Quantification of tissue-specific protein translation in whole C. elegans using O-propargyl-puromycin labeling and fluorescence microscopy.
| Autor: | Somers HM; Mount Desert Island Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Salisbury Cove, ME 04609, USA., Fuqua JH; Mount Desert Island Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Salisbury Cove, ME 04609, USA., Bonnet FXA; Mount Desert Island Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Salisbury Cove, ME 04609, USA., Rollins JA; Mount Desert Island Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Salisbury Cove, ME 04609, USA. |
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| Jazyk: | angličtina |
| Zdroj: | Cell reports methods [Cell Rep Methods] 2022 Apr 25; Vol. 2 (4), pp. 100203. Date of Electronic Publication: 2022 Apr 25 (Print Publication: 2022). |
| DOI: | 10.1016/j.crmeth.2022.100203 |
| Abstrakt: | The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in C. elegans , they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact C. elegans , we report the application of O-propargyl-puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors . Furthermore, we demonstrate that O-propargyl-puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age. Competing Interests: J.A.R. is a paid consultant for Biocomp Instruments (Fredericton, Canada). (© 2022 The Author(s).) |
| Databáze: | MEDLINE |
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