A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC

Abstract

Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC is poised to exert ultrasensitive control on the balance between respiration and anaplerosis/gluconeogenesis. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes

“…Another method to obtain single cell data from fluorescent nanosensor imaging is the single point calibration protocol (e.g., Sotelo-Hitschfeld et al, 2012; Fernández-Moncada and Barros, 2014;Arce-Molina et al, 2020). Only one nanosensor is needed for this approach, and k D and n H are derived from other experimental systems like in the dual nanosensor approach ( Table 1).…”

“…Fluorescent nanosensors for metabolites have strongly contributed to a much deeper knowledge on the metabolism and its dynamics of the mammalian brain (and of other organs and organisms; Deuschle et al, 2006;Tsuyama et al, 2013;Yaginuma et al, 2014;Masia et al, 2018;Volkenhoff et al, 2018;Nguyen et al, 2019;Takaine et al, 2019;Arce-Molina et al, 2020;Kioka et al, 2020). As these sensors are proteins which can be genetically encoded, they allow cell type specific expression using specific promoters as well as subcellular targeting using appropriate targeting sequences.…”

“…As these sensors are proteins which can be genetically encoded, they allow cell type specific expression using specific promoters as well as subcellular targeting using appropriate targeting sequences. Combined with different stateof-the art microscopy technologies, the dynamics of metabolites can be followed in cultured cells, in tissue preparations like brain slices or the isolated optic nerve, but also in vivo in living and even awake animals (Bittner et al, 2011;Ruminot et al, 2011;Mächler et al, 2016;Díaz-García et al, 2017, 2019Trevisiol et al, 2017;Köhler et al, 2018;Baeza-Lehnert et al, 2019;Gerkau et al, 2019;Lerchundi et al, 2019;Arce-Molina et al, 2020;Zuend et al, 2020). However, while these nanosensors readily allow for monitoring relative changes of the metabolite concentration, deduction of absolute concentrations and absolute concentration changes (i.e., in mol/l) during treatments has remained challenging as calibration of the signal of the nanosensors to the actual concentration of the metabolite is hampered by both, theoretical and practical problems (Barros et al, 2018a).…”

A sort-seq approach to the development of single fluorescent protein biosensors

“…Genetically-encoded single fluorescent protein biosensors (SFPBs) can unmask aspects of cellular signaling and metabolism that cannot be detected using traditional biochemical approaches (Okumoto et al, 2012). Biosensors can provide crucial information on the subcellular compartmentalization of analytes, resolve changes in concentration over time, and highlight cellular heterogeneity (Arce-Molina et al, 2020;Hou et al, 2011;Miyawaki and Niino, 2015;Ni et al, 2018;Okumoto et al, 2012;Pendin et al, 2017). However, each SFPB specifically measures a single analyte, requiring a unique biosensor for each target.…”

“…SFPBs can be created by inserting a fluorescent protein (FP) into a ligand-binding domain (LBD) such that ligand binding allosterically regulates fluorescence (Arce-Molina et al, 2020;Doi and Yanagawa, 1999;Marvin et al, 2013Marvin et al, , 2011Nadler et al, 2016). The nature of this allosteric domain coupling is not well understood and has proven difficult to design rationally (Nadler et al, 2016).…”