Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor

Haynes, Emily P; Rajendran, Megha; Henning, Chace K; Mishra, Abhipri; Lyon, Angeline M.; Tantama, Mathew

doi:10.1021/acs.analchem.9b01562

Cited by 9 publications

(15 citation statements)

References 105 publications

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“…Upon addition of cyanide to live-cell cultures, the sensor reproducibly reports an increase in bacterial ADP levels with an increase in the donor mTFP1 emission peak and a reciprocal decrease in the mVenus sensitized emission peak (Figure 4a). To validate that the observed spectral change represents a change in FRET, we carried out time-resolved spectroscopy on live-cell cultures expressing the sensor [20]. From the spectral response, ADP-binding to the sensor causes a decrease in FRET, which should manifest as an increase in the mTFP1 donor fluorescence lifetime when ADP levels increase [28].…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Trull

Miller

Tat

et al. 2019

Sensors

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Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems.

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Section: Resultsmentioning

confidence: 99%

“…Live E. coli suspensions were prepared as described above except that cultures were diluted to an OD 600 of ~0.5 and pre-starved for 30–60 min in the absence of glucose. Live-cell lifetime decays were collected using Fluoracle software, and empirical lifetimes were measured and analyzed in Matlab as previously described [20].…”

Section: Methodsmentioning

confidence: 99%

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Trull

Miller

Tat

et al. 2019

Sensors

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“…The observed phenotypes need to be further examined in future studies, e.g., with a fluorescence signal that is coupled to PMF (34), so that loss of capability or damage during or after pH stress can be analysed in more detail. Furthermore, detection of the cytoplasmic pH can be determined with pH sensitive fluorescent proteins (6).…”

Section: Glutamicum Growth At Single Ph Stress Pulse Experimentsmentioning

confidence: 99%

“…the enzyme activity, protein stability, solubility of trace elements and the proton motive force (PMF) (2)–(5). The regulation of the intracellular pH value is maintained by pH homeostasis (6). Corynebacterium glutamicum is a neutralophilic organism that can maintain the internal pH of 7.5 ± 0.5 in environmental pH fluctuations ranging between 5.5 and 9.0 (2).…”

Section: Introductionmentioning

confidence: 99%

Growth response and recovery ofCorynebacterium glutamicumcolonies on single-cell level upon defined pH stress pulses

Täuber

Blöbaum

Wendisch

et al. 2021

Preprint

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Bacteria respond to pH changes in their environment via pH homeostasis to keep the intracellular pH as constant as possible within a small range. A change of the intracellular pH value influences e.g., the enzyme activity, protein stability, solubility of trace elements and the proton motive force. Here, the species Corynebacterium glutamicum has been chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with an external pH between 5.5 and 9. In the recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell responding to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyse the physiological growth response of C. glutamicum upon precise pH stress pulses at a single-cell level. Analysis of the growth behaviour at the colony level by dMSCC exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in the viability with increasing stress duration. Colony regrowth was possible after increasing lag phases when stress durations were increased from 5 min to 9 h for all tested pH values. Furthermore, the single-cell analysis revealed heterogeneous regrowth of cells after pH stress, which can be distinguished into two distinct behaviours: firstly, cells continue to grow without interruption after the pH stress, and secondly, some cells rest for several hours after the pH stress before they start to grow again after this lag phase. This study provides the first insights into the single-cell response to acidic and alkaline pH stress adaptation of C. glutamicum.

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“…Relatively few fluorescent pH probes can respond to pH changes in a wide range, eg pH 3‒9, 41 pH 1‒8, 42 pH 2.5‒10, 43 pH 1–9, 44 pH 1–10, 45,46 pH 2–12, 47–49 pH 3.5–13.5, 50 pH 1–13, 51 pH 1–14, 52 or sensitive to pH changes in more than one pH interval, eg pH 2.88‒5 and pH 10–13.78, 53 pH 4.5–7.5 and pH 9–12 54 . The sensing of pH by using fluorescent probes are mainly based on the fluorescence mechanisms of photoinduced electron transfer (PET), 9,13,30,31,38,45 intramolecular charge transfer (ICT), 17,21,47 PET and ICT, 11,39 fluorescence resonance energy transfer (FRET), 22,33,36 protonation‐activable resonance charge transfer (PARCT), 40 through‐bond energy transfer (TBET), 53 and excited state intramolecular proton transfer (ESIPT) 48,49,55 . By far, a number of chemosensors bearing a reaction site for fluorometric detection of mercury, 56–60 hypochlorite, 61–66 nitric oxide, 67 and biothiols and peroxynitrite 68,69 were developed based on specific chemical reactions.…”

Section: Introductionmentioning

confidence: 99%

A novel reaction‐based fluorescent probe sensitive to pH changes in multiple intervals

Жен

Zheng

et al. 2020

Coloration Technology

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A new coumarin derivative containing both imine bond and amide moiety was synthesised. It was used as a reaction‐based pH probe and was sensitive to pH changes in the intervals 1.50–4.10 and 10.20–12.86. In the pH range 1.50–4.10, the ultraviolet‐visible (UV‒vis) absorption band of the probe gradually shifted from 353 nm to 340 nm with decrease in the pH value. The colour changed from pale yellow fluorescence at pH 4.10 to blue fluorescence at pH 1.50. Meanwhile the fluorescence emission band at 501 nm gradually shifted to 560 nm accompanying a gradual enhancing in the emission intensity. This pH response is caused by the hydrolysis of the imine bond in the probe under acidic conditions (pH 1.50–4.10). The colourless probe solution turned yellow at pH range 10.20–12.86. Bathochromic shift of the fluorescence band from 501 nm gradually to 550 nm was observed with the increase of pH value from 10.20 to 12.86 accompanying a gradual enhancement of the fluorescence emission intensity, indicating that the new compound serves as a fluorescent pH probe at pH range 10.20–12.86 through a deprotonation process. Under strongly alkaline conditions with pH larger than 12.86, hydrolysis of the lactone structure in the probe occurred to produce non‐fluorescent species.

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Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor

Cited by 9 publications

References 105 publications

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Growth response and recovery ofCorynebacterium glutamicumcolonies on single-cell level upon defined pH stress pulses

A novel reaction‐based fluorescent probe sensitive to pH changes in multiple intervals

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