Abstract
AbstractIntracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a GFP-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by mass spectrometry and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high throughput applications of variables that affect aspartate levels.
Publisher
Cold Spring Harbor Laboratory
Reference38 articles.
1. Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission;Nat Methods,2023
2. Metformin directly acts on mitochondria to alter cellular bioenergetics;Cancer Metab,2014
3. Arnold PK , Jackson BT , Paras KI , Brunner JS , Hart ML , Newsom OJ , Alibeckoff SP , Endress J , Drill E , Sullivan LB , Finley LWS . A non-canonical tricarboxylic acid cycle underlies cellular identity. Nature. 2022 Mar; p. 1–5.
4. Distinct modes of mitochondrial metabolism uncouple T cell differentiation and function
5. An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis