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Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.

Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H - Front Cell Dev Biol (2016)

Bottom Line: The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes.Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential.At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.

ABSTRACT
The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

No MeSH data available.


Related in: MedlinePlus

Compatibility of DiSC3(5) and GFP in microscopic single-cell experiments. (A) Phase contrast image (left panel) of cells expressing GFP-MinD (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the strong decrease in DiSC3(5) fluorescence and delocalization of MinD upon depolarization. (B) Phase contrast image (left panel) of cells expressing GFP-FtsZ (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the reduced septal signal of FtsZ upon depolarization. Strains used: B. subtilis KS64 (GFP-MinD), and B. subtilis 2020 (GFP-FtsZ).
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Figure 6: Compatibility of DiSC3(5) and GFP in microscopic single-cell experiments. (A) Phase contrast image (left panel) of cells expressing GFP-MinD (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the strong decrease in DiSC3(5) fluorescence and delocalization of MinD upon depolarization. (B) Phase contrast image (left panel) of cells expressing GFP-FtsZ (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the reduced septal signal of FtsZ upon depolarization. Strains used: B. subtilis KS64 (GFP-MinD), and B. subtilis 2020 (GFP-FtsZ).

Mentions: The speed and extent of membrane depolarization by an antimicrobial compound is frequently used in discussing pore formation as a potential mechanism of action (Silverman et al., 2003; Spindler et al., 2011). Since normal fluorometric assays only measure the cell population average, a cell-to-cell heterogeneity can result in seemingly slow depolarization kinetics even when full and rapid depolarization of individual cells takes place. By detecting such heterogeneity, the single cell analysis using DiSC3(5) provides a valuable control experiment to support conclusions drawn from fluorometric measurements. At last, the far-red fluorescence of DiSC3(5) is compatible with simultaneous detection of GFP. Changes in membrane potential upon antimicrobial challenge, and the consequences on cellular organization and protein localization can therefore be directly correlated on a single-cell level (Figure 6).


Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.

Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H - Front Cell Dev Biol (2016)

Compatibility of DiSC3(5) and GFP in microscopic single-cell experiments. (A) Phase contrast image (left panel) of cells expressing GFP-MinD (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the strong decrease in DiSC3(5) fluorescence and delocalization of MinD upon depolarization. (B) Phase contrast image (left panel) of cells expressing GFP-FtsZ (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the reduced septal signal of FtsZ upon depolarization. Strains used: B. subtilis KS64 (GFP-MinD), and B. subtilis 2020 (GFP-FtsZ).
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Related In: Results  -  Collection

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Show All Figures
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Figure 6: Compatibility of DiSC3(5) and GFP in microscopic single-cell experiments. (A) Phase contrast image (left panel) of cells expressing GFP-MinD (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the strong decrease in DiSC3(5) fluorescence and delocalization of MinD upon depolarization. (B) Phase contrast image (left panel) of cells expressing GFP-FtsZ (middle panel) and stained with DiSC3(5) (right panel) in the absence and presence of gramicidin (5 μM). Note the reduced septal signal of FtsZ upon depolarization. Strains used: B. subtilis KS64 (GFP-MinD), and B. subtilis 2020 (GFP-FtsZ).
Mentions: The speed and extent of membrane depolarization by an antimicrobial compound is frequently used in discussing pore formation as a potential mechanism of action (Silverman et al., 2003; Spindler et al., 2011). Since normal fluorometric assays only measure the cell population average, a cell-to-cell heterogeneity can result in seemingly slow depolarization kinetics even when full and rapid depolarization of individual cells takes place. By detecting such heterogeneity, the single cell analysis using DiSC3(5) provides a valuable control experiment to support conclusions drawn from fluorometric measurements. At last, the far-red fluorescence of DiSC3(5) is compatible with simultaneous detection of GFP. Changes in membrane potential upon antimicrobial challenge, and the consequences on cellular organization and protein localization can therefore be directly correlated on a single-cell level (Figure 6).

Bottom Line: The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes.Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential.At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Newcastle upon Tyne, UK.

ABSTRACT
The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

No MeSH data available.


Related in: MedlinePlus