Various Nucleolar Stress Inducers Result in Highly Distinct Changes in Water, Dry Mass and Elemental Content in Cancerous Cell Compartments: Investigation Using a Nano-Analytical Approach.

Numerous chemotherapeutic drugs that affect ribosome biogenesis in the nucleolus induce nucleolar stress. Improving our understanding of the effects of these drugs will require uncovering and comparing their impact on several biophysical parameters of the major cell compartments. Here, we quantified the water content and dry mass of cancerous cells treated with CX-5461, DRB or DAM to calculate macromolecular crowding and the volume occupied by free water, as well as elemental content.

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.

Targeted nano analysis of water and ions in the nucleus using cryo-correlative microscopy.

The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches.

Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach.

The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5 %, respectively.

Targeted nano analysis of water and ions using cryocorrelative light and scanning transmission electron microscopy.

Cryo fluorescence imaging coupled with the cryo-EM technique (cryo-CLEM) avoids chemical fixation and embedding in plastic, and is the gold standard for correlated imaging in a close to native state. This multi-modal approach has not previously included elementary nano analysis or evaluation of water content. We developed a new approach allowing analysis of targeted in situ intracellular ions and water measurements at the nanoscale (EDXS and STEM dark field imaging) within domains identified by examination of specific GFP-tagged proteins.