_short | Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2 |
title_sort | determining the target protein localization in 3d using the combination of fib-sem and apex2 |
topic | Method |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719812/ https://www.ncbi.nlm.nih.gov/pubmed/29238746 http://dx.doi.org/10.1007/s41048-017-0043-x |
work_keys_str_mv | AT shiyang determiningthetargetproteinlocalizationin3dusingthecombinationoffibsemandapex2 AT wangli determiningthetargetproteinlocalizationin3dusingthecombinationoffibsemandapex2 AT zhangjianguo determiningthetargetproteinlocalizationin3dusingthecombinationoffibsemandapex2 AT zhaiyujia determiningthetargetproteinlocalizationin3dusingthecombinationoffibsemandapex2 AT sunfei determiningthetargetproteinlocalizationin3dusingthecombinationoffibsemandapex2 |