Imaging Approaches for the Study of Metabolism in Real Time Using Genetically Encoded Reporters

Abstract

Metabolism comprises of two axes in order to serve homeostasis: anabolism and catabolism. Both axes are interbranched with the so-called bioenergetics aspect of metabolism. There is a plethora of analytical biochemical methods to monitor metabolites and reactions in lysates, yet there is a rising need to monitor, quantify and elucidate in real time the spatiotemporal orchestration of complex biochemical reactions in living systems and furthermore to analyze the metabolic effect of chemical compounds that are destined for the clinic. The ongoing technological burst in the field of imaging creates opportunities to establish new tools that will allow investigators to monitor dynamics of biochemical reactions and kinetics of metabolites at a resolution that ranges from subcellular organelle to whole system for some key metabolites. This article provides a mini review of available toolkits to achieve this goal but also presents a perspective on the open space that can be exploited to develop novel methodologies that will merge classic biochemistry of metabolism with advanced imaging. In other words, a perspective of “watching metabolism in real time.”

Metabolism Shapes Immune Responses to <i>Staphylococcus aureus</i>

“…However, these methods measure metabolism at the protein, not the metabolite level, which requires conventional mass spectrometry approaches. The development of sensors that can monitor metabolites is expanding [ 204 , 205 ] and real-time monitoring and spatiotemporal organization of metabolites during disease conditions will extend our understanding of metabolite shuttling and subsequent cellular responses. Recently, a correlative imaging approach, metaFISH [ 206 ], was developed by combining matrix-assisted laser desorption ionization-mass spectrometry imaging with fluorescence in situ hybridization, allowing the spatial distribution of metabolites to be identified in a tissue section.…”

Direct observation of fluorescent proteins in gels: a rapid cost-efficient, and quantitative alternative to immunoblotting

“…Various techniques have been implemented using GFP and its variants to visualize protein-protein interactions in cells, through bimolecular fluorescence complementation (Hu and Kerppola, 2003; Romei and Boxer, 2019), or fluorescence resonance energy transfer (FRET, BRET) (Hochreiter et al, 2015). This also opened the way to the development of various probes and biosensors to report on the quantity and/or localization of ions, metabolites and other organic molecules in cells and tissues (Chandris et al, 2021; Wang et al, 2023; Zacharias et al, 2002). The various folding kinetics of FPs have been exploited to report on protein stability and half-life (fluorescent timers) (Khmelinskii et al, 2012; Subach et al, 2009; Terskikh et al, 2000).…”

A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine’s Effect on Cellular Amino Acids via Inhibiting Cathepsin L

“…To study the uptake, storage, and utilization of amino acids, it would be useful to be able to visualize and monitor amino acid pools in live cells at high resolution. There are a number of genetically encoded biosensors available that utilize fluorescence resonance energy transfer (FRET) or fluorescent protein permutations to monitor individual metabolites . One system, named OLIVe (optical biosensor for leucine, isoleucine, and valine), uses YFP/CFP FRET technology to sense branched chain amino acids.…”