Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy
Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments[superscript 1] or require light and can be difficult to use[superscript 2]. Here we report the development of 'APEX...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2015
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| Online Access: | http://hdl.handle.net/1721.1/95743 https://orcid.org/0000-0002-8277-5226 |
| _version_ | 1826196528822222848 |
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| author | Deerinck, Thomas J. Sancak, Yasemin Poulos, Thomas L. Mootha, Vamsi K. Sosinsky, Gina E. Ellisman, Mark H. Martell, Jeffrey Daniel Ting, Alice Y. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Deerinck, Thomas J. Sancak, Yasemin Poulos, Thomas L. Mootha, Vamsi K. Sosinsky, Gina E. Ellisman, Mark H. Martell, Jeffrey Daniel Ting, Alice Y. |
| author_sort | Deerinck, Thomas J. |
| collection | MIT |
| description | Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments[superscript 1] or require light and can be difficult to use[superscript 2]. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed[superscript 3, 4]. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen. |
| first_indexed | 2024-09-23T10:28:03Z |
| format | Article |
| id | mit-1721.1/95743 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:28:03Z |
| publishDate | 2015 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/957432022-09-27T09:40:25Z Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy Deerinck, Thomas J. Sancak, Yasemin Poulos, Thomas L. Mootha, Vamsi K. Sosinsky, Gina E. Ellisman, Mark H. Martell, Jeffrey Daniel Ting, Alice Y. Massachusetts Institute of Technology. Department of Chemistry Martell, Jeffrey Daniel Ting, Alice Y. Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments[superscript 1] or require light and can be difficult to use[superscript 2]. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed[superscript 3, 4]. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen. National Institutes of Health (U.S.) (Grant DP1 OD003961) National Science Foundation (U.S.). Graduate Research Fellowship Program United States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships) 2015-03-03T15:25:56Z 2015-03-03T15:25:56Z 2012-10 2012-06 Article http://purl.org/eprint/type/JournalArticle 1087-0156 1546-1696 http://hdl.handle.net/1721.1/95743 Martell, Jeffrey D, Thomas J Deerinck, Yasemin Sancak, Thomas L Poulos, Vamsi K Mootha, Gina E Sosinsky, Mark H Ellisman, and Alice Y Ting. “Engineered Ascorbate Peroxidase as a Genetically Encoded Reporter for Electron Microscopy.” Nature Biotechnology 30, no. 11 (October 21, 2012): 1143–1148. https://orcid.org/0000-0002-8277-5226 en_US http://dx.doi.org/10.1038/nbt.2375 Nature Biotechnology Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Nature Publishing Group PMC |
| spellingShingle | Deerinck, Thomas J. Sancak, Yasemin Poulos, Thomas L. Mootha, Vamsi K. Sosinsky, Gina E. Ellisman, Mark H. Martell, Jeffrey Daniel Ting, Alice Y. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy |
| title | Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy |
| title_full | Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy |
| title_fullStr | Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy |
| title_full_unstemmed | Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy |
| title_short | Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy |
| title_sort | engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy |
| url | http://hdl.handle.net/1721.1/95743 https://orcid.org/0000-0002-8277-5226 |
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