Stage-Specific Changes in the Water, Na(+), Cl(-) and K(+) Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach

Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na(+) and Cl(-) contents of the mitochondria and a strong decrease in mitochondrial K(+) content. During the first step in apoptotic volume decrease (AVD), Na(+) and Cl(-) levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na(+) and Cl(-) levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K(+) content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K(+) contents similar to those of cells entering apoptosis but lower Na(+) and Cl(-) contents than both cells entering apoptosis and control cells.