Osmotic water permeability measurements using confocal laser scanning microscopy

Area of Science:

• Cell Biology
• Biophysics
• Membrane Transport

Background:

• Accurate measurement of plasma membrane water permeability (P(f)) is crucial for understanding cell volume regulation and transport.
• Existing methods may have limitations in precision or applicability to intact cells.

Purpose of the Study:

• To develop and validate a novel method for quantifying P(f) in intact adherent cells using laser scanning confocal microscopy.
• To investigate the factors influencing water permeability in MDCK cells and the role of aquaporins.

Main Methods:

• Utilized laser scanning confocal microscopy to record fluorescence intensity changes in calcein-loaded cells during osmotic shock.
• Calculated P(f) based on the time constant of fluorescence change, cell surface-to-volume ratio, and osmotically active cell volume.
• Applied the method to Madin-Darby Canine Kidney (MDCK) cells, including those transfected with aquaporin 2.

Main Results:

• MDCK cells behaved as linear osmometers between 100-350 mosM, with 57% of cell volume being osmotically inactive.
• Water permeability was temperature-dependent and significantly increased by amphotericin B and DMSO, but not HgCl2.
• Aquaporin 2 transfection increased P(f) by an order of magnitude; nuclear membrane water permeability was unlimited.

Conclusions:

• Confocal microscopy offers a sensitive and reproducible method for measuring P(f) in various adherent cell types and potentially tissue preparations.
• The findings highlight the significant role of aquaporins in cellular water transport and provide a valuable tool for further research.