4D confocal microscopy of time dependent cell and nuclear osmotic response reveals differential osmoregulation and transport kinetics
A cell's ability to maintain its volume is a prerequisite for its essential physiological functions. Changes in extracellular osmolarity induce the flux of water and solutes across the cell and nuclear membrane and, therefore, alters cell and nuclear volumes. Here, we investigated the equilibrium and dynamic volumetric responses of cells and nuclei in adherent human hepatoma (HepG2) cell monolayers using live three-dimensional imaging. Through image segmentation, 3D reconstruction, and volume rendering, precise cell and nuclear volumes were quantified. We found that during hypoosmotic stress, cells swell and lose osmolytes through active volume regulation, and nuclei undergo greater relative expansion than cells. During hyperosmotic stress, we found that both cells and nuclei shrink rapidly, develop irregular morphologies and undergo passive volume regulation. Furthermore, we explored the osmotic responses in these cells by restoring isosmotic conditions following osmotic stress and found that nuclei returned to volumes greater than their initial isosmotic volume, whereas cytoplasmic volume returned to a value below its initial isosmotic volume following swelling. By fitting coupled transport models water and solute permeability estimates for both cells and nuclei were made from their dynamic responses to different anisosmotic media with and without the presence of permeating solutes dimethyl sulfoxide and ethylene glycol. Together, these quantitative measurements of cell, cytoplasm, and nuclear volumetric responses and associated transport parameters have important implications for understanding cell physiology and the mechanism of osmoregulation.