Red fluorescent proteins engineered from green fluorescent proteins.

Fluorescent proteins (FPs) form a fluorophore through autocatalysis from three consecutive amino acid residues within a polypeptide chain. The two major groups, green FPs (GFPs) and red FPs (RFPs), have distinct fluorophore structures; RFPs have an extended π-conjugation system with an additional double bond. However, due to the low sequence homology between the two groups, amino acid residues essential for determining the different fluorophore structures were unclear. Therefore, engineering a GFP into an RFP has been challenging, and the exact mechanism of how GFPs and RFPs achieve different autocatalytic reactions remained elucidated. Here, we show the conversion of two coral GFPs, AzamiGreen (AG) and mcavGFP, into RFPs by defined mutations. Structural comparison of AG and AzamiRed1.0, an AG-derived RFP, revealed that the mutations triggered drastic rearrangements in the interaction networks between amino acid residues around the fluorophore, suggesting that coordinated multisite mutations are required for the green-to-red conversion. As a result of the structural rearrangements, a cavity suitable for the entry of an oxygen molecule, which is necessary for the double bond formation of the red fluorophores, is created in the proximity of the fluorophore. We also show that a monomeric variant of AzamiRed1.0 can be used for labeling organelles and proteins in mammalian cells. Our results provide a structural basis for understanding the red fluorophore formation mechanism and demonstrate that protein engineering of GFPs is a promising way to create RFPs suitable for fluorescent tags.

Microbial energy metabolism fuels an intestinal macrophage niche in solitary isolated lymphoid tissues through purinergic signaling.

Maintaining macrophage (MΦ) heterogeneity is critical to ensure intestinal tissue homeostasis and host defense. The gut microbiota and host factors are thought to synergistically guide intestinal MΦ development, although the exact nature, regulation, and location of such collaboration remain unclear. Here, we report that microbial biochemical energy metabolism promotes colony-stimulating factor 2 (CSF2) production by group 3 innate lymphoid cells (ILC3s) within solitary isolated lymphoid tissues (SILTs) in a cell-extrinsic, NLRP3/P2X7R-dependent fashion in the steady state. Tissue-infiltrating monocytes accumulating around SILTs followed a spatially constrained, distinct developmental trajectory into SILT-associated MΦs (SAMs). CSF2 regulated the mitochondrial membrane potential and reactive oxygen species production of SAMs and contributed to the antimicrobial defense against enteric bacterial infections. Collectively, these findings identify SILTs and CSF2-producing ILC3s as a microanatomic niche for intestinal MΦ development and functional programming fueled by the integration of commensal microbial energy metabolism.

The SGLT2 inhibitor empagliflozin improves cardiac energy status via mitochondrial ATP production in diabetic mice.

Empagliflozin, a sodium-glucose co-transporter 2 inhibitor developed, has been shown to reduce cardiovascular events in patients with type 2 diabetes and established cardiovascular disease. Several studies have suggested that empagliflozin improves the cardiac energy state which is a partial cause of its potency. However, the detailed mechanism remains unclear. To address this issue, we used a mouse model that enabled direct measurement of cytosolic and mitochondrial ATP levels. Empagliflozin treatment significantly increased cytosolic and mitochondrial ATP levels in the hearts of db/db mice. Empagliflozin also enhanced cardiac robustness by maintaining intracellular ATP levels and the recovery capacity in the infarcted area during ischemic-reperfusion. Our findings suggest that empagliflozin enters cardiac mitochondria and directly causes these effects by increasing mitochondrial ATP via inhibition of NHE1 and Nav1.5 or their common downstream sites. These cardioprotective effects may be involved in the beneficial effects on heart failure seen in clinical trials.

High and stable ATP levels prevent aberrant intracellular protein aggregation in yeast.

Adenosine triphosphate (ATP) at millimolar levels has recently been implicated in the solubilization of cellular proteins. However, the significance of this high ATP level under physiological conditions and the mechanisms that maintain ATP remain unclear. We herein demonstrated that AMP-activated protein kinase (AMPK) and adenylate kinase (ADK) cooperated to maintain cellular ATP levels regardless of glucose levels. Single-cell imaging of ATP-reduced yeast mutants revealed that ATP levels in these mutants underwent stochastic and transient depletion, which promoted the cytotoxic aggregation of endogenous proteins and pathogenic proteins, such as huntingtin and α-synuclein. Moreover, pharmacological elevations in ATP levels in an ATP-reduced mutant prevented the accumulation of α-synuclein aggregates and its cytotoxicity. The present study demonstrates that cellular ATP homeostasis ensures proteostasis and revealed that suppressing the high volatility of cellular ATP levels prevented cytotoxic protein aggregation, implying that AMPK and ADK are important factors that prevent proteinopathies, such as neurodegenerative diseases.

Cells use a chemical called adenosine triphosphate (ATP) as a controllable source of energy. Like a battery, each ATP molecule contains a specific amount of energy that can be released when needed. Cells just need enough ATP to survive, but most cells store a lot more than they need. It is unclear why cells keep so much ATP, or whether this excess ATP has any other purpose. To answer these questions, Takaine et al. identified mutants of the yeast Saccharomyces cerevisiae that had low levels of ATP, and studied how these cells differ from normal yeast The results showed that, in S. cerevisiae cells with lower and variable levels of ATP, proteins stick together, forming clumps. Proteins are molecules that perform diverse roles, keeping cells alive. When they clump together, they stop working and can cause cells to die. Further experiments showed that reducing the levels of ATP just for a short time increased the rate at which proteins stick together. Taken together, Takaine et al.'s results suggest that ATP plays a role in stopping proteins from sticking together, explaining why cells may store excess ATP, since it could aid survival. Protein clumps, also called aggregates, are a key feature of various illnesses, including neurodegenerative diseases such as Alzheimer's. Takaine et al. provide a possible cause for why proteins aggregate in these diseases, which may be worth further study.

Lactate is an energy substrate for rodent cortical neurons and enhances their firing activity.

Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with KCNJ11 and ABCC8 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

Shear stress activates mitochondrial oxidative phosphorylation by reducing plasma membrane cholesterol in vascular endothelial cells.

Vascular endothelial cells (ECs) sense and respond to hemodynamic shear stress, which is critical for circulatory homeostasis and the pathophysiology of vascular diseases. The mechanisms of shear stress mechanotransduction, however, remain elusive. We previously demonstrated a direct role of mitochondria in the purinergic signaling of shear stress: shear stress increases mitochondrial adenosine triphosphate (ATP) production, triggering ATP release and Ca2+ signaling via EC purinoceptors. Here, we showed that shear stress rapidly decreases cholesterol in the plasma membrane, thereby activating mitochondrial ATP production. Imaging using domain 4 mutant-derived cholesterol biosensors showed that the application of shear stress to cultured ECs markedly decreased cholesterol levels in both the outer and inner plasma membrane bilayers. Flow cytometry showed that the cholesterol levels in the outer bilayer decreased rapidly after the onset of shear stress, reached a minimum (around 60% of the control level) at 10 min, and plateaued thereafter. After the shear stress ceased, the decreased cholesterol levels returned to those seen in the control. A biochemical analysis showed that shear stress caused both the efflux and the internalization of plasma membrane cholesterol. ATP biosensor imaging demonstrated that shear stress significantly increased mitochondrial ATP production. Similarly, the treatment of cells with methyl-ß-cyclodextrin (MßCD), a membrane cholesterol-depleting agent, increased mitochondrial ATP production. The addition of cholesterol to cells inhibited the increasing effects of both shear stress and MßCD on mitochondrial ATP production in a dose-dependent manner. These findings indicate that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs.

Spatiotemporal ATP Dynamics during AKI Predict Renal Prognosis.

BACKGROUND: Depletion of ATP in renal tubular cells plays the central role in the pathogenesis of kidney diseases. Nevertheless, inability to visualize spatiotemporal in vivo ATP distribution and dynamics has hindered further analysis. METHODS: A novel mouse line systemically expressing an ATP biosensor (an ATP synthase subunit and two fluorophores) revealed spatiotemporal ATP dynamics at single-cell resolution during warm and cold ischemic reperfusion (IR) with two-photon microscopy. This experimental system enabled quantification of fibrosis 2 weeks after IR and assessment of the relationship between the ATP recovery in acute phase and fibrosis in chronic phase. RESULTS: Upon ischemia induction, the ATP levels of proximal tubule (PT) cells decreased to the nadir within a few minutes, whereas those of distal tubule (DT) cells decreased gradually up to 1 hour. Upon reperfusion, the recovery rate of ATP in PTs was slower with longer ischemia. In stark contrast, ATP in DTs was quickly rebounded irrespective of ischemia duration. Morphologic changes of mitochondria in the acute phase support the observation of different ATP dynamics in the two segments. Furthermore, slow and incomplete ATP recovery of PTs in the acute phase inversely correlated with fibrosis in the chronic phase. Ischemia under conditions of hypothermia resulted in more rapid and complete ATP recovery with less fibrosis, providing a proof of concept for use of hypothermia to protect kidney tissues. CONCLUSIONS: Visualizing spatiotemporal ATP dynamics during IR injury revealed higher sensitivity of PT cells to ischemia compared with DT cells in terms of energy metabolism. The ATP dynamics of PTs in AKI might provide prognostic information.

Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis.

ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a 'programmed' process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP.

Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep-wake states.

Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5'-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

Measurement of ATP concentrations in mitochondria of living cells using luminescence and fluorescence approaches.

Adenosine 5'-triphosphate (ATP) is the central metabolite in the energy metabolism of cells and is hydrolyzed to ADP and inorganic phosphate to provide free energy in various cellular processes. ATP also functions as an intracellular signaling molecule. Thus, it is important to know the ATP concentration within cells to understand cellular activities. Here, we describe two methods to detect ATP concentrations in the cytoplasm and mitochondrial matrix using genetically encoded luminescent or fluorescent biosensors. These methods enable quantitative investigation of ATP concentration dynamics in living cells, single cells and cell populations.

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

Most eukaryotic cells generate adenosine triphosphate (ATP) through the oxidative phosphorylation system (OXPHOS) to support cellular activities. In cultured cell-based experiments, we recently identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS, and showed that G0s2 protects cultured cardiomyocytes from hypoxia. In this study, we examined the in vivo protective role of G0s2 against hypoxia by generating both loss-of-function and gain-of-function models of g0s2 in zebrafish. Zebrafish harboring transcription activator-like effector nuclease (TALEN)-mediated knockout of g0s2 lost hypoxic tolerance. Conversely, cardiomyocyte-specific transgenic zebrafish hearts exhibited strong tolerance against hypoxia. To clarify the mechanism by which G0s2 protects cardiac function under hypoxia, we introduced a mitochondrially targeted FRET-based ATP biosensor into zebrafish heart to visualize ATP dynamics in in vivo beating hearts. In addition, we employed a mosaic overexpression model of g0s2 to compare the contraction and ATP dynamics between g0s2-expressing and non-expressing cardiomyocytes, side-by-side within the same heart. These techniques revealed that g0s2-expressing cardiomyocyte populations exhibited preserved contractility coupled with maintained intra-mitochondrial ATP concentrations even under hypoxic condition. Collectively, these results demonstrate that G0s2 provides ischemic tolerance in vivo by maintaining ATP production, and therefore represents a promising therapeutic target for hypoxia-related diseases.

Higd1a improves respiratory function in the models of mitochondrial disorder.

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]mito) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP]mito under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease; notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal velocity of the reaction between CcO and cytochrome c without changing the affinity between them, indicating that Higd1a is a positive modulator of CcO. These results corroborate that Higd1a, or its mimic, provides therapeutic options for the treatment of mitochondrial diseases.

OLIVe: A Genetically Encoded Fluorescent Biosensor for Quantitative Imaging of Branched-Chain Amino Acid Levels inside Single Living Cells.

Branched-chain amino acids (BCAAs) are essential amino acids, controlling cellular metabolic processes as signaling molecules; therefore, utilization of intracellular BCAAs may be regulated by the availability of nutrients in the environment. However, spatial and temporal regulation of intracellular BCAA concentration in response to environmental conditions has been unclear due to the lack of suitable methods for measuring BCAA concentrations inside single living cells. Here, we developed a Förster resonance energy transfer (FRET)-based genetically encoded biosensor for BCAAs, termed optical biosensor for leucine-isoleucine-valine (OLIVe). The biosensor showed approximately 2-fold changes in FRET values corresponding to BCAA concentrations. Importantly, FRET signals from HeLa cells expressing OLIVe in the cytoplasm and nucleus correlated with bulk intracellular BCAA concentrations determined from populations of cells by a biochemical method, and were decreased by knockdown of L-type amino acid transporter 1 (LAT1), a transporter for BCAAs, indicating that OLIVe can reliably report intracellular BCAA concentrations inside single living cells. We also succeeded in imaging BCAA concentrations in the mitochondria using mitochondria-targeted OLIVe. Using the BCAA imaging technique, we found apparently correlated concentrations between the cytoplasm and the mitochondria. We also found that extracellular non-BCAA amino acids affected intracellular BCAA concentrations. Of these amino acids, extracellular glutamine markedly increased intracellular BCAA concentrations in a LAT1-dependent manner. Unexpectedly, extracellular pyruvate was also found to have significant positive effects on maintaining intracellular BCAA concentrations, suggesting that the cells have pyruvate-dependent systems to import BCAAs and/or to regulate BCAA metabolism.

NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival.

DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.

A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2.

Oxidative phosphorylation generates most of the ATP in respiring cells. ATP is an essential energy source, especially in cardiomyocytes because of their continuous contraction and relaxation. Previously, we reported that G0/G1 switch gene 2 (G0S2) positively regulates mitochondrial ATP production by interacting with FOF1-ATP synthase. G0S2 overexpression mitigates ATP decline in cardiomyocytes and strongly increases their hypoxic tolerance during ischemia. Here, we show that G0S2 protein undergoes proteasomal degradation via a cytosolic molecular triage system and that inhibiting this process increases mitochondrial ATP production in hypoxia. First, we performed screening with a library of siRNAs targeting ubiquitin-related genes and identified RING finger protein 126 (RNF126) as an E3 ligase involved in G0S2 degradation. RNF126-deficient cells exhibited prolonged G0S2 protein turnover and reduced G0S2 ubiquitination. BCL2-associated athanogene 6 (BAG6), involved in the molecular triage of nascent membrane proteins, enhanced RNF126-mediated G0S2 ubiquitination both in vitro and in vivo Next, we found that Glu-44 in the hydrophobic region of G0S2 acts as a degron necessary for G0S2 polyubiquitination and proteasomal degradation. Because this degron was required for an interaction of G0S2 with BAG6, an alanine-replaced G0S2 mutant (E44A) escaped degradation. In primary cultured cardiomyocytes, both overexpression of the G0S2 E44A mutant and RNF126 knockdown effectively attenuated ATP decline under hypoxic conditions. We conclude that the RNF126/BAG6 complex contributes to G0S2 degradation and that interventions to prevent G0S2 degradation may offer a therapeutic strategy for managing ischemic diseases.

Spindle pole body movement is affected by glucose and ammonium chloride in fission yeast.

The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition.

Reliable imaging of ATP in living budding and fission yeast.

Adenosine triphosphate (ATP) is a main metabolite essential for all living organisms. However, our understanding of ATP dynamics within a single living cell is very limited. Here, we optimized the ATP-biosensor QUEEN and monitored the dynamics of ATP with good spatial and temporal resolution in living yeasts. We found stable maintenance of ATP concentration in wild-type yeasts, regardless of carbon sources or cell cycle stages, suggesting that mechanism exists to maintain ATP at a specific concentration. We further found that ATP concentration is not necessarily an indicator of metabolic activity, as there is no clear correlation between ATP level and growth rates. During fission yeast meiosis, we found a reduction in ATP levels, suggesting that ATP homeostasis is controlled by differentiation. The use of QUEEN in yeasts offers an easy and reliable assay for ATP dynamicity and will answer several unaddressed questions about cellular metabolism in eukaryotes.

PPARα-Mediated Positive-Feedback Loop Contributes to Cold Exposure Memory.

Fluctuations in food availability and shifts in temperature are typical environmental changes experienced by animals. These environmental shifts sometimes portend more severe changes; e.g., chilly north winds precede the onset of winter. Such telltale signs may be indicators for animals to prepare for such a shift. Here we show that HEK293A cells, cultured under starvation conditions, can "memorize" a short exposure to cold temperature (15 °C), which was evidenced by their higher survival rate compared to cells continuously grown at 37 °C. We refer to this phenomenon as "cold adaptation". The cold-exposed cells retained high ATP levels, and addition of etomoxir, a fatty acid oxidation inhibitor, abrogated the enhanced cell survival. In our standard protocol, cold adaptation required linoleic acid (LA) supplementation along with the activity of Δ-6-desaturase (D6D), a key enzyme in LA metabolism. Moreover, supplementation with the LA metabolite arachidonic acid (AA), which is a high-affinity agonist of peroxisome proliferator-activated receptor-alpha (PPARα), was able to underpin the cold adaptation, even in the presence of a D6D inhibitor. Cold exposure with added LA or AA prompted a surge in PPARα levels, followed by the induction of D6D expression; addition of a PPARα antagonist or a D6D inhibitor abrogated both their expression, and reduced cell survival to control levels. We also found that the brief cold exposure transiently prevents PPARα degradation by inhibiting the ubiquitin proteasome system, and starvation contributes to the enhancement of PPARα activity by inhibiting mTORC1. Our results reveal an innate adaptive positive-feedback mechanism with a PPARα-D6D-AA axis that is triggered by a brief cold exposure in cells. "Cold adaptation" could have evolved to increase strength and resilience against imminent extreme cold temperatures.

A Trace Amount of Galactose, a Major Component of Milk Sugar, Allows Maturation of Glycoproteins during Sugar Starvation.

Milk sugar is composed of glucose and galactose. Galactose is less suitable as an energy source than glucose. Thus, it has been a puzzle as to why mammals utilize galactose as a major component of milk sugar. Here we show that in hypoglycemic conditions, the presence of a trace amount of galactose, but not glucose, is able to maintain the production of mature glycoproteins and to abolish cell-death-inducing endoplasmic reticulum stress. In severely sugar-limited conditions, both glucose and galactose enter into the glycolytic pathway, but galactose is not able to raise the phosphofructokinase 1 activity, leading to the accumulation of fructose-6-phosphate, which in turn is utilized for the maturation of glycoproteins (e.g., growth factor receptors) and allows the activation of their intracellular signaling and prevents cell death from hypoglycemic conditions. Thus trace amounts of galactose may play unexpectedly important roles in the growth of infants and their protection during starvation.