Dataset Information

Fluorescent Visualization of Cellular Proton Fluxes.

ABSTRACT: Cells use plasma membrane proton fluxes to maintain cytoplasmic and extracellular pH and to mediate the co-transport of metabolites and ions. Because proton-coupled transport often involves movement of multiple substrates, traditional electrical measurements provide limited information about proton transport at the cell surface. Here we visualize voltage-dependent proton fluxes over the entire landscape of a cell by covalently attaching small-molecule fluorescent pH sensors to the cell's glycocalyx. We found that the extracellularly facing sensors enable real-time detection of proton accumulation and depletion at the plasma membrane, providing an indirect readout of channel and transporter activity that correlated with whole-cell proton current. Moreover, the proton wavefront emanating from one cell was readily visible as it crossed over nearby cells. Given that any small-molecule fluorescent sensor can be covalently attached to a cell's glycocalyx, our approach is readily adaptable to visualize most electrogenic and non-electrogenic transport events at the plasma membrane.

SUBMITTER: Zhang L

PROVIDER: S-EPMC5182157 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Fluorescent Visualization of Cellular Proton Fluxes.

Zhang Lejie L Bellve Karl K Fogarty Kevin K Kobertz William R WR

Cell chemical biology 20161201 12

Cells use plasma membrane proton fluxes to maintain cytoplasmic and extracellular pH and to mediate the co-transport of metabolites and ions. Because proton-coupled transport often involves movement of multiple substrates, traditional electrical measurements provide limited information about proton transport at the cell surface. Here we visualize voltage-dependent proton fluxes over the entire landscape of a cell by covalently attaching small-molecule fluorescent pH sensors to the cell's glycoca ...[more]

PMID: 27916567

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Project description:Fluorescent imaging, which is an important interdisciplinary field bridging research from organic chemistry, biochemistry and cell biology has been applied for multi-dimensional detection, visualization and characterization of biological structures and processes. Especially valuable is the possibility to monitor cellular processes in real time using fluorescent probes. In this work, conjugated oligoelectrolytes and neutral derivatives with the distyrylnaphthalene core (SN-COEs) were designed, synthetized and tested for biological properties as membrane-specific fluorescent dyes for the visualization of membrane-dependent cellular processes. The group of tested compounds includes newly synthesized distyrylnaphthalene derivatives (DSNNs): a trimethylammonium derivative (DSNN-NMe3+), a phosphonate derivative (DSNN-P), a morpholine derivative (DSNN-Mor), a dihydroxyethylamine derivative (DSNN-DEA), a phosphonate potassium salt (DSNN-POK), an amino derivative (DSNN-NH2) and pyridinium derivative (DSNN-Py+). All compounds were tested for their biological properties, including cytotoxicity and staining efficiency towards mammalian cells. The fluorescence intensity of SN-COEs incorporated into cellular structures was analyzed by fluorescence activated cell sorting (FACS) and photoluminescence spectroscopy. The cytotoxicity results have shown that all tested SN-COEs can be safely used in the human and animal cell studies. Fluorescence and confocal microscopy observations confirm that tested COEs can be applied as fluorescent probes for the visualization of intracellular membrane components in a wide range of different cell types, including adherent and suspension cells. The staining procedure may be performed under both serum free and complete medium conditions. The presented studies have revealed the interesting biological properties of SN-COEs and confirmed their applicability as dyes for staining the membranous structures of eukaryotic cells, which may be useful for visualization of wide range of biological processes dependent of the extra-/intracellular communications and/or based on the remodeling of cellular membranes.

Project description:Photophysical data and orbital energy levels (from electrochemistry) were compared for molecules with the same BODIPY acceptor part (red) and perpendicularly oriented xanthene or BODIPY donor fragments (green). Transfer of energy, hence the photophysical properties of the cassettes, including the pH dependent fluorescence in the xanthene-containing molecules, correlates with the relative energies of the frontier orbitals in these systems. Intracellular sensing of protons is often achieved via sensors that switch off completely at certain pH values, but probes of this type are not easy to locate inside cells in their "off-state". A communication from these laboratories (J. Am. Chem. Soc., 2009, 131, 1642-3) described how the energy transfer cassette 1 could be used for intracellular imaging of pH. This probe is fluorescent whatever the pH, but its exact photophysical properties are governed by the protonation states of the xanthene donors. This work was undertaken to further investigate correlations between structure, photophysical properties, and pH for energy transfer cassettes. To achieve this, three other cassettes 2-4 were prepared: another one containing pH-sensitive xanthene donors (2) and two "control cassettes" that each have two BODIPY-based donors (3 and 4). Both the cassettes 1 and 2 with xanthene-based donors fluoresce red under slightly acidic conditions (pH < ?6) and green when the medium is more basic (>?7), whereas the corresponding cassettes with BODIPY donors give almost complete energy transfer regardless of pH. The cassettes that have BODIPY donors, by contrast, show no significant fluorescence from the donor parts, but the overall quantum yields of the cassettes when excited at the donor (observation of acceptor fluorescence) are high (ca. 0.6 and 0.9). Electrochemical measurements were performed to elucidate orbital energy level differences between the pH-fluorescence profiles of cassettes with xanthene donors, relative to the two with BODIPY donors. These studies confirm energy transfer in the cassettes is dramatically altered by analytes that perturb relative orbital levels. Energy transfer cassettes with distinct fluorescent donor and acceptor units provide a new, and potentially useful, approach to sensors for biomedical applications.

Dataset Information

Fluorescent Visualization of Cellular Proton Fluxes.

Publications

Fluorescent Visualization of Cellular Proton Fluxes.

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