Methods for detection of cardiac glycogen-autophagy
Glycogen-autophagy (‘glycophagy’) is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg...
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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 |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/27694127.2024.2405331 |
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| author | Parisa Koutsifeli Lorna J. Daniels Joshua Neale Sarah Fong Upasna Varma Marco Annandale Xun Li Yohanes Nursalim James R. Bell Kate L. Weeks Aleksandr Stotland David J. Taylor Roberta A. Gottlieb Lea M.D. Delbridge Kimberley M. Mellor |
| author_facet | Parisa Koutsifeli Lorna J. Daniels Joshua Neale Sarah Fong Upasna Varma Marco Annandale Xun Li Yohanes Nursalim James R. Bell Kate L. Weeks Aleksandr Stotland David J. Taylor Roberta A. Gottlieb Lea M.D. Delbridge Kimberley M. Mellor |
| author_sort | Parisa Koutsifeli |
| collection | DOAJ |
| description | Glycogen-autophagy (‘glycophagy’) is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease. |
| format | Article |
| id | doaj-art-896be00534814dda8ffacc5947942049 |
| institution | Kabale University |
| issn | 2769-4127 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Autophagy Reports |
| spelling | doaj-art-896be00534814dda8ffacc59479420492024-12-09T07:19:32ZengTaylor & Francis GroupAutophagy Reports2769-41272024-12-013110.1080/27694127.2024.2405331Methods for detection of cardiac glycogen-autophagyParisa Koutsifeli0Lorna J. Daniels1Joshua Neale2Sarah Fong3Upasna Varma4Marco Annandale5Xun Li6Yohanes Nursalim7James R. Bell8Kate L. Weeks9Aleksandr Stotland10David J. Taylor11Roberta A. Gottlieb12Lea M.D. Delbridge13Kimberley M. Mellor14Department of Physiology, University of Auckland, New ZealandDepartment of Physiology, University of Auckland, New ZealandDepartment of Physiology, University of Auckland, New ZealandDepartment of Physiology, University of Auckland, New ZealandDepartment of Anatomy & Physiology, University of Melbourne, AustraliaDepartment of Physiology, University of Auckland, New ZealandDepartment of Physiology, University of Auckland, New ZealandDepartment of Physiology, University of Auckland, New ZealandDepartment of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, AustraliaDepartment of Anatomy & Physiology, University of Melbourne, AustraliaDepartment of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USADepartment of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USADepartment of Cardiology, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USADepartment of Physiology, University of Auckland, New ZealandDepartment of Physiology, University of Auckland, New ZealandGlycogen-autophagy (‘glycophagy’) is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.https://www.tandfonline.com/doi/10.1080/27694127.2024.2405331Atg8autophagyGABARAPL1glycogenglycophagy flux |
| spellingShingle | Parisa Koutsifeli Lorna J. Daniels Joshua Neale Sarah Fong Upasna Varma Marco Annandale Xun Li Yohanes Nursalim James R. Bell Kate L. Weeks Aleksandr Stotland David J. Taylor Roberta A. Gottlieb Lea M.D. Delbridge Kimberley M. Mellor Methods for detection of cardiac glycogen-autophagy Autophagy Reports Atg8 autophagy GABARAPL1 glycogen glycophagy flux |
| title | Methods for detection of cardiac glycogen-autophagy |
| title_full | Methods for detection of cardiac glycogen-autophagy |
| title_fullStr | Methods for detection of cardiac glycogen-autophagy |
| title_full_unstemmed | Methods for detection of cardiac glycogen-autophagy |
| title_short | Methods for detection of cardiac glycogen-autophagy |
| title_sort | methods for detection of cardiac glycogen autophagy |
| topic | Atg8 autophagy GABARAPL1 glycogen glycophagy flux |
| url | https://www.tandfonline.com/doi/10.1080/27694127.2024.2405331 |
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