Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy
Autophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either comple...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-04-01
|
| Series: | Cells |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4409/12/8/1134 |
| _version_ | 1797605978774962176 |
|---|---|
| author | Natalia Jimenez-Moreno Carla Salomo-Coll Laura C. Murphy Simon Wilkinson |
| author_facet | Natalia Jimenez-Moreno Carla Salomo-Coll Laura C. Murphy Simon Wilkinson |
| author_sort | Natalia Jimenez-Moreno |
| collection | DOAJ |
| description | Autophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either complex, low throughput or not sensitive enough for reliable quantitative results. Recently, ER-phagy has emerged as a physiologically relevant pathway to maintain ER homeostasis but the process is poorly understood, highlighting the need for tools to monitor ER-phagy flux. In this study, we validate the use of the signal-retaining autophagy indicator (SRAI), a fixable fluorescent probe recently generated and described to detect mitophagy, as a versatile, sensitive and convenient probe for monitoring ER-phagy. This includes the study of either general selective degradation of the endoplasmic reticulum (ER-phagy) or individual forms of ER-phagy involving specific cargo receptors (e.g., FAM134B, FAM134C, TEX264 and CCPG1). Crucially, we present a detailed protocol for the quantification of autophagic flux using automated microscopy and high throughput analysis. Overall, this probe provides a reliable and convenient tool for the measurement of ER-phagy. |
| first_indexed | 2024-03-11T05:08:49Z |
| format | Article |
| id | doaj.art-2c2f1751a9654ff0ad3ec4766cba7042 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T05:08:49Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-2c2f1751a9654ff0ad3ec4766cba70422023-11-17T18:43:00ZengMDPI AGCells2073-44092023-04-01128113410.3390/cells12081134Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-PhagyNatalia Jimenez-Moreno0Carla Salomo-Coll1Laura C. Murphy2Simon Wilkinson3Cancer Research UK Scotland Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XR, UKCancer Research UK Scotland Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XR, UKMRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UKCancer Research UK Scotland Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XR, UKAutophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either complex, low throughput or not sensitive enough for reliable quantitative results. Recently, ER-phagy has emerged as a physiologically relevant pathway to maintain ER homeostasis but the process is poorly understood, highlighting the need for tools to monitor ER-phagy flux. In this study, we validate the use of the signal-retaining autophagy indicator (SRAI), a fixable fluorescent probe recently generated and described to detect mitophagy, as a versatile, sensitive and convenient probe for monitoring ER-phagy. This includes the study of either general selective degradation of the endoplasmic reticulum (ER-phagy) or individual forms of ER-phagy involving specific cargo receptors (e.g., FAM134B, FAM134C, TEX264 and CCPG1). Crucially, we present a detailed protocol for the quantification of autophagic flux using automated microscopy and high throughput analysis. Overall, this probe provides a reliable and convenient tool for the measurement of ER-phagy.https://www.mdpi.com/2073-4409/12/8/1134autophagyfluorescent proteinreporterSRAIautophagy fluxlysosome |
| spellingShingle | Natalia Jimenez-Moreno Carla Salomo-Coll Laura C. Murphy Simon Wilkinson Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy Cells autophagy fluorescent protein reporter SRAI autophagy flux lysosome |
| title | Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy |
| title_full | Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy |
| title_fullStr | Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy |
| title_full_unstemmed | Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy |
| title_short | Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy |
| title_sort | signal retaining autophagy indicator as a quantitative imaging method for er phagy |
| topic | autophagy fluorescent protein reporter SRAI autophagy flux lysosome |
| url | https://www.mdpi.com/2073-4409/12/8/1134 |
| work_keys_str_mv | AT nataliajimenezmoreno signalretainingautophagyindicatorasaquantitativeimagingmethodforerphagy AT carlasalomocoll signalretainingautophagyindicatorasaquantitativeimagingmethodforerphagy AT lauracmurphy signalretainingautophagyindicatorasaquantitativeimagingmethodforerphagy AT simonwilkinson signalretainingautophagyindicatorasaquantitativeimagingmethodforerphagy |