A photocross-linking fluorescent indicator of mitochondrial membrane potential.

Ionic dyes that distribute across membranes according to electrical potential have proven valuable as fluorescent indicators of mitochondrial energetics in living cells. Applications have been limited, however, as potential-dependent staining is lost during cell fixation. We have produced a membrane potential indicator whose potential-dependent distribution can be made permanent, to enable correlation of membrane potential with cytochemical information from immunofluorescence. A carbocyanine dye was derivatized with a photoreactive nitrophenylazide moiety so that irradiation would induce nonspecific, covalent attachment to nearby molecules. Photo-induced cross-linking was observed in paper chromatography, when irradiation caused immobilization of the dye. The new dye, named PhoCy (photofixable cyanine), showed specific staining of mitochondria in living Swiss 3T3 fibroblasts. When living cells were stained, irradiated, and fixed with formaldehyde, mitochondrial staining was retained owing to cross-linking with mitochondrial components. Omission of irradiation eliminated mitochondrial staining in fixed cells. Labeling, irradiation, and fixation procedures were optimized to produce bright specific staining with minimal background. The indicator's sensitivity to mitochondrial potential was demonstrated by treating cells with 2,4-dinitrophenol, an uncoupler of mitochondrial electron transport, which decreased mitochondrial staining in living cells and in the corresponding fixed specimens.

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Faculty Opinions recommendation of Fluorescent indicators for imaging protein phosphorylation in single living cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1005065.59105 ◽

2002 ◽

Author(s):

Steve Ward

Keyword(s):

Protein Phosphorylation ◽

Living Cells ◽

Fluorescent Indicators ◽

Single Living Cells ◽

Single Living

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On the Wireless Microwave Sensing of Bacterial Membrane Potential in Microfluidic-Actuated Platforms

Sensors ◽

10.3390/s21103420 ◽

2021 ◽

Vol 21 (10) ◽

pp. 3420

Author(s):

Marc Jofre ◽

Lluís Jofre ◽

Luis Jofre-Roca

Keyword(s):

Membrane Potential ◽

Living Cells ◽

Membrane Potentials ◽

Electromagnetic Properties ◽

Bacterial Membrane ◽

Microfluidic Platforms ◽

Bacterial Membranes ◽

Wireless Monitoring ◽

Bacterial Dynamics ◽

Engineered Systems

The investigation of the electromagnetic properties of biological particles in microfluidic platforms may enable microwave wireless monitoring and interaction with the functional activity of microorganisms. Of high relevance are the action and membrane potentials as they are some of the most important parameters of living cells. In particular, the complex mechanisms of a cell’s action potential are comparable to the dynamics of bacterial membranes, and consequently focusing on the latter provides a simplified framework for advancing the current techniques and knowledge of general bacterial dynamics. In this work, we provide a theoretical analysis and experimental results on the microwave detection of microorganisms within a microfluidic-based platform for sensing the membrane potential of bacteria. The results further advance the state of microwave bacteria sensing and microfluidic control and their implications for measuring and interacting with cells and their membrane potentials, which is of great importance for developing new biotechnologically engineered systems and solutions.

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Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.3.c747 ◽

1999 ◽

Vol 276 (3) ◽

pp. C747-C757 ◽

Cited By ~ 50

Author(s):

Sujatha Jayaraman ◽

Joachim Biwersi ◽

A. S. Verkman

Keyword(s):

First Generation ◽

Spectral Characteristics ◽

Dual Wavelength ◽

Reduced Form ◽

Excitation Wavelength ◽

Transmembrane Conductance Regulator ◽

Fluorescent Indicators ◽

3T3 Fibroblasts ◽

Ratio Imaging ◽

Ratiometric Measurement

The fluorescence of quinolinium-based Cl− indicators such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl− by a collisional mechanism without change in spectral shape. A series of “chimeric” dual-wavelength Cl− indicators were synthesized by conjugating Cl−-sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl−-sensitive moiety [excitation wavelength (λex) 350 nm, emission wavelength (λem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl−-insensitive moiety because of its different spectral characteristics (λex 380 nm, λem 546 nm), insensitivity to Cl−, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl−-sensitive indicators were α-MQ-α′-dimethyl-AQ-xylene dichloride and trans-1,2-bis(4-[1-α′-MQ-1′-α′-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl− sensitive; Stern-Volmer constants 82 and 98 M−1) and 565 nm (Cl−insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 μM, 10 min). Changes in Cl− concentration in response to Cl−/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl− indicators for ratiometric measurement of Cl− concentration.

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Signaling mechanism by the Staphylococcus aureus two-component system LytSR: role of acetyl phosphate in bypassing the cell membrane electrical potential sensor LytS

F1000Research ◽

10.12688/f1000research.6213.2 ◽

2016 ◽

Vol 4 ◽

pp. 79 ◽

Cited By ~ 2

Author(s):

Kevin Patel ◽

Dasantila Golemi-Kotra

Keyword(s):

Staphylococcus Aureus ◽

Membrane Potential ◽

Cell Membrane ◽

Electrical Potential ◽

Cell Membrane Potential ◽

Acetyl Phosphate ◽

Two Component System ◽

Component System ◽

Two Component ◽

Membrane Electrical Potential

The two-component system LytSR has been linked to the signal transduction of cell membrane electrical potential perturbation and is involved in the adaptation of Staphylococcus aureus to cationic antimicrobial peptides. It consists of a membrane-bound histidine kinase, LytS, which belongs to the family of multiple transmembrane-spanning domains receptors, and a response regulator, LytR, which belongs to the novel family of non-helix-turn-helix DNA-binding domain proteins. LytR regulates the expression of cidABC and lrgAB operons, the gene products of which are involved in programmed cell death and lysis. In vivo studies have demonstrated involvement of two overlapping regulatory networks in regulating the lrgAB operon, both depending on LytR. One regulatory network responds to glucose metabolism and the other responds to changes in the cell membrane potential. Herein, we show that LytS has autokinase activity and can catalyze a fast phosphotransfer reaction, with 50% of its phosphoryl group lost within 1 minute of incubation with LytR. LytS has also phosphatase activity. Notably, LytR undergoes phosphorylation by acetyl phosphate at a rate that is 2-fold faster than the phosphorylation by LytS. This observation is significant in lieu of the in vivo observations that regulation of the lrgAB operon is LytR-dependent in the presence of excess glucose in the medium. The latter condition does not lead to perturbation of the cell membrane potential but rather to the accumulation of acetate in the cell. Our study provides insights into the molecular basis for regulation of lrgAB in a LytR-dependent manner under conditions that do not involve sensing by LytS.

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Shedding light on the mitochondrial matrix through a functional membrane transporter

Chemical Science ◽

10.1039/c9sc04852a ◽

2020 ◽

Vol 11 (4) ◽

pp. 1052-1065 ◽

Cited By ~ 1

Author(s):

Alberto Blázquez-Moraleja ◽

Ines Sáenz-de-Santa María ◽

María D. Chiara ◽

Delia Álvarez-Fernández ◽

Inmaculada García-Moreno ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane ◽

Living Cells ◽

Membrane Transporter ◽

Mitochondrial Matrix ◽

Functional Membrane

A BODIPY derivative of carnitine enters mitochondria regardless of their membrane potential and in an enantioselective way through a specific mitochondrial membrane transporter in living cells.

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