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

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Abstract

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. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83 % (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128–462 mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.

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Acknowledgments

We received funding from: the Agence Nationale pour la Recherche (ANR-07 Nano-CESIWIN), Europe Community (FEDER) and Région Champagne Ardennes.

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Authors and Affiliations

  1. Laboratoire de Recherche en Nanosciences, Université de Reims Champagne Ardenne, Reims, France

    Frédérique Nolin, Jean Michel, Laurence Wortham, Gérard Balossier & Vincent Banchet

  2. CNRS FRE 3481, Université de Reims Champagne Ardenne, Reims, France

    Pavel Tchelidze, Hélène Bobichon, Nathalie Lalun & Dominique Ploton

  3. Plate-forme IBISA, SFR CAP-SANTE, Université de Reims Champagne Ardenne, Reims, France

    Christine Terryn

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  1. Frédérique Nolin
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Correspondence to Dominique Ploton.

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J. Michel and D. Ploton are co-senior authors.

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18_2013_1267_MOESM3_ESM.tif

Online Resource 3 Percentage water in control and actinomycin D-treated HeLa NOP 52-GFP cells. Quantification was carried out for regions of interest in several compartments: DFC/GC (nucleolar dense fibrillar component and granular component); FC/NLC (nucleolar fibrillar centers and nucleolar light caps); NPL (nucleoplasm); CY (cytosol); MIT (mitochondria). Data are means ± SD from triplicate experiments (n = 40 for each condition). (TIFF 2389 kb)

18_2013_1267_MOESM4_ESM.tif

Online Resource 4 Elements (N, P, S, K, Cl, Mg) were identified and quantified by EDXS in control and actinomycin D-treated HeLa NOP 52-GFP cells. Analyses were performed on regions of interest in several compartments: DFC/GC (nucleolar dense fibrillar component and granular component); FC/NLC (nucleolar fibrillar centers and nucleolar light caps); NPL (nucleoplasm); CYT (cytosol); MIT (mitochondria). Data are means ± SD from triplicate experiments (n = 40 for each condition). (TIFF 6637 kb)

18_2013_1267_MOESM5_ESM.doc

Online Resource 5 Elements (N, P, S, K, Cl, Mg) were identified and quantified by EDXS in control and actinomycin D-treated HeLa NOP 52-GFP cells. Analyses were performed on regions of interest in several compartments: DFC/GC (nucleolar dense fibrillar component and granular component); NPL (nucleoplasm); CYT (cytosol); MIT (mitochondria). Data are means ± SD from triplicate experiments (n = 40 for each condition) and are presented as spiderweb diagrams for each cell compartment. (DOC 21 kb)

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Nolin, F., Michel, J., Wortham, L. et al. Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach. Cell. Mol. Life Sci. 70, 2383–2394 (2013). https://doi.org/10.1007/s00018-013-1267-7

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