Membrane Potential Estimation by Flow Cytometry

doi:10.1006/meth.2000.1007

Methods

Volume 21, Issue 3, July 2000, Pages 271-279

https://doi.org/10.1006/meth.2000.1007 Get rights and content

Abstract

Membrane potential (ΔΨ) is generated and maintained by concentration gradients of ions such as sodium, potassium, chloride, and hydrogen. Changes in cytoplasmic ΔΨ in the course of surface-receptor-mediated processes related to the development, function, and pathology of many cell types often play a role in transmembrane signaling. Cytoplasmic ΔΨ is also reduced to zero when the membrane is ruptured by chemical or physical agents. Mitochondrial ΔΨ is reduced when energy metabolism is disrupted, notably in apoptosis. In bacteria, which lack mitochondria, ΔΨ reflects both the state of energy metabolism and the physical integrity of the cytoplasmic membrane. Flow cytometry can be used to estimate membrane potential in eukaryotic cells, mitochondria in situ, isolated mitochondria, and bacteria. Older methods, using lipophilic cationic dyes such as the cyanines and rhodamine 123 or lipophilic anionic dyes such as the oxonols can detect relatively large changes in ΔΨ and identify heterogeneity of response in subpopulations comprising substantial fractions of a cell population. Newer ratiometric techniques allow precise measurement of ΔΨ to within 10 mV or less. Among other factors, action of efflux pumps, changes in membrane structure, and changes in protein or lipid concentration in the medium in which cells are suspended can produce changes in cellular fluorescence which may be misinterpreted as changes in ΔΨ. Techniques for estimation and measurement of ΔΨ therefore typically require careful control of cell and reagent concentrations and incubation times and selection of appropriate controls if they are to provide accurate information.

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The experimental error of MBC values was 6–7%. The transmembrane potential (TM) development assessment in M. smegmatis was performed based on the ratiometric approach [42–44]. Aliquots of the bacterial samples − 500 μl (OD600 ∼ 0.2) were placed into the flow-cytometry plastic tubes.
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Mitochondria are real sensors of the physiological status of tissues. After the death of an animal, they maintain physiological activity for several days. This activity is highly dependent on the availability of nutrients in the tissue. In this study, flow cytometry was used to measure the membrane potential of mitochondria isolated from European seabass (Dicentrarchus labrax) red muscle stored in ice for seven days in order to characterize fish freshness. Two probes, TMRM and Rhodamine 123, were used to measure mitochondrial potential. During the first few days (D0 to D3), isolated mitochondria maintained high potential, and then lost their potential (from D3 to D5), but were always re-polarizable after addition of substrates (glutamate, malate and succinate). From D7, the mitochondria were more strongly depolarized and were difficult to repolarize by the substrates. Using flow cytometry, we demonstrated that mitochondria were an excellent marker to confirm seabass freshness.
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Antimicrobial peptides (AMPs) are host defense peptides, and unlike conventional antibiotics, they possess potent broad spectrum activities and, induce little or no antimicrobial resistance. They are attractive lead molecules for rational development to improve their therapeutic index. Our current studies examined dimerization of the de novo designed proline-rich AMP (PrAMP), Chex1-Arg20 hydrazide, via C-terminal thiol addition to a series of bifunctional benzene or phenyl tethers to determine the effect of orientation of the peptides and linker length on antimicrobial activity. Antibacterial assays confirmed that dimerization per se significantly enhances Chex1-Arg20 hydrazide action. Greatest advantage was conferred using perfluoroaromatic linkers (tetrafluorobenzene and octofluorobiphenyl) with the resulting dimeric peptides and exhibiting potent action against Gram-negative bacteria, especially the World Health Organization's critical priority-listed multidrug-resistant (MDR)/extensively drug-resistant (XDR) Acinetobacter baumannii as well as preformed biofilms. Mode of action studies indicated these lead PrAMPs can interact with both outer and inner bacterial membranes to affect the membrane potential and stress response. Additionally, and possess potent immunomodulatory activity and neutralise inflammation via nitric oxide production in macrophages. Molecular dynamics simulations of adsorption and permeation mechanisms of the PrAMP on a mixed lipid membrane bilayer showed that a rigid, planar tethered dimer orientation, together with the presence of fluorine atoms that provide increased bacterial membrane interaction, is critical for enhanced dimer activity. These findings highlight the advantages of use of such bifunctional tethers to produce first-in-class, potent PrAMP dimers against MDR/XDR bacterial infections.
Natural flavonoid morin showed anti-bacterial activity against Vibrio cholera after binding with cell division protein FtsA near ATP binding site
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Increasing antibiotic-resistance in bacterial strains has boosted the need to find new targets for drug delivery. FtsA, a major bacterial divisome protein can be a potent novel drug-target.
This study finds, morin (3,5,7,2′,4′-pentahydroxyflavone), a bio-available flavonoid, had anti-bacterial activities against Vibrio cholerae, IC₅₀ (50 μM) and MIC (150 μM). Morin (2 mM) kills ~20% of human lung fibroblast (WI38) and human intestinal epithelial (HIEC-6) cells in 24 h in-vitro. Fluorescence studies showed morin binds to VcFtsA (FtsA of V. cholerae) with a K_d of 4.68 ± 0.4 μM, inhibiting the protein's polymerization by 72 ± 7% at 25 μM concentration. Morin also affected VcFtsA's ATPase activity, recording ~80% reduction at 20 μM concentration. The in-silico binding study indicated binding sites of morin and ATP on VcFtsA had overlapping amino acids. Mant-ATP, a fluorescent ATP-derivative, showed increased fluorescence on binding to VcFtsA in absence of morin, but in its presence, Mant-ATP fluorescence decreased. VcFtsA-S40A mutant protein did not bind to morin.
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Collectively, these findings explore the anti-bacterial effect of morin on V. cholerae cells targeting VcFtsA, encouraging it to become a potent anti-bacterial agent. Low cytotoxicity of morin against human cells (host) is therapeutically advantageous. This study will also help in synthesizing novel derivatives that can target VcFtsA more efficiently.
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Early reports in the fungus Ustilago maydis suggest that the amphipathic fungicide dodine disrupts the fungal plasma membrane (PM), thereby killing this corn smut pathogen. However, a recent study in the wheat pathogen Zymoseptoria tritici does not support such mode of action (MoA). Instead, dodine inhibits mitochondrial ATP-synthesis, both in Z. tritici and U. maydis. This casts doubt on an fungicidal activity of dodine at the PM. Here, we use a cell biological approach and investigate further the effect of dodine on the plasma membrane in both fungi. We show that dodine indeed breaks the integrity of the PM in U. maydis, indicated by a concentration-dependent cell depolarization. In addition, the fungicide reduces PM fluidity and arrests endocytosis by inhibiting the internalization of endocytic vesicles at the PM. This is likely due to impaired recruitment of the actin-crosslinker fimbrin to endocytic actin patches. However, quantitative data reveal that the effect on mitochondria represents the primary MoA in U. maydis. None of these plasma membrane-associated effects were found in dodine-treated Z. tritici cells. Thus, the physiological effect of an anti-fungal chemistry can differ between pathogens. This merits consideration when characterizing a given fungicide.

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Regular ArticleMembrane Potential Estimation by Flow Cytometry

Abstract

Biophys. J.

Biochem. Biophys. Acta

Biochem. Pharmacol.

J. Photochem. Photobiol. B.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Chem. Biol.

Proc. Natl. Acad. Sci. USA

J. Gen. Physiol.

Rev. Physiol. Biochem. Pharmacol.

Biochemistry

J. Physiol.

Cytometry

Cytometry

J. Cell Biol.

Regular Article
Membrane Potential Estimation by Flow Cytometry