Related Experiment Videos
Intracellular pH in single motile cells
Abstract:
Cytoplasmic pH in single living specimens of Chaos carolinensis is determined microfluorometrically by measuring the ratio of fluorescence intensity of microinjected fluorescein-thiocarbamyl (FTC)-ovalbumin at two different excitation wavelengths. The probe is evenly distributed throughout, and confined to, the cytoplasm, and the fluorescence intensity ratio depends only upon pH. It is independent of pathlength, concentration of probe, divalent cations, and ionic strength. Ratios are calibrated with a standard curve generated in situ by adjusting internal pH of FTC-ovalbumin-containing amebae with weak acid and weak base or by injection of strong buffers. With this technique, the average cytoplasmic pH of freely moving ameba is found to be 6.75 (SD +/- 0.3). The pH of a given spot relative to the morphology of a moving ameba remains fairly constant (+/- 0.05 U), whereas the pH of two different spots in the same cell may differ by as much as 0.4 U, and average pH in different amebae ranges from 6.3 to 7.4, with a suggestion of clustering about pH 6.5 and 6.8. During wound healing, there is a local, transient drop in pH (as great as 0.35 U) at the wound site upon puncture, proportional in extent to the degree of damage. Comparison of tails and advancing pseudopod tips reveals no significant difference in cytoplasmic pH at this level of spatial (50 microns diameter spot) and temporal (1.3 s) resolution. Fluctuations in intracellular pH and/or intracellular free Ca++ may be involved in regulation of cytoplasmic structure and contractility.
Insights
This study measured the cytoplasmic pH of Chaos carolinensis using microfluorometry. The average pH was found to be 6.75, with localized variations and transient drops during wound healing, suggesting pH
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Understanding cytoplasmic pH is crucial for cellular function.
- Previous methods for measuring intracellular pH in single-celled organisms were limited.
Purpose of the Study:
- To develop and apply a microfluorometric technique for determining cytoplasmic pH in living Chaos carolinensis.
- To investigate spatial and temporal variations in cytoplasmic pH within individual amebae and during cellular processes like wound healing.
Main Methods:
- Microfluorometry using fluorescein-thiocarbamyl (FTC)-ovalbumin as a pH-sensitive probe.
- In situ calibration of fluorescence ratios against known pH values.
- Measurement of pH in freely moving amebae, during wound healing, and in different cellular regions.
Main Results:
- The average cytoplasmic pH of Chaos carolinensis was determined to be 6.75.
- Significant spatial variations in pH were observed within single amebae (up to 0.4 U difference).
- A transient pH drop (up to 0.35 U) occurred at wound sites during healing.
Conclusions:
- Cytoplasmic pH in Chaos carolinensis is tightly regulated but exhibits localized heterogeneity.
- Intracellular pH fluctuations may play a role in regulating cytoplasmic structure and contractility.
- The developed microfluorometric method provides a reliable tool for studying intracellular pH dynamics.