Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes
Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin m...
| Main Authors: | , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
eLife Sciences Publications, Ltd.
2016
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| Online Access: | http://hdl.handle.net/1721.1/101427 https://orcid.org/0000-0002-6199-6855 |
| _version_ | 1826205717778923520 |
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| author | Katz, Zachary B English, Brian P Lionnet, Timothee Yoon, Young J Monnier, Nilah Ovryn, Ben Bathe, Mark Singer, Robert H |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Katz, Zachary B English, Brian P Lionnet, Timothee Yoon, Young J Monnier, Nilah Ovryn, Ben Bathe, Mark Singer, Robert H |
| author_sort | Katz, Zachary B |
| collection | MIT |
| description | Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. |
| first_indexed | 2024-09-23T13:17:29Z |
| format | Article |
| id | mit-1721.1/101427 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:17:29Z |
| publishDate | 2016 |
| publisher | eLife Sciences Publications, Ltd. |
| record_format | dspace |
| spelling | mit-1721.1/1014272022-10-01T14:20:07Z Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes Katz, Zachary B English, Brian P Lionnet, Timothee Yoon, Young J Monnier, Nilah Ovryn, Ben Bathe, Mark Singer, Robert H Massachusetts Institute of Technology. Department of Biological Engineering Monnier, Nilah Bathe, Mark Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. National Science Foundation (U.S.). Office of the Director (Grant PoLS PHY 1305537) 2016-03-03T02:23:58Z 2016-03-03T02:23:58Z 2016-01 2015-07 Article http://purl.org/eprint/type/JournalArticle 2050-084X http://hdl.handle.net/1721.1/101427 Katz, Zachary B, Brian P English, Timothee Lionnet, Young J Yoon, Nilah Monnier, Ben Ovryn, Mark Bathe, and Robert H Singer. “Mapping Translation ‘Hot-Spots’ in Live Cells by Tracking Single Molecules of mRNA and Ribosomes.” eLife 5 (January 13, 2016). https://orcid.org/0000-0002-6199-6855 en_US http://dx.doi.org/10.7554/eLife.10415 eLife Creative Commons Attribution http://creativecommons.org/licenses/by/4.0/ application/pdf eLife Sciences Publications, Ltd. eLife Sciences Publications, Ltd. |
| spellingShingle | Katz, Zachary B English, Brian P Lionnet, Timothee Yoon, Young J Monnier, Nilah Ovryn, Ben Bathe, Mark Singer, Robert H Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes |
| title | Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes |
| title_full | Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes |
| title_fullStr | Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes |
| title_full_unstemmed | Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes |
| title_short | Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes |
| title_sort | mapping translation hot spots in live cells by tracking single molecules of mrna and ribosomes |
| url | http://hdl.handle.net/1721.1/101427 https://orcid.org/0000-0002-6199-6855 |
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