Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells
Adenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent AT...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-02-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/12/5/695 |
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| author | Donnell White Lothar Lauterboeck Parnia Mobasheran Tetsuya Kitaguchi Antoine H. Chaanine Qinglin Yang |
| author_facet | Donnell White Lothar Lauterboeck Parnia Mobasheran Tetsuya Kitaguchi Antoine H. Chaanine Qinglin Yang |
| author_sort | Donnell White |
| collection | DOAJ |
| description | Adenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease. |
| first_indexed | 2024-03-11T07:28:25Z |
| format | Article |
| id | doaj.art-26dafbd0c1c44a6d8d2038e88311693c |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-11T07:28:25Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-26dafbd0c1c44a6d8d2038e88311693c2023-11-17T07:26:56ZengMDPI AGCells2073-44092023-02-0112569510.3390/cells12050695Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured CellsDonnell White0Lothar Lauterboeck1Parnia Mobasheran2Tetsuya Kitaguchi3Antoine H. Chaanine4Qinglin Yang5Cardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USACardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USACardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USALaboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Kanagawa, JapanHealthPartners Group, Regions Hospital, Saint Paul, MN 55101, USACardiovascular Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USAAdenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.https://www.mdpi.com/2073-4409/12/5/695mitochondriaATPbiosensorfluorescencemetabolism |
| spellingShingle | Donnell White Lothar Lauterboeck Parnia Mobasheran Tetsuya Kitaguchi Antoine H. Chaanine Qinglin Yang Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells Cells mitochondria ATP biosensor fluorescence metabolism |
| title | Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells |
| title_full | Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells |
| title_fullStr | Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells |
| title_full_unstemmed | Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells |
| title_short | Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells |
| title_sort | real time visualization of cytosolic and mitochondrial atp dynamics in response to metabolic stress in cultured cells |
| topic | mitochondria ATP biosensor fluorescence metabolism |
| url | https://www.mdpi.com/2073-4409/12/5/695 |
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