Hiromi Imamura

Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators

Adenosine 5′-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial FoF1-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of FoF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

Evidence for rotation of V1-ATPase

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V1-ATPase, a catalytic subcomplex of VoV1-ATPase. V1-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that VoV1-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase.

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-Å resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V1 (soluble) and the Vo (membrane bound) domains. Subunit C, attached to the Vo domain, seems to have a socket like function in attaching the central-stalk subunits of the V1 domain. This architecture seems essential for the reversible association/dissociation of the V1 and the Vo domains, unique for V-ATPase activity regulation.

Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells.

Leucine Zipper EF Hand-containing Transmembrane Protein 1 (Letm1) and Uncoupling Proteins 2 and 3 (UCP2/3) Contribute to Two Distinct Mitochondrial Ca2+ Uptake Pathways

Cytosolic Ca2+ signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca2+ domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca2+/H+ antiporter that achieved mitochondrial Ca2+ sequestration at small Ca2+ increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca2+ uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca2+ but strongly diminished the transfer of entering Ca2+ into mitochondria, subsequently, resulting in a reduction of store-operated Ca2+ entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca2+ signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca2+ uptake at low Ca2+ conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca2+ uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca2+ uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca2+ uptake pathways in intact endothelial cells.

Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators.

Fluorescent protein (FP)-based Forster 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 Aequorea FPs 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.

Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

Adenosine 5′-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

Ca2+ Regulation of Mitochondrial ATP Synthesis Visualized at the Single Cell Level

Intracellular Ca(2+) levels play a crucial role in the control of ATP synthesis. However, the spatiotemporal correlation between ATP and Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) and mitochondrial ATP levels. The increment of mitochondrial ATP levels was proportional to that of Ca(2+). This finding suggests that cellular Ca(2+) levels might precisely control mitochondrial ATP synthesis in response to the increased ATP consumption triggered by Ca(2+). 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.

Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation

F1-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α3β3 ring. The crystal structures of F1, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F1 has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F1, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F1 from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F1, the corresponding residues in the ADP-bound β (βDP) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F1, α3β2β(E190D/E391C)γ(R84C), was prepared. In single-molecule rotation assay, chimera F1 showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F1 paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that βDP is the catalytically active form. The former point was again confirmed in experiments where F1 rotation was inhibited by adenosine-5′-(β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F1.

Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation

Significance We developed a sensitive method to assess the activity of oxidative phosphorylation in living cells using a FRET-based ATP biosensor. We then revealed that G0/G1 switch gene 2, a protein rapidly induced by hypoxia, increases mitochondrial ATP production by interacting with FoF1-ATP synthase and protects cells from a critical energy crisis. The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.