AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells

• Published in: Cell chemical biology Vol. 31; no. 8; pp. 1529 - 1541.e12
• Main Authors: Hellweg, Lars, Pfeifer, Martin, Tarnawski, Miroslaw, Thing-Teoh, Shao, Chang, Lena, Bergner, Andrea, Kress, Jana, Hiblot, Julien, Wiedmer, Tabea, Superti-Furga, Giulio, Reinhardt, Jürgen, Johnsson, Kai, Leippe, Philipp
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 15.08.2024
• Subjects: Aspartic Acid - metabolism; Biosensing Techniques; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; HeLa Cells; Humans; Mitochondria - metabolism
• ISSN: 2451-9456; 2451-9456; 2451-9448
• DOI: 10.1016/j.chembiol.2024.05.002

Aspartate is crucial for nucleotide synthesis, ammonia detoxification, and maintaining redox balance via the malate-aspartate-shuttle (MAS). To disentangle these multiple roles of aspartate metabolism, tools are required that measure aspartate concentrations in real time and in live cells. We introduce AspSnFR, a genetically encoded green fluorescent biosensor for intracellular aspartate, engineered through displaying and screening biosensor libraries on mammalian cells. In live cells, AspSnFR is able to precisely and quantitatively measure cytosolic aspartate concentrations and dissect its production from glutamine. Combining high-content imaging of AspSnFR with pharmacological perturbations exposes differences in metabolic vulnerabilities of aspartate levels based on nutrient availability. Further, AspSnFR facilitates tracking of aspartate export from mitochondria through SLC25A12, the MAS’ key transporter. We show that SLC25A12 is a rapidly responding and direct route to couple Ca2+ signaling with mitochondrial aspartate export. This establishes SLC25A12 as a crucial link between cellular signaling, mitochondrial respiration, and metabolism. [Display omitted] •AspSnFR enables real-time measurement of intracellular aspartate levels•AspSnFR facilitates monitoring of amino acid uptake and metabolism•AspSnFR serves as a fluorescent readout of the redox state and mitochondrial respiration•SLC25A12’s coupling of Ca2+ signals to aspartate levels is showcased in live cells Hellweg et al. introduce AspSnFR, a genetically encoded green fluorescent biosensor designed for the real-time measurement of aspartate concentrations in live cells. AspSnFR enables studies of amino acid uptake, mitochondrial metabolism, and Ca2+-regulated aspartate export from mitochondria by SLC25A12.