An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos
Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive and reliable procedures to visualize sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at sub...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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The Company of Biologists
2018-12-01
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| Series: | Biology Open |
| Subjects: | |
| Online Access: | http://bio.biologists.org/content/7/12/bio039362 |
| _version_ | 1818859753203302400 |
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| author | Inês Garcez Palha Isabelle Anselme Sylvie Schneider-Maunoury François Giudicelli |
| author_facet | Inês Garcez Palha Isabelle Anselme Sylvie Schneider-Maunoury François Giudicelli |
| author_sort | Inês Garcez Palha |
| collection | DOAJ |
| description | Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive and reliable procedures to visualize sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique (
Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. The addition of a cell-permeable protease inhibitor instantly stabilizes newly produced tagged protein allowing us to distinguish recently synthesized proteins from pre-existing ones. After a brief protease inhibitor treatment, the ratio of tagged versus non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged proteins it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development. |
| first_indexed | 2024-12-19T09:17:11Z |
| format | Article |
| id | doaj.art-d95e8bb4a24a41d1aa72d030a529a5fb |
| institution | Directory Open Access Journal |
| issn | 2046-6390 |
| language | English |
| last_indexed | 2024-12-19T09:17:11Z |
| publishDate | 2018-12-01 |
| publisher | The Company of Biologists |
| record_format | Article |
| series | Biology Open |
| spelling | doaj.art-d95e8bb4a24a41d1aa72d030a529a5fb2022-12-21T20:28:05ZengThe Company of BiologistsBiology Open2046-63902018-12-0171210.1242/bio.039362039362An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryosInês Garcez Palha0Isabelle Anselme1Sylvie Schneider-Maunoury2François Giudicelli3 Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du développement (LBD), F-75005 Paris, France Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du développement (LBD), F-75005 Paris, France Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du développement (LBD), F-75005 Paris, France Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du développement (LBD), F-75005 Paris, France Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive and reliable procedures to visualize sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique ( Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. The addition of a cell-permeable protease inhibitor instantly stabilizes newly produced tagged protein allowing us to distinguish recently synthesized proteins from pre-existing ones. After a brief protease inhibitor treatment, the ratio of tagged versus non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged proteins it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.http://bio.biologists.org/content/7/12/bio039362SPoTTimeStampLocal translationNeuronZebrafish |
| spellingShingle | Inês Garcez Palha Isabelle Anselme Sylvie Schneider-Maunoury François Giudicelli An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos Biology Open SPoT TimeStamp Local translation Neuron Zebrafish |
| title | An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos |
| title_full | An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos |
| title_fullStr | An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos |
| title_full_unstemmed | An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos |
| title_short | An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos |
| title_sort | in vivo translation reporter system for the study of protein synthesis in zebrafish embryos |
| topic | SPoT TimeStamp Local translation Neuron Zebrafish |
| url | http://bio.biologists.org/content/7/12/bio039362 |
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