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

Rationale: 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. Methods: HeLa-H2B-GFP cells were treated with CX-5461, DRB or DAM. Water content and dry mass were measured in numerous regions of interest of ultrathin cryo-sections by quantitative scanning transmission electron microscope dark-field imaging and the elements quantified by energy dispersive X-ray spectrometry. The data were used to calculate macromolecular crowding and the volume occupied by free water in all cell compartments of control and treated cells. Hydrophobic and unfolded proteins were revealed by 8-Anilinonaphtalene-1-sulfonic acid (ANS) staining and imaging by two-photon microscopy. Immunolabeling of UBF, pNBS1 and pNF-κB was carried out and the images acquired with a confocal microscope for 3D imaging to address whether the localization of these proteins changes in treated cells. Results: Treatment with CX-5461, DRB or DAM induced completely different changes in macromolecular crowding and elemental content. Macromolecular crowding and elemental content were much higher in CX-5461-treated, moderately higher in DRB-treated, and much lower in DAM-treated cells than control cells. None of the drugs alone induced nucleolar ANS staining but it was induced by heat-shock of control cells and cells previously treated with DAM. UBF and pNBS1 were systematically co-localized in the nucleolus of CX-5461- and DAM-treated cells. pNF-κB only localized to the nucleolar caps of pre-apoptotic DAM-treated cells. Conclusion: We directly quantified water and ion content in cell compartments using cryo-correlative electron microscopy. We show that different chemotherapeutic nucleolar stress inducers result in distinctive, thus far-unrecognized changes in macromolecular crowding and elemental content which are known to modify cell metabolism. Moreover we were able to correlate these changes to the sensitivity of treated cells to heat-shock and the behavior of nucleolar pNBS1 and pNF-κB.