A high throughput screen of real-time ATP levels in individual cells reveals mechanisms of energy failure
Bryce A. Mendelsohn1, 2, Neal K. Bennett1, Maxwell Darch1, Katharine Yu1, Max Horlbeck3, Luke Gilbert3, William Hyun4, Martin Kampmann5, Jean L. Nakamura6 and Ken Nakamura*1,7 1Gladstone Institute of Neurological Disease, San Francisco, CA, 94158, USA; 2Department of Pediatrics, 3Department of Cellular and Molecular Pharmacology, 4Department of Laboratory Medicine, 5Department of Biochemistry and Biophysics and Institute for Neurodegenerative Diseases, 6Department of Radiation Oncology, and 7Department of Neurology, University of California, San Francisco, CA 94158, USA
Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, yet no systematic understanding exists of critical pathways for regulating ATP levels. We combined a pooled CRISPRi library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting in a high-throughput genetic screen assaying ATP concentrations in live human cells. With this approach, we identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels specifically under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which CoQ10 supplementation rescued ATP deficits caused by knockdown, including a subset of CoQ10 biosynthetic genes associated with human disease and at least one gene not linked to CoQ10 biosynthesis. Validation studies are ongoing. This screening paradigm shows that FRET and FACS can be used to screen a metabolite based on the real-time level, and reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.
This work was supported by a Burroughs-Wellcome Fund Award, NIH RO1NS091902, NIH/NCI U54CA196519, NIH R01 DA036858, NIH/NIGMS DP2 GM119139 and the Pediatric Scientist Development Program (PSDP) award.
Credits: None available.
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