Dataset Information

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

ABSTRACT: Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 ns lifetime change from pH 5.5 to 9.0. Critically, we find that the sensor maintains a p Ka of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are robustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time-resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent sensor, we also demonstrate that mCherryTYG is spectrally compatible with the ATeam sensor and EGFP for simultaneous dual-color measurements of intracellular pH, ATP, and extracellular pH. In a proof-of-concept, we quantify acute respiration-dependent pH homeostasis that exhibits a stoichiometric relationship with the ATP-generating capacity of the carbon fuel choice in E. coli. Broadly speaking, our work presents a previously unemployed methodology that will greatly facilitate continuous pH quantification.

SUBMITTER: Haynes EP

PROVIDER: S-EPMC6623984 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Haynes Emily P EP Rajendran Megha M Henning Chace K CK Mishra Abhipri A Lyon Angeline M AM Tantama Mathew M

Analytical chemistry 20190619 13

Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first repor ...[more]

PMID: 31247720

Similar Datasets

Project description:Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets