A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy
Autophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensi...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2024-12-01
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| Series: | Autophagy Reports |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/27694127.2024.2371736 |
| _version_ | 1826945199892332544 |
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| author | Zachary D. Dawson Hemalatha Sundaramoorthi Suk Regmi Bo Zhang Stephanie Morrison Sara M. Fielder Jessie R. Zhang Hieu Hoang David H. Perlmutter Cliff J. Luke Gary A. Silverman Stephen C. Pak |
| author_facet | Zachary D. Dawson Hemalatha Sundaramoorthi Suk Regmi Bo Zhang Stephanie Morrison Sara M. Fielder Jessie R. Zhang Hieu Hoang David H. Perlmutter Cliff J. Luke Gary A. Silverman Stephen C. Pak |
| author_sort | Zachary D. Dawson |
| collection | DOAJ |
| description | Autophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensive and not readily amenable to high-throughput procedures. Here we describe a fluorescent reporter, GFP::LGG-1::mKate2, which is useful for monitoring autophagic flux in live animals. In the intestine, the fusion protein is processed by endogenous ATG-4 to generate GFP::LGG-1 and mKate2 proteins. We provide data indicating that the GFP:mKate ratio is a suitable readout for measuring cellular autophagic flux. Using this reporter, we measured autophagic flux in L1 larvae to day 7 adult animals. We show that basal autophagic flux is relatively low during larval development but increases markedly in reproductive adults before decreasing with age. Furthermore, we show that wild-type, eat-2, and daf-2 mutant animals have distinct autophagic flux profiles through post-embryonic development. Finally, we demonstrate the utility of this reporter by performing a high-content small molecule screen to identify compounds that alter autophagic flux in C. elegans. |
| first_indexed | 2025-02-17T20:51:07Z |
| format | Article |
| id | doaj.art-50031cc4024141b38ff18745781f19c2 |
| institution | Directory Open Access Journal |
| issn | 2769-4127 |
| language | English |
| last_indexed | 2025-02-17T20:51:07Z |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Autophagy Reports |
| spelling | doaj.art-50031cc4024141b38ff18745781f19c22024-12-09T07:19:32ZengTaylor & Francis GroupAutophagy Reports2769-41272024-12-013110.1080/27694127.2024.2371736A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagyZachary D. Dawson0Hemalatha Sundaramoorthi1Suk Regmi2Bo Zhang3Stephanie Morrison4Sara M. Fielder5Jessie R. Zhang6Hieu Hoang7David H. Perlmutter8Cliff J. Luke9Gary A. Silverman10Stephen C. Pak11Department of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USADepartment of Pediatrics, Washington University in St Louis School of Medicine, St Louis, Washington 63110, USAAutophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensive and not readily amenable to high-throughput procedures. Here we describe a fluorescent reporter, GFP::LGG-1::mKate2, which is useful for monitoring autophagic flux in live animals. In the intestine, the fusion protein is processed by endogenous ATG-4 to generate GFP::LGG-1 and mKate2 proteins. We provide data indicating that the GFP:mKate ratio is a suitable readout for measuring cellular autophagic flux. Using this reporter, we measured autophagic flux in L1 larvae to day 7 adult animals. We show that basal autophagic flux is relatively low during larval development but increases markedly in reproductive adults before decreasing with age. Furthermore, we show that wild-type, eat-2, and daf-2 mutant animals have distinct autophagic flux profiles through post-embryonic development. Finally, we demonstrate the utility of this reporter by performing a high-content small molecule screen to identify compounds that alter autophagic flux in C. elegans.https://www.tandfonline.com/doi/10.1080/27694127.2024.2371736Biomarkerhigh-content screeningLC3LGG-1probesmall molecule |
| spellingShingle | Zachary D. Dawson Hemalatha Sundaramoorthi Suk Regmi Bo Zhang Stephanie Morrison Sara M. Fielder Jessie R. Zhang Hieu Hoang David H. Perlmutter Cliff J. Luke Gary A. Silverman Stephen C. Pak A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy Autophagy Reports Biomarker high-content screening LC3 LGG-1 probe small molecule |
| title | A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy |
| title_full | A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy |
| title_fullStr | A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy |
| title_full_unstemmed | A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy |
| title_short | A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy |
| title_sort | fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during c elegans post embryonic development and identifies compounds that modulate autophagy |
| topic | Biomarker high-content screening LC3 LGG-1 probe small molecule |
| url | https://www.tandfonline.com/doi/10.1080/27694127.2024.2371736 |
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