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

PLoS Biology
Bryce A MendelsohnKen Nakamura

Abstract

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.

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Citations

Dec 15, 2018·PLoS Biology·Jianhan ZhangYihan Lin
Apr 26, 2019·Journal of Inherited Metabolic Disease·Kyle ThompsonRobert W Taylor
Apr 5, 2019·Molecular Diagnosis & Therapy·Petros PatsaliCarsten W Lederer
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Apr 4, 2021·Cancers·Laia Castells-RocaJordi Surrallés
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Jul 31, 2021·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Yuanyuan Li, Li-Quan Zhou
Oct 15, 2021·Nature Communications·Debasmita BhattacharyaAnthony Scimè

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Methods Mentioned

BETA
biosensors
fluorescence
fluorescence-activated cell sorting
FRET
FACS
flow cytometry
flow
gene knockdown
reverse transcription PCR
dissection

Key Resources (RRID) Mentioned

CVCL_0004
CVCL_0224

Software Mentioned

Image Studio Lite
GraphPad Prism
FlowJo
GeneCards

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