NADH Autofluorescence—A Marker on its Way to Boost Bioenergetic Research

Schaefer, Patrick; Kalinina, Sviatlana; Rueck, Angelika C.; Arnim, Christine A. F. Von; Einem, Bjoern von

doi:10.1002/cyto.a.23597

Cited by 169 publications

(177 citation statements)

References 88 publications

(148 reference statements)

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“…While the steady-state NADH fluorescence before KCl addition is not large enough to be detected, a step increase after KCl addition invariably points to the drop in O 2 concentration. Cytosolic NADH is produced at detectable levels within a few seconds after O 2 consumption in the light reaction and similar such observations were reported in earlier work [55]. This result also suggests that the steadystate aldehyde concentration in cells is sufficient to carry out the light reaction for a long duration (at least for a few tens of minutes) because NADH production rate due to O 2 drop is faster than rate of NADH consumption for aldehyde synthesis.…”

Section: Nadh Assaysupporting

confidence: 89%

“…We do not know if the BRET pair interacts with the VSD domain and its rearrangement caused the voltage shift. Earlier work showed evidences for the interaction between fluorescent protein and the VSD domain but the mechanism of fluorescence modulation by VSD is still not fully understood [55]. We speculate the interaction between the YPET and the VSD domains could be a possible reason for the observed shift.…”

Section: Voltage Switching Characterizationmentioning

confidence: 70%

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An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Srinivasan

Griffin

Joshi

et al. 2019

Preprint

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1.AbstractGenetically encoded reporters have greatly increased our understanding of biology, especially in neuroscience. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach we have engineered Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential. Unlike other luciferase-luciferin bioluminescent systems, AMBER encodes the genes to express both the luciferase and luciferin. AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When AMBER is co-expressed with the luciferin producing genes it reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods we show that AMBER modulates its enzymatic activity as a function of the membrane potential. AMBER shows several-fold increase in the luminescent (ΔL/L) signal upon switching from the off to on state when the cell is depolarized. In vivo expression of AMBER in C. elegans allowed detecting pharyngeal pumping action and mechanosensory neural activity from multiple worms simultaneously. AMBER reports neural activity of multiple animals at the same time and can be used in social behavior assays to elucidate the role of membrane potential underlying behavior.2.Significance StatementThere have been many exciting advances in the development of genetically encoded voltage indicators to monitor intracelluar voltage changes. Most sensors employ fluorescence, which requires external light, potentially causing photobleaching or overheating. Consequently, there has been interest in developing luminescence reporters. However, they require addition of an exogenous substrate to produce light intracellularly. Here, we engineered a genetically encoded bioluminescent voltage indicator, AMBER, which unlike other bioluminescent activity indicators, does not require addition of an exogenous substrate. AMBER allows a large differential signal, a high signal-to-noise ratio, and causes minimal metabolic demand on cells. We used AMBER to record voltage activity in freely-moving C. elegans, demonstrating that AMBER is a important new tool for monitoring neuronal activity during social behavior.

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Section: Nadh Assaysupporting

confidence: 89%

Section: Voltage Switching Characterizationmentioning

confidence: 70%

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Srinivasan

Griffin

Joshi

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…Treatment with 2-DG did not alter the intracellular pH during lifetime measurements, a factor known to distort lifetimes of fluorescent proteins (S2B Fig). Furthermore, two-photon FLIM, a well-established method to decipher the dynamics of NAD(P)H metabolism in living cells [39], showed robust decreases in the ratio of NAD(P)H free vs. Our approach emphasizing fluorescent lifetimes of metabolic biosensors allowed us to calculate relative ATP:ADP ratios or relative lactate concentrations (see Methods) prior to infection, and whether or not these calculated parameters could be predictive of cellular permissiveness to HIV-1 infection, as measured by β-galactosidase activity [40]. Briefly, the βgalactosidase assay utilizes an X-gal stain to detect virus-infected cells as TZM-bls harbour the Tat-responsive reporter LacZ reporter for the detection of Tat-dependent β-galactosidase activities during viral infection.…”

Section: Basal Metabolic State Is a Determinant Of Hiv-1 Infection Inmentioning

confidence: 99%

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion

et al. 2020

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There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.

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“…This result suggests, in accordance with what is reported in ref. 66 , that NLCs treatment induces a lower mitochondrial respiratory function. Furthermore an increase of the NADH/NAD + ratio (fluorescent and not fluorescent form, respectively) could be speculated, owing to its ability to influence the activities of the PARP-1, which uses NAD + as substrate 67 .…”

Section: Discussionmentioning

confidence: 99%

Synergic pro-apoptotic effects of Ferulic Acid and nanostructured lipid carrier in glioblastoma cells assessed through molecular and Delayed Luminescence studies

Grasso

Dell’Albani

Carbone

et al. 2020

Sci Rep

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Herein, we assessed the effect of Ferulic Acid (FA), a natural antioxidant with anti-cancer effect, on the human glioblastoma cells through molecular and Delayed Luminescence (DL) studies. DL, a phenomenon of ultra-week emission of optical photons, was used to monitor mitochondrial assessment. The effect of FA loaded in nanostructured lipid carriers (NLCs) was also assessed. To validate NLCs as a drug delivery system for glioblastoma treatment, particular attention was focused on their effect. We found that free FA induced a significant decrease in c-Myc and Bcl-2 expression levels accompanied by the apoptotic pathway activation. Blank NLCs, even if they did not induce cytotoxicity and caspase-3 cleavage, decreased Bcl-2, ERK1/2, c-Myc expression levels activating PARP-1 cleavage. The changes in DL intensity and kinetics highlighted a possible effect of nanoparticle matrix on mitochondria, through the involvement of the NADH pool and ROS production that, in turn, activates ERK1/2 pathways. All the effects on protein expression levels and on the activation of apoptotic pathway appeared more evident when the cells were exposed to FA loaded in NLCs. We demonstrated that the observed effects are due to a synergic pro-apoptotic influence exerted by FA, whose bioavailability increases in the glioblastoma cells, and NLCs formulation.Glioblastoma multiforme (GBM), also known as grade IV astrocytoma, represents the most prevalent and aggressive brain cancer. It is characterized by glial cells and has finger-like tentacles that infiltrate the brain, which make them very difficult to remove with surgical procedures. GBM exhibits a high level of resistance to conventional chemotherapy and radiotherapy, also due to the existence of blood-brain barrier (BBB), glioma stem cells and complex network of multiple modified signalling pathways 1 . The most frequent aberrant expression is represented by the dysregulation of extracellular signal-regulated protein kinase (ERK), which is associated with poor survival of the patients. The ERK isoforms (p42/44 or ERK1/ERK2) by interacting with specific phosphorylation substrates, play a pivotal role in the control of several cellular processes involved in proliferation, as well as activation of transcription factors, apoptosis and the control of cellular process 2,3 . In addition, the transcription factor c-Myc has been recognized as an important regulator of stem cell biology implicated with GBM malignancy and stemness 4 , as it contributes to proliferation, growth and survival of GBM stem cells 5 . GBM has been also related to the impairment of mitochondrial metabolic capacity, which leads to the alteration in energy production 6,7 and is characterized by an overexpression of Bcl-2 8 . This protein can regulate transition pores permeability of the outer mitochondrial membrane and block pro-apoptotic proteins 9 . Furthermore, it has been identified a novel www.nature.com/scientificreports www.nature.com/scientificreports/ interaction between Bcl-2 and (ADP-ribose) polymerase (PARP) 10 , t...

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NADH Autofluorescence—A Marker on its Way to Boost Bioenergetic Research

Cited by 169 publications

References 88 publications

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion

Synergic pro-apoptotic effects of Ferulic Acid and nanostructured lipid carrier in glioblastoma cells assessed through molecular and Delayed Luminescence studies

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