Cytoplasmic ATP Research Articles

Mitochondrial ATP synthesis is essential for efficient gametogenesis in Plasmodium falciparum.

Plasmodium male and female gametocytes are the gatekeepers of human-to-mosquito transmission, therefore essential for propagation of malaria within a population. Whilst dormant in humans, their divergent roles during transmission become apparent soon after mosquito feeding with a rapid transformation into gametes - males forming eight motile sperm-like cells aiming to fertilise a single female gamete. Little is known about how the parasite fuels this abrupt change, and the potential role played by their large and elaborate cristate mitochondrion. Using a sex-specific antibody and functional mitochondrial labelling, we show that the male gametocyte mitochondrion is less active than that of female gametocytes and more sensitive to antimalarials targeting mitochondrial energy metabolism. Rather than a vestigial organelle discarded during male gametogenesis, we demonstrate that mitochondrial ATP synthesis is essential for its completion. Additionally, using a genetically encoded ratiometric ATP sensor, we show that gametocytes can maintain cytoplasmic ATP homeostasis in the absence of mitochondrial respiration, indicating the essentiality of the gametocyte mitochondrion for transmission alone. Together, this reveals how gametocytes responsively balance the conflicting demands of a dormant and active lifestyle, highlighting the mitochondria as a rich source of transmission-blocking targets for future drug development.

Generation of two isogenic human iPSC lines (ZIPi013-B-1, ZIPi013-B-2) carrying a CRISPR/Cas9-mediated deletion of TRPM4

Two isogenic hiPSC lines, ZIPi013-B-1 and ZIPi013-B-2, were generated by CRISPR/Cas9-mediated indels in the TRPM4 gene of the previously published ZIPi013-B. TRPM4 belongs to the evolutionarily conserved family of transient receptor potential (TRP) channels. It is expressed ubiquitously and its activity is regulated by intracellular calcium binding, changes in membrane potential, phosphoinositide lipids in the plasma membrane and the local concentration of cytoplasmic ATP and ADP. TRPM4 has been implicated in various diseases, including neurological and immune system disorders, cardiac diseases and cancer. Both new cell lines offer the opportunity to model human diseases and test therapeutic modalities addressing these

A simulation study on the role of mitochondria-sarcoplasmic reticulum Ca2+ interaction in cardiomyocyte energetics during exercise.

Previous studies demonstrated that the mitochondrial Ca2+ uniporter MCU and the Na+-Ca2+ exchanger NCLX exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria-SR Ca2+ interaction. However, the physiological relevance of the mitochondria-SR Ca2+ interaction has remained unsolved. Furthermore, although mitochondrial Ca2+ has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH-producing dehydrogenases, the contribution of the Ca2+-dependent regulatory mechanisms to cellular functions under physiological conditions has been controversial. In this study, we constructed a new integrated model of human ventricular myocyte with excitation-contraction-energetics coupling and investigated systematically the contribution of mitochondria-SR Ca2+ interaction, especially focusing on cardiac energetics during dynamic workload transitions in exercise. Simulation analyses revealed that the spatial coupling of mitochondria and SR, particularly via mitochondrial Ca2+ uniport activity-RyR, was the primary determinant of mitochondrial Ca2+ concentration, and that the Ca2+-dependent regulatory mechanism facilitated mitochondrial NADH recovery during exercise and contributed to the stability of NADH in the workload transition by about 40%, while oxygen consumption rate and cytoplasmic ATP level were not influenced. We concluded that the mitochondria-SR Ca2+ interaction, created via the uneven distribution of Ca2+ handling proteins, optimizes the contribution of the mitochondrial Ca2+-dependent regulatory mechanism to stabilizing NADH during exercise. KEY POINTS: The mitochondrial Ca2+ uniporter protein MCU and the Na+-Ca2+ exchanger protein NCLX are reported to exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria-SR Ca2+ interaction in cardiomyocytes. Mitochondrial Ca2+ (Ca2+ mit) has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH-producing dehydrogenases. Here we constructed an integrated model of a human ventricular myocyte with excitation-contraction-energetics coupling and investigated the role of the mitochondria-SR Ca2+ interaction in cardiac energetics during exercise. Simulation analyses revealed that the spatial coupling particularly via mitochondrial Ca2+ uniport activity-RyR is the primary determinant of Ca2+ mit concentration, and that the activation of NADH-producing dehydrogenases by Ca2+ mit contributes to NADH stability during exercise. The mitochondria-SR Ca2+ interaction optimizes the contribution of Ca2+ mit to the activation of NADH-producing dehydrogenases.

FLASH Proton Radiation Therapy Mitigates Inflammatory and Fibrotic Pathways and Preserves Cardiac Function in a Preclinical Mouse Model of Radiation-Induced Heart Disease

PurposeStudies during the past 9 years suggest that delivering radiation at dose rates exceeding 40 Gy/s, known as “FLASH” radiotherapy, enhances the therapeutic index of radiation therapy (RT) by decreasing normal tissue damage while maintaining tumor response compared to conventional (or standard) RT. This study demonstrates the cardioprotective benefits of FLASH proton RT (F-PRT) compared to standard (conventional) proton RT (S-PRT), as evidenced by reduced acute and chronic cardiac toxicities. Methods and MaterialsMice were imaged using cone beam computed tomography to precisely determine the heart's apex as the beam isocenter. Irradiation was conducted utilizing a shoot-through technique with a 5mm diameter circular collimator. Bulk RNA-sequencing was performed on non-irradiated (NR) samples, as well as apexes treated with F-PRT or S-PRT, at 2 weeks following a single 40 Gy dose. Inflammatory responses were assessed through multiplex cytokine/chemokine microbead assay and immunofluorescence analyses. Levels of perivascular fibrosis were quantified using Masson's Trichrome and Picrosirius red staining. Additionally, cardiac tissue functionality was evaluated by 2D echocardiograms at 8 and 30 weeks post-PRT. ResultsRadiation damage was specifically localized to the heart's apex. RNA profiling of cardiac tissues treated with PRT revealed that S-PRT uniquely upregulated pathways associated with DNA damage response, induction of tumor necrosis factor superfamily and inflammatory response, while F-PRT primarily affected cytoplasmic translation, mitochondrion organization and ATP synthesis. Notably, F-PRT led to a milder inflammatory response, accompanied by significantly attenuated changes in TGF-β1 and αSMA levels. Critically, F-PRT decreased collagen deposition, and better preserved cardiac functionality compared to S-PRT. ConclusionsThis study demonstrated that F-PRT reduces the induction of an inflammatory environment with lower expression of inflammatory cytokines and profibrotic factors. Importantly, the results indicate that F-PRT better preserves cardiac functionality, as confirmed by echocardiography analysis, while also mitigating the development of long-term fibrosis.

TR-57 Treatment of SUM159 Cells Induces Mitochondrial Dysfunction without Affecting Membrane Potential.

Recent works identified ClpXP, mitochondrial caseinolytic protease, as the only target of imipridones, a new class of antitumor agents. Our study of the mechanism of imipridone derivative TR-57 action in SUM159 human breast cancer cells demonstrated mitochondrial fragmentation, degradation of mitochondrial mtDNA and mitochondrial dysfunction due to inhibition of Complex I and Complex II activity. Complete inhibition of oxidative phosphorylation accompanied 90, 94, 88 and 87% decreases in the content of Complex I, II, III and IV proteins, respectively. The content of the FOF1-ATPase subunits decreased sharply by approximately 35% after 24 h and remained unchanged up to 72 h of incubation with TR-57. At the same time, a disappearance of the ATPIF1, the natural inhibitor of mitochondrial FOF1-ATPase, was observed after 24 h exposure to TR-57. ATPase inhibitor oligomycin did not affect the mitochondrial membrane potential in intact SUM159, whereas it caused a 65% decrease in TR-57-treated cells. SUM159 cells incubated with TR57 up to 72 h retained the level of proteins facilitating the ATP transfer across the mitochondrial membranes: VDAC1 expression was not affected, while expression of ANT-1/2 and APC2 increased by 20% and 40%, respectively. Thus, our results suggest that although TR-57 treatment leads to complete inhibition of respiratory chain activity of SUM159 cells, hydrolysis of cytoplasmic ATP by reversal activity of FOF1-ATPase supports mitochondrial polarization.

Effect of maternal age on ATP content and distribution of mitochondria in bovine oocytes.

Our objective was to understand how maternal age influences the mitochondrial population and ATP content of in vivo matured bovine oocytes. We hypothesized that in vivo matured oocytes from older cows would have altered mitochondrial number and distribution patterns and lower cytoplasmic ATP content compared to the oocytes obtained from younger cows. Follicles ≥5mm were ablated in old cows (13 to 22 yrs, Old Group, n = 7) and their younger daughters (4 to 10 years old, Young Group; n = 7) to induce the emergence of a new follicular wave. Cows were treated twice daily with eight doses of FSH starting 24 hr after ablation (Day 0, day of wave emergence). Prostaglandin F2alpha (PGF) was given on Days 3 and 3.5, LH on Day 4.5, and cumulus-oocyte-complexes were collected 18-20 hours post-LH by ultrasound-guided follicular aspiration. Oocytes were either processed for staining with MitoTracker Deep Red FM or for ATP assay. Stained oocytes were imaged with a Zeiss LSM 710 confocal microscope, and mitochondria were segmented in the oocyte volume sets using Imaris Pro 7.4. In vivo matured oocytes obtained from old cows were similar in morphological grades to those from young cows. However, the oocytes of COC from older cows had 23% less intracellular ATP (27.4±1.9 vs 35.7±2.2 pmol per oocyte, P = 0.01) than those of young cows. Furthermore, the average volume of individual mitochondria, indicated by the number of image voxels, was greater (P<0.05) in oocytes from older cows than in those from younger cows. Oocytes from older cows also tended to have a greater number of mitochondrial clusters (P = 0.06) and an increased number of clusters in the central region of the oocytes (P = 0.04) compared to those from younger cows. In conclusion, our study demonstrated that maternal age was associated with a decrease in the cytoplasmic ATP content of in vivo mature oocytes and an altered distribution of mitochondrial structures. These findings suggest that maternal age may negatively influence the developmental competence of oocytes from older cows.

Mitochondrial ATP concentration decreases immediately after glucose administration to glucose-deprived hepatocytes.

Hepatocytes can switch their metabolic processes in response to nutrient availability. However, the dynamics of metabolites (such as lactate, pyruvate, and ATP) in hepatocytes during the metabolic switch remain unknown. In this study, we visualized metabolite dynamics in primary cultured hepatocytes during recovery from glucose-deprivation. We observed a decrease in the mitochondrial ATP concentration when glucose was administered to hepatocytes under glucose-deprivation conditions. In contrast, there was slight change in the cytoplasmic ATP concentration. A decrease in mitochondrial ATP concentration was associated with increased protein synthesis rather than glycogen synthesis, activation of urea cycle, and production of reactive oxygen species. These results suggest that mitochondrial ATP is important in switching metabolic processes in the hepatocytes.

Anti-Warburg effect via generation of ROS and inhibition of PKM2/β-catenin mediates apoptosis of lambertianic acid in prostate cancer cells.

To elucidate the underlying antitumor mechanism of lambertianic acid (LA) derived from Pinus koraiensis, the role of cancer metabolism related molecules was investigated in the apoptotic effect of LA in DU145 and PC3 prostate cancer cells. MTT assay for cytotoxicity, RNA interference, cell cycle analysis for sub G1 population, nuclear and cytoplasmic extraction, lactate, Glucose and ATP assay by ELISA, Measurement of reactive oxygen species (ROS) generation, Western blotting, and immunoprecipitation assay were conducted in DU145 and PC3 prostate cancer cells. Herein LA exerted cytotoxicity, increased sub G1 population and attenuated the expression of pro-Caspase3 and pro-poly (ADP-ribose) polymerase (pro-PARP) in DU145 and PC3 cells. Also, LA reduced the expression of lactate dehydrogenase A (LDHA), glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2 (PKM2) with reduced production of lactate in DU145 and PC3 cells. Notably, LA decreased phosphorylation of PKM2 on Tyr105 and inhibited the expression of p-STAT3, cyclin D1, C-Myc, β-catenin, and p-GSK3β with the decrease of nuclear translocation of p-PKM2. Furthermore, LA disturbed the binding of p-PKM2 and β-catenin in DU145 cells, which was supported by Spearman coefficient (0.0463) of cBioportal database. Furthermore, LA generated ROS in DU145 and PC3 cells, while ROS scavenger NAC (N-acetyl L-cysteine) blocked the ability of LA to reduce p-PKM2, PKM2, β-catenin, LDHA, and pro-caspase3 in DU145 cells. Taken together, these findings provide evidence that LA induces apoptosis via ROS generation and inhibition of PKM2/β-catenin signaling in prostate cancer cells.

Dependence of cardiomyocyte force, velocity, and power generation on ATP concentration

Theoretical analysis and computer modeling of myocardial energy metabolism under physiological and heart failure conditions predicts that cytoplasmic ATP and ADP levels both fall roughly in proportion to reductions in total adenine nucleotide pool observed in heart failure. Assessing sub-maximal force and velocity in loaded myocardial muscle at MgATP levels in the range of 2 to 8 mM, Beard et al. ( Biophys. J. 2022. 121: 3213–3223) observed effects of MgATP on myocardial power generation that suggest that although maximal force and velocity are not substantially sensitive to differences in [MgATP] that occur in the normal versus failing myocardium, maximal power generation, which occurs at submaximal force and velocity, is sensitive to [MgATP]. Previously developed computational models are not able to reproduce the observed dependence on MgATP. The goals of the present study are to construct and analyze a computational model of muscle dynamics to analyze data on the dependence of maximal force, velocity, and power generation on [MgATP] in order to determine a potential mechanism to explain the MgATP-dependent phenomena. Model development and identification was guided by dynamic measurements on permeabilized mouse trabecular fibers. The data include force-velocity relation and time constant of tension development. The mathematical model used to simulate cardiomyocyte crossbridge mechanics was based on the previous crossbridge and calcium models from Tewari et al. (J Mol Cell Cardiol, 2016. 96: 11-25) and Campbell et al. (Biophys J, 2018. 115: 543-553). The developed model includes strain- and metabolite-dependent transition between attachment states, and ATP-dependent destabilization of the super-relaxed (SRX) myosin state. Model-based analysis of force and velocity dyanmics is consistent with the hypothesis that ATP assocition with the myosin protein influence the kinetics and thermodynamics of transitions between the SRX and disorded (DRX) states. Funding: Department of Veterans Affairs Merit Review Award I01BX000740 (A.J.B.) and National Heart, Lung, and Blood Institute grant R01 HL154624 (A.J.B., D.A.B.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Self-assembly CuO-loaded nanocomposite involving functionalized DNA with dihydromyricetin for water-based efficient and controllable antibacterial action.

With the antibiotic crisis intensifies, the defense and treatment of pathogen infections in safe and effective fashion has become a critical issue. Herein, we report a novel and advanced type of sterilization agent designed via the functionalization DNA nanocarriers based on dihydromyricetin and CuO-loaded nanoparticles (DNA/DMY-CuO). Firstly, a pure dihydromyricetin (DMY) isolated from Ampelopsis grossedentata is used as a bridge to the stimulate the construction of DNA cross-linking networks by hydrogen bonding. Subsequently, a 3D spherical CuO-loaded nanocomposite (204.39nm) is customized using the DNA/DMY network as a biological template through a simple coordination-assisted self-assembly method, which exhibits a high dispersibility, water-solubility and physiological stability. The reversible physical interactions in nanocarriers allows the selective separation and automatic release of CuO NPs from DNA/DMY-CuO in neutral and wound exudate environments, thereby extending the survival period of CuO NPs by nearly 24h. Meanwhile, the nanocarriers system relied on the strong binding ability of DMY to the outer membrane of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) achieves controlled drug delivery onto the pathogen wall. The advanced antibacterial action of DNA/DMY-CuO also reflected in membrane destruction, cytoplasmic constituent leakages and ATP synthetic pathway cessation, thereby halting cytosolic metalloregulatory mechanisms and minimizing drug-resistant bacteria. In summary, such multi-functional CuO-loaded nanocomposite provides a water-dispersibility, controllable, low cytotoxicity and long-effective platform to address the ever-growing threats of bacterial infections.

Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

'Carbon-Monoxide-Releasing Molecule-2 (CORM-2)' Is a Misnomer: Ruthenium Toxicity, Not CO Release, Accounts for Its Antimicrobial Effects.

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological ‘gasotransmitter’, in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (<0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 μM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 μM; His, Kd 130 μM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 μM; GSSG, Kd 25,000 μM). The toxicity of low, but potent, levels (15 μM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.

Alteration of the F1Fo ATP Synthase Causes Metabolic Remodeling in Breast Cancer Cells

Abstract Objectives The F1Fo ATP synthase is a multienzyme complex that produces mitochondrial ATP. Aberrant expression or assembly of F1Fo ATP synthase subunits leads to alterations in energy metabolism. We recently found that breast cancer cells exposed to fluid shear stress (FSS) have significantly enhanced metastatic behavior including chemoresistance and cell proliferation. Chemoresistance depends upon active transport systems, and cell division and growth require ATP. Therefore, we hypothesized that circulating breast cancer cells undergo altered energy metabolism via FSS-induced changes in F1Fo ATP synthase subunits and subsequent mitochondrial remodeling. Methods Non-metastatic MCF7 and metastatic MDA-MB-231 human breast cancer cells were treated with or without FSS and cultured. Cellular proliferation was assayed by measuring cell number and gap distance. Metabolic profile including intracellular ATP and oxygen consumption rate were analyzed. We also quantified abundance of F1Fo ATP synthase subunits using immunoblotting. Results Treatment with FSS significantly increased proliferation of both MCF7 and MDA-MB-231 human breast cancer cells. FSS significantly increased intracellular ATP in MDA-MB-231 breast cancer cells while ATP levels in MCF7 were not significantly changed. MDA-MB-231 cells retained increased ATP after treatment with the uncoupler FCCP, indicating remodeling and decreased reliance on mitochondrial energy metabolism. Interestingly, oxygen consumption rate was significantly increased in both MCF7 and MDA-MB-231 by FSS. We further quantified the abundance of F1Fo ATP synthase subunits in both cell lines. The β- and c-subunits of the F1Fo ATP synthase were significantly depleted in both lines of FSS-treated breast cancer cells. Conclusions Our data show that FSS alters abundance of the F1Fo ATP synthase subunits leading to metabolic remodeling. We suggest that FSS may influence non-metastatic (MCF7) and metastatic cancer cells (MDA-MB-231) differently. Underlying changes in mitochondrial and cytoplasmic ATP production in these cells is still under investigation. However, it is possible that reactive oxygen species generated during FSS may signal a switch to cytoplasmic intracellular energy metabolism. Funding Sources Alabama Life Research Institute Pilot Project (University of Alabama)

Abstract 1834: Real-time visualization and quantification of flexible, multi-parameter indicators of cancer cell heath and metabolism using live-cell analysis

Abstract The ability to perform concurrent, dynamic measurements of multiple parameters of tumor cell health can provide valuable mechanistic insight. For example, a single compound may exhibit cytotoxic and/or cytostatic properties, depending on the tumor cell type being targeted as well as concentration and duration of treatment. In addition, evaluating immune-cancer cell interactions or metabolic exchange between cancer and stromal cells can be a critical component of elucidating mechanism of action. However, standard methods of evaluating compound effects on tumor cell health are largely limited to single parameter, population-based, endpoint measurements. Here we describe a new live-cell imaging solution providing flexibility in fluorescent readouts of cancer cell health, metabolism, and cell-cell interactions. Phase and fluorescent images were acquired and analyzed using an IncuCyte live cell analysis system. Changeable filter sets enabled selection of fluorescent readouts in designated combinations of green, orange, red, and/or NIR spectra as well as a dual excitation, single emission filter set to provide ratiometric measurements of cytoplasmic ATP. Using this approach, we demonstrate concentration- and time-dependent effects of compounds on cell cycle and cell death. Kazusamycin A (0.08-20 nM) treatment of HT-1080 cells resulted in a concentration-dependent cell cycle arrest as measured by a fluorescence ubiquitination-based cell cycle indicator. An increase in the percentage of cells in G1 (22% vs 31% of vehicle or kazusamycin A-treated cells, respectively) was observed as early as four hours after treatment and increased over the 3-day time course. Cell death was evaluated concurrently using an Annexin V reagent. Apoptosis was observed only at high concentrations (6.67-20 nM) and after 18 hours of incubation. Similar results were observed in MDA-MB-231 cells. Incorporation of genetically encoded and/or live-cell immunocytochemistry reagents enables monitoring subpopulations of cells in co-culture models. We utilized these techniques to monitor dynamic readouts in target and effector populations in an immune cell killing assay. MDA-MB-231 cells were transduced to express a fluorescent nuclear marker and were co-cultured with PBMCs with and without activation (ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator + IL-2). A decrease in nuclear counts accompanied by apoptosis (Annexin V-positive cells) of MDA-MB-231 target cells was observed within 27 hours in the presence of activated PBMCs. Measurements of target cell proliferation and apoptosis were unaffected by the presence of PBMCs without activation cocktail. Additional example datasets of cancer cell metabolism, proliferation, and death readouts in mono- and co-culture models will be shared to illustrate the value of flexible, multi-parameter live cell analysis. Citation Format: Cicely L. Schramm, John N. Rauch, Libuse Oupicka, Laura A. Skerlos, Nicola J. Bevan, Gillian F. Lovell, Sandra Perez-Garrido, Grigory S. Filonov, Yong X. Chen, Ilya Kovalenko, Susan Foltin, Hinnah Campwala, Timothy J. Dale, Daniel M. Appledorn. Real-time visualization and quantification of flexible, multi-parameter indicators of cancer cell heath and metabolism using live-cell analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1834.

Magnesium Links Starvation-Mediated Antibiotic Persistence to ATP.

Bacterial persisters emerge and increase in numbers over time as a bacterial culture grows from log phase to stationary phase. However, the underlying basis of the inevitable tendency is unclear. In this study, we investigated the role of nutrients in starvation-mediated persister formation of Staphylococcus aureus By screening of nutrient components, we found that starvation-induced persister formation of log-phase cultures could be reversed by addition of magnesium (Mg2+) but not amino acids, nucleotides, or other salts. Further, deprivation of extracellular Mg2+ reduced cytoplasmic ATP, inducing persistence without affecting cytoplasmic Mg2+ or membrane potential. Finally, we showed that Mg2+ reduced expression of stationary cell marker genes, cap5A and arcA These findings indicate a connection between Mg2+ levels and ATP, which represents metabolic status and mediates antibiotic persistence during growth.IMPORTANCE Various genes have been identified to be involved in bacterial persister formation regardless of the presence or absence of persister genes. Despite recent discoveries of the roles of ATP and membrane potential in persister formation, the key element that triggers change of ATP or membrane potential remains elusive. Our work demonstrates that Mg2+ instead of other ions or nutrient components is the key element for persistence by inducing a decrease of cytoplasmic ATP, which subsequently induces persister formation. In addition, we observed tight regulation of genes for Mg2+ transport in different growth phases in S. aureus These findings indicate that despite being a key nutrient, Mg2+ also served as a key signal in persister formation during growth.

ADGRL4/ELTD1 Silencing in Endothelial Cells Induces ACLY and SLC25A1 and Alters the Cellular Metabolic Profile.

Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1′s full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1′s role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of ACLY (fatty acid and cholesterol synthesis or acetylation). Silencing induced KIT expression and affected the Notch signalling pathway, upregulating Delta Like Canonical Notch Ligand 4 (DLL4) and suppressing Jagged Canonical Notch Ligand 1 (JAG1) and Hes Family BHLH Transcription Factor 2 (HES2). The effect of ADGRL4/ELTD1 silencing on the cellular metabolic profile was modest but several metabolites were significantly affected. Cis-aconitic acid, uridine diphosphate (UDP)-glucoronate, fructose 2,6-diphosphate, uridine 5-diphosphate, and aspartic acid were all elevated as a result of silencing and phosphocreatine, N-acetylglutamic acid, taurine, deoxyadenosine triphosphate, and cytidine monophosphate were depleted. Metabolic pathway analysis implicated ADGRL4/ELTD1 in pyrimidine, amino acid, and sugar metabolism. In summary, this study shows that ADGRL4/ELTD1 impacts core components of endothelial metabolism and regulates genes involved in endothelial differentiation/homeostasis and Notch signalling.

Functional characterization of activating mutations in the sulfonylurea receptor 1 (ABCC8) causing neonatal diabetes mellitus in Asian Indian children.

Gain-of-function of ATP-sensitive K+ (KATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in KATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known KATP inhibitors in intact cells were determined by 86 Rb efflux assays. Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP4- inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the KATP channel inhibitors glibenclamide and carbamazepine. The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect KATP channel function to cause the disease. The reduced ATP4- sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.

Nitric oxide triggers the assembly of \u201ctype II\u201d stress granules linked to decreased cell viability

We show that 3-morpholinosydnonimine (SIN-1)-induced nitric oxide (NO) triggers the formation of SGs. Whereas the composition of NO-induced SGs is initially similar to sodium arsenite (SA)-induced type I (cytoprotective) SGs, the progressive loss of eIF3 over time converts them into pro-death (type II) SGs. NO-induced SG assembly requires the phosphorylation of eIF2α, but the transition to type II SGs is temporally linked to the mTOR-regulated displacement of eIF4F complexes from the m7 guanine cap. Whereas SA does not affect mitochondrial morphology or function, NO alters mitochondrial integrity and function, resulting in increased ROS production, decreased cytoplasmic ATP, and plasma membrane permeabilization, all of which are supported by type II SG assembly. Thus, cellular energy balance is linked to the composition and function of NO-induced SGs in ways that determine whether cells live or die.

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.