Application of FRET-Based Biosensor “ATeam” for Visualization of ATP Levels in the Mitochondrial Matrix of Living Mammalian Cells

Abstract

Genetically encoded biosensors utilizing the Förster resonance energy transfer (FRET) are powerful tools for live cell imaging of various cellular processes. Our group has previously developed a series of FRET-based biosensors, named "ATeam," for visualization of ATP levels inside a single living cell. ATeam not only provides a window of insight into a single cell but also allows for visualization of ATP levels in mitochondrial matrix of a single living cell. This novel tool is able to monitor alterations in cellular ATP in response to various treatments in real time. Here we present a method for the evaluation of ATP levels in mitochondria in living cells by using ATeam. At the end of this chapter, an example of experimental results is described for a better understanding of the presented procedure.

Adaptive F-Actin Polymerization and Localized ATP Production Drive Basement Membrane Invasion in the Absence of MMPs

“…For sensitized emission, excitation and emission parameters and laser power were kept constant throughout imaging and were matched across channels. Lines that genetically encode the fluorescent ATP biosensor (ATeam, (Imamura et al, 2009;Yoshida et al, 2017) optimized for use in C. elegans (AT1.03L, (Tsuyama et al, 2013) were created by PCR fusion to an AC specific promoter (cdh-3>ATeam). The ATeam reporter is composed of the epsilon subunit of the bacterial F 0 F 1 -ATP synthase located between Venus and Cyan Fluorescent Protein.…”

Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells

“…Recently, genetically encoded fluorescent probes for real-time imaging of specific cellular metabolites have been developed (e.g., Bilan et al, 2014 ; San Martín et al, 2014 ; Takanaga et al, 2008 ). Among these tools are Förster resonance energy transfer (FRET)-based ATP probes, referred to as “ATeams” ( Imamura et al, 2009 ; Vishnu et al, 2014 ; Yoshida et al, 2017 ). ATeams are approved tools that enable visualizing spatiotemporal dynamics of intracellular ATP fluctuations and, thus, give insight into the metabolic activities of individual cells.…”

In vivo real‐time ATP imaging in zebrafish hearts reveals G0s2 induces ischemic tolerance

“…Therefore, in vivo real‐time metabolic imaging is needed for the investigations of energy metabolism along with in vivo tissue function. For this purpose, we introduced the ATP‐sensing FRET biosensor Mit‐ATeam into zebrafish heart and successfully established a Mit‐ATeam zebrafish line. It should be noted that the heart is an ideal tissue for ATP imaging because we can simultaneously evaluate tissue function (ie, contraction of the heart muscle) as well as ATP dynamics.…”