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ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an ex vivoIR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an ex vivoIR-mimicking model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

ATP is the major energy source for myotube contraction, and is quickly produced to compensate ATP consumption and to maintain sufficient ATP level. ATP is consumed mainly in cytoplasm and produced in mitochondria during myotube contraction. To understand the mechanism of ATP homeostasis during myotube contraction, it is essential to monitor mitochondrial ATP at single‐cell level, and examine how ATP is produced and consumed in mitochondria. We established C2C12 cell line stably expressing fluorescent probe of mitochondrial ATP, and induced differentiation into myotubes. We gave electric pulse stimulation to the differentiated myotubes, and measured mitochondrial ATP. We constructed mathematical model of mitochondrial ATP at single‐cell level, and analyzed kinetic parameters of ATP production and consumption. We performed hierarchical clustering analysis of time course of mitochondrial ATP, which resulted in two clusters. Cluster 1 showed strong transient increase, whereas cluster 2 showed weak transient increase. Mathematical modeling at single‐cell level revealed that the ATP production rate of cluster 1 was larger than that of cluster 2, and that both regulatory pathways of ATP production and consumption of cluster 1 were faster than those of cluster 2. Cluster 1 showed larger mitochondrial mass than cluster 2, suggesting that cluster 1 shows the similar property of slow muscle fibers, and cluster 2 shows the similar property of fast muscle fibers. Cluster 1 showed the stronger mitochondrial ATP increase by larger ATP production rate, but not smaller consumption. Cluster 1 might reflect the larger oxidative capacity of slow muscle fiber. Author summary:ATP is the major energy source for myotube contraction, and is quickly produced to compensate ATP consumption. We performed live cell imaging of mitochondrial ATP level in differentiated C2C12 myotubes by electrical stimulation. Myotubes were clustered into two populations with larger and smaller increase of mitochondrial ATP. We analyzed the dynamics of each myotube, and found that the former population shows larger ATP production rate and larger mitochondrial mass than the latter, suggesting that the former shows the similar property of slow muscle fibers, and the later shows the similar property of fast muscle fibers.

We show that the phasor-FLIM approach serves as a unique tool to monitor damage-induced metabolic changes in real time, revealing cell-wide NADH dynamics in response to nuclear DNA damage and that NAD+ consumption by PARP1 triggers an increase in metabolic reliance to oxidative phosphorylation, which is critical for damaged cell survival.

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

Real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor within the lumen of the ER reveal a local Ca2+-controlled ATP signal that is restored during energy stress. The data highlight a novel Ca2+-controlled process that supplies the ER with additional energy upon cell stimulation.

Intracellular Ca2+levels play a crucial role in the control of ATP synthesis. However, the spatiotemporal correlation between ATP and Ca2+remains unclear due to the inability to visualize these factors within same individual cells. A Förster resonance energy transfer (FRET)-based fluorescent ATP probe, named ATeam, was recently developed for ATP imaging in single living cells. However, the spectra of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) used as the FRET donor and the acceptor, respectively, significantly overlap with the ultraviolet-excitable Ca2+probe, fura-2. In the present work, we developed new red-shifted ATP probes, GO-ATeams, in which green fluorescent protein (GFP) and orange fluorescent protein (OFP) was used as the FRET pair to minimize spectral overlap with the fura-2 emission. The dynamics of intracellular Ca2+and mitochondrial ATP levels in single histamine-stimulated HeLa cells were successfully visualized by using fura-2 and GO-ATeam. The experiments showed that histamine induced increases of both intracellular Ca2+and mitochondrial ATP levels. The increment of mitochondrial ATP levels was proportional to that of Ca2+. This finding suggests that cellular Ca2+levels might precisely control mitochondrial ATP synthesis in response to the increased ATP consumption triggered by Ca2+. In addition, GO-ATeam has several advantages over the original ATeam. The GO-ATeam signal was more stable against acidification, which would allow ATP imaging inside acidic intracellular compartments. Also, the GO-ATeam excitation wavelength is much less phototoxic to cells, making the probe suitable for long-time observation.

Fluorescent protein (FP)-based Förster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of AequoreaFPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.

The prokaryotic V-type ATPase/synthases (prokaryotic V-ATPases) have simpler subunit compositions than eukaryotic V-ATPases, and thus are useful subjects for studying chemical, physical and structural properties of V-ATPase. In this review, we focus on the results of recent studies on the structure/function relationships in the V-ATPase from the eubacterium Thermus thermophilus. First, we describe single-molecule analyses of T. thermophilusV-ATPase. Using the single-molecule technique, it was established that the V-ATPase is a rotary motor. Second, we discuss arrangement of subunits in V-ATPase. Third, the crystal structure of the C-subunit (homolog of eukaryotic d-subunit) is described. This funnel-shape subunit appears to cap the proteolipid ring in the V0domain in order to accommodate the V1central stalk. This structure seems essential for the regulatory reversible association/dissociation of the V1and the V0domains. Last, we discuss classification of the V-ATPase family. We propose that the term prokaryotic V-ATPases should be used rather than the term archaeal-type ATPase (A-ATPase).

Amylase A from Dictyoglomus thermophilumis a thermophilic enzyme and has about 40% identity with 4-α-glucanotransferase (GTase) from Thermococcus litoralis, and both of these enzymes belong to family 57 glycosyl hydrolase. Since the transglycosylation activity of T. litoralisGTase has been well characterized, the substrate specificity and reaction products of amylase A from D. thermophilumwere examined. α-1,4 Glucan was produced from maltooligosaccharides, and glucoamylase-resistant molecules (cycloamyloses) were produced from longer chain amylose (average molecular mass 200 kDa). It has been reported that amylase A from D. thermophilumhydrolyzes starch, but in this study it was found that the enzyme was also able to use maltooligosaccharides and long chain amylose as substrate and has transglycosylation activity.

The Thermococcus litoralis4‐α‐glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the GTase gene in E. coliresulted in low protein production and the accumulation of inclusion bodies. However, simultaneous expression of GTase with tRNAAGA, tRNAAGGand GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5‐fold. This new E. coliexpression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.

The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobussp. strain 7, a thermoacidophilic archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNAArg, cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obatined after removal of heat denatured E. coliproteins.

The Thermococcus litoralis4-α-glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the GTase gene in E. coliresulted in low protein production and the accumulation of inclusion bodies. However, simultaneous expression of GTase with tRNA AGA, tRNA AGGand GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5-fold. This new E. coliexpression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.

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