The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Growth Conditions
2.2. Membrane Potential Estimations, Fluorescence of ThT
2.3. Fluorescence Measured with a Multi-Well Plate Reader
2.4. Microscopy
2.5. Membrane Potential Measurements by the Accumulation of ThT
- Δψ is the plasma membrane potential difference, measured in volts (V);
- R is the universal gas constant, (8.314 J·K−1·mol−1);
- T is the temperature in Kelvin degrees (30 °C = 303 K);
- z is the number of elementary charges of thioflavin T (ThT) (=1);
- F is the Faraday constant, equal to 96,485 J·V−1·mol−1;
- [int] is the intracellular concentration of ThT;
- [ext] is the extracellular concentration of ThT;
- 2.3026 RT/F = 60.12 mV (considering our experiments at 30 °C).
2.6. Flow Cytometry
2.7. K+ Uptake
2.8. Oxygen Consumption
2.9. Statistical Analysis
3. Results
3.1. Estimation of the PMP by Changes in the Fluorescence of ThT with Different Yeast Strains
3.2. Results Obtained by Fluorescence in a Multi-Well Plate Reader
3.3. Fluorescence Images of the Cells
3.4. Values of PMP Obtained from the Accumulation of ThT
3.5. ThT Fluorescence as a Monitor of PMP, Can also Be Measured by Flow Cytometry
3.6. K+ Uptake and Oxygen Consumption
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain | Genotype | Source |
---|---|---|
Candida albicans | ATCC 10231 | |
Debaryomyces hansenii Y7426 | US Department of Agriculture, Peoria, IL, | |
Meyerozyma guilliermondii | [33] | |
Rhodotorula mucilaginosa | ATCC 66034 | |
Saccharomyces cerevisiae Azteca | Commercial strain | |
Saccharomyces cerevisiae FY833 | MATa (his3Δ200 leu2Δ1 lys2Δ202 trp1Δ63 ura3-52) | [34] |
trk1Δ | MATa (his3Δ200 leu2Δ1 lys2Δ202 trp1Δ63 ura3-52) trk1Δ::kanMX | [35] |
trk2Δ | MATa (his3Δ200 leu2Δ1 lys2Δ202 trp1Δ63 ura3-52) trk2Δ::HIS3 | [35] |
trk1Δ trk2Δ | MATa (his3Δ200 leu2Δ1 lys2Δ202 trp1Δ63 ura3-52) trk1Δ::TRP1-trk2Δ::kanMX | [36] |
Saccharomyces cerevisiae W303-1A | MATa (ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 mal10) | [37] |
TOW (tok1Δ) | MATa (ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 mal 10) tok1Δ::kanMX | [38] |
% Dry Weight | Internal Cell Volume mL/g (w. w.) | Vacuole/Cell Volume Ratio | Total Vacuole Water/g Cells (mL) | Cytoplasm Volume/g Cells (mL) | Cytoplasm Volume/150 mg Cells (mL) | |
---|---|---|---|---|---|---|
S. cerevisiae Azteca | 22.2 | 0.430 | 0.117 | 0.050 | 0.380 | 0.0569 |
S. cerevisiae FY833 | 18.0 | 0.424 | 0.276 | 0.117 | 0.307 | 0.0460 |
S. cerevisiae W303-1A | 22.4 | 0.440 | 0.252 | 0.111 | 0.329 | 0.0494 |
C. albicans | 27.0 | 0.410 | 0.150 | 0.061 | 0.349 | 0.0523 |
D. hansenii | 21.0 | 0.440 | 0.091 | 0.040 | 0.400 | 0.0600 |
M. guilliermondii | 28.2 | 0.390 | 0.099 | 0.039 | 0.351 | 0.0527 |
R. mucilaginosa | 14.5 | 0.420 | 0.071 | 0.030 | 0.390 | 0.0585 |
trk1Δ | 19.9 | 0.460 | 0.124 | 0.057 | 0.403 | 0.0605 |
trk2Δ | 18.3 | 0.478 | 0.265 | 0.127 | 0.352 | 0.0528 |
trk1Δ trk2Δ | 18.4 | 0.463 | 0.142 | 0.066 | 0.397 | 0.0595 |
TOW | 15.4 | 0.529 | 0.264 | 0.140 | 0.390 | 0.0585 |
Non-Corrected | Corrected | |
---|---|---|
Δψ (mV) ± SD | Δψ (mV) ± SD | |
S.c. Azteca | ||
Glucose | −201.6 ± 3.4 | −192.8 ± 22.3 |
CCCP | −166.2 ± 9.9 | −148.3 ± 13.9 |
CCCP + KCl | −100.5 ± 6.1 | −72.5 ± 8.0 |
S. c. FY833 | ||
Glucose | −164.3 ± 12.6 b | −153.51 ± 12.3 b |
CCCP | −130.4 ± 11.1 | −115.46 ± 12.8 |
CCCP + KCl | −36.9 ± 15.2 | −71.66 * |
S.c. W303-1A | ||
Glucose | −179.3 ± 15.6 b | −160.7 ± 16.5 b |
CCCP | −154.3 ± 16.4 | −133.1 ± 15.8 |
CCCP + KCl | −112.1 ± 24.1 | −96.2 ± 27.7 |
C. albicans | ||
Glucose | −248.8 ± 14.9 | −228.2 ± 19.0 |
CCCP | −185.7 ± 16.5 | −153.2 ± 15.5 |
CCCP + KCl | −142.0 ± 18.1 | −140.4 ± 19.9 |
D. hansenii | ||
Glucose | −154.4 ± 12.1 a,b | −116.9 ± 17.1 a,b |
CCCP | −120.7 ± 12.2 | −56.7 ± 9.7 |
CCCP + KCl | −101.5 ± 10.6 | −73.3 ± 18.4 |
M. guilliermondii | ||
Glucose | −167.1 ± 13.1 b | −135.7 ± 8.6 a,b |
CCCP | −135.2 ± 10.9 | −96.9 ± 12.6 |
CCCP + KCl | −111.8 ± 13.5 | −115.5 ± 19.0 |
R. mucilaginosa | ||
Glucose | −148.3 ± 20.3 a,b | −139.0 ± 13.8 a,b |
CCCP | −70.5 ± 10.8 | −32.4 ± 2.6 |
CCCP + KCl | −75.1 ± 8.3 | −72.7 ± 8.8 |
Non-Corrected | Corrected | |
---|---|---|
Δψ (mV) ± SD | Δψ (mV) ± SD | |
FY833 | ||
Glucose | −164.3 ± 12.6 | −153.51 ± 12.3 |
CCCP | −130.4 ± 11.1 | −115.46 ± 12.8 |
CCCP + KCl | −36.9 ± 15.2 | −71.66 * |
trk1Δ | ||
Glucose | −186.4 ± 5.7 a | −174.9 ± 4.8 a |
CCCP | −143.1 ± 7.3 | −128.1 ± 8.3 |
CCCP + KCl | −134.9 ± 4.8 | −126.6 ± 8.3 |
trk2Δ | ||
Glucose | −157.0 ± 8.6 ns | −147.9 ± 7.3 ns |
CCCP | −129.9 ± 4.2 | −118.8 ± 5.5 |
CCCP + KCl | −54.4 ± 4.9 | −21.17 ** |
trk1Δ trk2Δ | ||
Glucose | −193.8 ± 12.7 a | −179.7 ± 13.9 a |
CCCP | −158.9 ± 10.6 | −143.2 ± 11.8 |
CCCP + KCl | −163.8 ± 9.7 | −155.9 ± 9.2 |
W303-1A | ||
Glucose | −186.8 ± 7.0 | −167.7 ± 11.8 |
CCCP | −162.9 ± 4.4 | −143.8 ± 6.4 |
CCCP + KCl | −117.9 ± 21.7 | −121.2 ± 3.5 |
TOW | ||
Glucose | −172.8 ± 9.4 ns | −159.4 ± 6.7 ns |
CCCP | −141.1 ± 7.3 | −125.1 ± 4.5 |
CCCP + KCl | −96.6 ± 9.3 | −82.1 ± 9.5 |
Glucose | CCCP | CCCP + KCl | |
---|---|---|---|
10 µM CCCP | −167.1 | −135.2 | −111.8 |
5 µM CCCP | −121.81 | −113.9 | −97.4 |
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Peña, A.; Sánchez, N.S.; Padilla-Garfias, F.; Ramiro-Cortés, Y.; Araiza-Villanueva, M.; Calahorra, M. The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains. J. Fungi 2023, 9, 948. https://doi.org/10.3390/jof9090948
Peña A, Sánchez NS, Padilla-Garfias F, Ramiro-Cortés Y, Araiza-Villanueva M, Calahorra M. The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains. Journal of Fungi. 2023; 9(9):948. https://doi.org/10.3390/jof9090948
Chicago/Turabian StylePeña, Antonio, Norma Silvia Sánchez, Francisco Padilla-Garfias, Yazmín Ramiro-Cortés, Minerva Araiza-Villanueva, and Martha Calahorra. 2023. "The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains" Journal of Fungi 9, no. 9: 948. https://doi.org/10.3390/jof9090948