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Intracellular pH in Dictyostelium discoideum: a 31P nuclear magnetic resonance study
Abstract:
We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum. We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils. Cells continued to develop normally in this set-up. Spectra acquired under these conditions typically show two peaks in the inorganic phosphate region corresponding to pH values of 7.16 +/- 0.03 and 6.48 +/- 0.02. These peaks are believed to represent the mitochondrial and cytosolic compartments respectively, based on a comparison of these values with published data and the collapse of the two compartments upon addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. Dictyostelium cells show a remarkable degree of intracellular pH homeostasis. Both mitochondrial and cytosolic pH remained unchanged as extracellular pH was varied from 4.3 to 8.1. There was also no apparent change in the pH of either compartment after up to 13.5 hours' development in suspension.
Insights
Researchers used phosphorus-31 nuclear magnetic resonance (NMR) to measure intracellular pH in Dictyostelium discoideum. The study found remarkable pH homeostasis, with internal pH remaining stable despite external changes.
Area of Science:
- Cell Biology
- Biophysics
- Biochemistry
Background:
- Intracellular pH is a critical regulator of cellular processes.
- Accurate measurement of intracellular pH in dense cell suspensions presents technical challenges.
Purpose of the Study:
- To determine the intracellular pH of Dictyostelium discoideum using phosphorus-31 nuclear magnetic resonance (31P NMR).
- To develop a method for maintaining cell viability and normal development during NMR measurements.
- To investigate the intracellular pH homeostasis of Dictyostelium discoideum.
Main Methods:
- Utilized 31P NMR spectroscopy to measure inorganic phosphate signals.
- Developed an air-lift circulator for continuous, well-oxygenated, and stirred cell suspension.
- Applied mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone to validate compartment assignments.
Main Results:
- Identified two distinct inorganic phosphate peaks, indicating mitochondrial (pH 7.16) and cytosolic (pH 6.48) compartments.
- Demonstrated remarkable intracellular pH homeostasis in Dictyostelium discoideum.
- Observed stable intracellular pH across a wide extracellular pH range (4.3–8.1) and during 13.5 hours of development.
Conclusions:
- Dictyostelium discoideum exhibits robust intracellular pH regulation.
- The developed air-lift circulator method is suitable for studying cellular physiology in dense suspensions.
- 31P NMR is a valuable tool for dissecting compartment-specific pH in developing cellular slime molds.