A high-throughput screen of real-time ATP levels in individual cells reveals mechanisms of energy failure

• Published in: PLoS biology Vol. 16; no. 8; p. e2004624
• Main Authors: Mendelsohn, Bryce A, Bennett, Neal K, Darch, Maxwell A, Yu, Katharine, Nguyen, Mai K, Pucciarelli, Daniela, Nelson, Maxine, Horlbeck, Max A, Gilbert, Luke A, Hyun, William, Kampmann, Martin, Nakamura, Jean L, Nakamura, Ken
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27-08-2018; Public Library of Science (PLoS)
• Subjects: Adenosine Triphosphate - analysis; Aging; ATP; Biology and Life Sciences; Biosensors; Biosynthesis; Cell cycle; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Coenzyme Q10; CRISPR; Degenerative diseases; Disease; Energy; Energy metabolism; Energy Metabolism - physiology; Flow cytometry; Fluorescence; Funding; Genes; Genetic screening; Genomes; High-throughput screening (Biochemical assaying); High-Throughput Screening Assays - methods; Humans; Metabolism; Metabolites; Mitochondria; Mitochondria - genetics; Mitochondria - metabolism; Mitochondrial DNA; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Proteins; Research and Analysis Methods; Ribosomal proteins; Sensors; Single-Cell Analysis - methods; Supervision; Supplements; Ubiquinone - analogs & derivatives; Ubiquinone - metabolism; United States > US; San Francisco California; California
• ISSN: 1545-7885; 1544-9173; 1545-7885
• DOI: 10.1371/journal.pbio.2004624

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, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.