No abstract

Intracellular protein concentration is an essential cell characteristic, which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on biological cells. Therefore, we have developed a calibration procedure that involves successive imaging of cells in solutions containing different amounts of added protein. This allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/L). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (μm ) and 1.0 is an empirical coefficient. We used calibrated TIE imaging to characterize the regulatory volume increase (RVI) in adherent HeLa cells placed in a hyperosmotic solution. We found that while no RVI occurs over the first 30-60 min, the protein concentration fully recovers after 20 h. Because interpretation of such long experiments may depend on whether protein concentration varies significantly throughout the cell cycle, we measured this parameter in three cell lines: HeLa, MDCK and DU145. Our data indicate that protein concentration remains relatively stable in these cells. © 2017 International Society for Advancement of Cytometry.

Potassium loss and persistent shrinkage have both been implicated in apoptosis but their relationship and respective roles remain controversial. We approached this problem by clamping intracellular sodium and potassium in HeLa or MDCK cells using a combination of ionophores. Although ionophore treatment caused significant cell swelling, the initial volume could be restored and further reduced by application of sucrose. The swollen cells treated with ionophores remained viable for at least 8 h without any signs of apoptosis. Application of sucrose and the resulting shrinkage caused volumedependent intrinsic apoptosis with all its classical features: inversion of phosphatidylserine, caspase activation and Bcl-2-dependent release of cytochrome c from mitochondria. In other experiments, apoptosis was induced by addition of the protein kinase inhibitor staurosporine at various degrees of swelling. Our results show that: (1) persistent shrinkage can cause apoptosis regardless of intracellular sodium or potassium composition or of the state of actin cytoskeleton; (2) strong potassium dependence of caspase activation is only observed in swollen cells with a reduced density of cytosolic proteins. We conclude that macromolecular crowding can be an important factor in determining the transition of cells to apoptosis.