Dissecting the regulation and function of ATP at the single-cell level

Abstract

Regulation of cellular ATP level is critical for diverse biological processes and may be defective in diseases such as cancer and mitochondrial disorders. While mitochondria play critical roles in ATP level regulation, we still lack a systematic and quantitative picture of how individual mitochondrial-related genes contribute to cellular ATP level and how dysregulated ATP levels may affect downstream cellular processes. Advances in genetically encoded ATP biosensors have provided new opportunities for addressing these issues. ATP biosensors allow researchers to quantify the changes of ATP levels in real time at the single-cell level and characterize corresponding effects at the cellular, tissue, and organismal level. Along this direction, several recent single-cell studies using ATP biosensors, including the work by Mendelsohn and colleagues, have started to uncover the principles for how genetic and nongenetic parameters may modulate ATP levels to affect cellular functions and human health.

Fig 1. The mitochondrion is a multifunctional organelle.

During mitochondrial bioenergetics, acetyl-CoA converted from food molecules is oxidized to produce ATP. In addition to ATP production, mitochondria play essential roles in cell signaling including nutrient sensing, redox signaling, cell death, and many other functions [3, 7]. Note that biosynthetic pathways in mitochondria [4] are omitted in the diagram. ADP, adenosine diphosphate; AMP, adenosine monophosphate; CoA, coenzyme A; ETC, electron transport chain; TCA, tricarboxylic acid.

Fig 2. Combining real-time ATP biosensor with CRISPRi enables a single-cell–based high-throughput screening assay.

(A) Assay design and results (schematics) [24]. (Left) Individual genes targeted by the sgRNA library were knocked down in cells expressing the improved ATP biosensor. Types of conditions are indicated by solid or dashed lines. (Middle) Gene knockdowns often result in opposite responses in cellular ATP levels for the respiratory versus the glycolytic condition. Experimentally, sgRNA-transfected cells were subjected to two different conditions, and the resulting effects on ATP level of different gene knockdowns were quantified. Here, types of genes are indicated by line colors, and solid or dashed lines indicate corresponding conditions on the left panel. (Right) The altered ATP levels affect cell-growth rates in a metabolic-context–dependent manner. Note that the ATP levels were not quantified in real time in the actual experiments. (B) The effects of CoQ10 supplementation (schematics) [24]. (Left) Screen design and screen conditions. Types of conditions are indicated by solid or dashed lines. (Middle) High CoQ10 supplementation can rescue respiratory ATP levels for both deficiencies in CoQ10 biosynthesis genes and a few non-CoQ10 biosynthesis-related genes. (Right) These results show that CoQ10 supplementation could rescue diverse mitochondrial respiration defects, suggesting its potential therapeutic applications in mitochondrial disorders. CoQ10, coenzyme Q10; CRISPR, clustered regularly interspaced short palindromic repeats; CRISPRi, CRISPR interference; OXPHOS, oxidative phosphorylation; sgRNA, single guide RNA.