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. 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.

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. This method allows both water and ions- fundamental to cell biology- to be located and quantified at the subcellular level. We illustrate the potential of this approach by investigating changes in water and ion content in nuclear domains identified by GFP-tagged proteins in cells stressed by Actinomycin D treatment and controls. The resolution of our approach was sufficient to distinguish clumps of condensed chromatin from surrounding nucleoplasm by fluorescence imaging and to perform nano analysis in this targeted compartment.

Long acting beta2-agonist and corticosteroid restore airway glandular cell function altered by bacterial supernatant.

BACKGROUND: Staphylococcus aureus releases virulence factors (VF) that may impair the innate protective functions of airway cells. The aim of this study was to determine whether a long-acting beta2 adrenergic receptor agonist (salmeterol hydroxynaphthoate, Sal) combined with a corticosteroid (fluticasone propionate, FP) was able to regulate ion content and cytokine expression by airway glandular cells after exposure to S. aureus supernatant. METHODS: A human airway glandular cell line was incubated with S. aureus supernatant for 1 h and then treated with the combination Sal/FP for 4 h. The expression of actin and CFTR proteins was analyzed by immunofluorescence. Videomicroscopy was used to evaluate chloride secretion and X-ray microanalysis to measure the intracellular ion and water content. The pro-inflammatory cytokine expression was assessed by RT-PCR and ELISA. RESULTS: When the cells were incubated with S. aureus supernatant and then with Sal/FP, the cellular localisation of CFTR was apical compared to the cytoplasmic localisation in cells incubated with S. aureus supernatant alone. The incubation of airway epithelial cells with S. aureus supernatant reduced by 66% the chloride efflux that was fully restored by Sal/FP treatment. We also observed that Sal/FP treatment induced the restoration of ion (Cl and S) and water content within the intracellular secretory granules of airway glandular cells and reduced the bacterial supernatant-dependent increase of pro-inflammatory cytokines IL8 and TNFalpha. CONCLUSIONS: Our results demonstrate that treatment with the combination of a corticosteroid and a long-acting beta2 adrenergic receptor agonist after bacterial infection restores the airway glandular cell function. Abnormal mucus induced by defective ion transport during pulmonary infection could benefit from treatment with a combination of beta2 adrenergic receptor agonist and glucocorticoid.

Salmeterol restores secretory functions in cystic fibrosis airway submucosal gland serous cells.

The activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) can be mediated by surface G protein-coupled receptors such as the beta(2)-adrenergic receptor. In this study, we explored the effect of a long-acting beta(2)-adrenergic agonist, salmeterol, on the CFTR-dependent secretory capacity of a human CF tracheal gland serous cell line (CF-KM4), homozygous for the delF508 mutation. We showed that, compared with the untreated CF serous cells, a 24-hour pre-incubation period with 200 nM salmeterol induced an 83% increase in delF508-CFTR-mediated chloride efflux. The restoration of the bioelectric properties is associated with increased apical surface pool of delF508-CFTR. Salmeterol induced a decrease in ion concentration and an increase in the level of hydration of the mucus packaged inside the CF secretory granules. The effects of salmeterol are not associated with a persistent production of cAMP. Western blotting on isolated secretory granules demonstrated immunoreactivity for CFTR and lysozyme. In parallel, we measured by atomic force microscopy an increased size of secretory granules isolated from CF serous cells compared with non-CF serous cells (MM39 cell line) and showed that salmeterol was able to restore a CF cell granule size similar to that of non-CF cells. To demonstrate that the salmeterol effect was a CFTR-dependent mechanism, we showed that the incubation of salmeterol-treated CF serous cells with CFTR-inh172 suppressed the restoration of normal secretory functions. The capacity of salmeterol to restore the secretory capacity of glandular serous cells suggests that it could also improve the airway mucociliary clearance in patients with CF.

Cryo-X-ray analysis-A novel tool to better understand the physicochemical reactions at the bioglass/biological fluid interface.

The present study deals with the short-term physicochemical reactions at the interface between bioactive glass particles [55SiO(2)-20CaO-9P(2)O(5)-12Na(2)O-4MgO. mol%] and biological fluid (Dulbecco Modified Eagle's Medium (DMEM)). The physicochemical reactions within the interface are characterized by scanning transmission electron microscopy (TEM) (STEM) associated with Energy-dispersive X-ray spectroscopy (EDXS). Microanalysis of diffusible ions such as sodium, potassium, or oxygen requires a special care. In the present investigation the cryo-technique was adopted as a suitable tool for the specimen preparation and characterization. Cryosectioning is essential for preserving the native distribution of ions so that meaningful information about the local concentrations can be obtained by elemental microanalysis. The bioglass particles immersed in biological fluid for 24 h revealed five reaction stages: (i) dealkalization of the surface by cationic exchange (Na(+), Ca(2+) with H(+) or H(3)O(+)); (ii) loss of soluble silica in the form of Si(OH)(4) to the solution resulting from the breakdown of Si--O--Si bonds (iii); repolymerization of Si(OH)(4) leading to condensation of SiO(2)); (iv) migration of Ca(2+) and PO(4) (3-) to the surface through the SiO(2)-rich layer to form CaO-P(2)O(5) film; (v) crystallization of the amorphous CaO-P(2)O(5) by incorporating OH-- or CO(3) (2-) anions with the formation of three different surface layers on the bioactive glass periphery. The thickness of each layer is approximately 300 nm and from the inner part to the periphery they consist of Si--OH, which permits the diffusion of Ca(2+) and PO(4) (3-) ions and the formation of the middle Ca--P layer, and finally the outer layer composed of Na--O, which acts as an ion exchange layer between Na(+) ions and H(+) or H(3)O(+) from the solution.

Development and in vitro characterization of sol-gel derived CaO-P2O5-SiO2-ZnO bioglass.

A CaO-P(2)O(5)-SiO(2)-ZnO bioglass was formed by the sol-gel technique and characterized by Raman spectroscopy, X-ray diffraction, energy dispersive X-ray analysis (EDXA) and scanning electron microscopy (SEM). The surface reactivity of the resultant glass-ceramic specimens was analyzed by immersion studies in simulated body fluid (SBF). SEM-EDXS and inductively coupled plasma atomic emission spectrometry techniques were used to monitor changes in the glass surface and SBF composition. Osteoblast cell culture experiments were performed to assess the biocompatibility and the alkaline phosphatase activity. Cell counts of the osteoblasts cultured on the bioglass samples were studied and compared with the polystyrene plates. The cells cultured on the bioglass disks consistently showed a higher alkaline phosphatase activity and cell counts compared to cells cultured on either polystyrene plates or the base CaO-P(2)O(5)-SiO(2) bioglass. This was due to cell proliferation and differentiation promoted by the zinc-substituted bioglass.

The distribution of light elements in biological cells measured by electron probe X-ray microanalysis of cryosections.

The intracellular distribution of the elements carbon, nitrogen, and oxygen was measured in cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of 100-nm-thick freeze-dried cryosections. Electron irradiation with a dose up to 106 e/nm2 caused no or merely negligible mass loss in mitochondria and in cytoplasm. Cell nuclei lost carbon, nitrogen, and-to a clearly higher extent-oxygen with increasing electron irradiation. Therefore, electron doses less than 3 x 105 e/nm2 were used to measure the subcellular compartmentation of carbon, nitrogen, and oxygen in cytoplasm, mitochondria, and nuclei of the cells. The subcellular distribution of carbon, nitrogen, and oxygen reflects the intracellular compartmentation of various biomolecules. Cells exposed to inorganic mercury before cryofixation showed an increase of oxygen in nuclei and cytoplasm. Concomitantly the phosphorus/nitrogen ratio decreased in mitochondria. The data suggest mercury-induced production of ribonucleic acid (RNA) and decrease of adenosine triphosphate (ATP). Although biomolecules cannot be identified by X-ray microanalysis, measurements of the whole element spectrum including the light elements carbon, nitrogen, and oxygen can be useful to study specific biomolecular activity in cellular compartments depending on the functional state of the cell.

Effects of bioactive glass particles and their ionic products on intracellular concentrations.

Numerous studies have described the bioactive properties of glass particles in the SiO(2)-CaO-Na(2)O-P(2)O(5) system. This kind of material is capable of developing a direct contact with bone through dissolution and physicochemical reactions. We have investigated the influence of bioactive particles, and ionic products from the same particles, on the intracellular concentrations in monocyte cells, which are among the first cells to colonize implantation sites. The only way to access these concentrations and particularly diffusible ionic concentrations (potassium, sodium, and chlorine) is to use cryomethods coupled to electron probe microanalysis. We have paid particular attention to the potassium:sodium ratio, the most sensitive criterion of viability. We have cultured cells with bioactive glass particles and in a conditioned medium obtained from the dissolution of the glass particles in the standard medium. Our study demonstrates that cells cultured in a conditioned medium are more active than cells cultured in a standard medium, or cells exposed to bioactive particles, and particles are more toxic for cells than are ionic products.

Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro.

Calcium phosphate bioceramics have been applied as bone substitutes for several decades. Aseptic loosening after total joint arthroplasty is a major problem in orthopaedic surgery. Hydroxyapatite particles from materials wear have been reported as the main cause of implant failure. For this reason, an investigation into possible wear particles from materials used in the implant may lead to longevity after arthroplasty. Monocytes are among the first cells to colonize the inflammatory site. In the present study, we have evaluated the inflammatory response after exposition to particles with different characteristics (size, sintering temperature and shape). Our data demonstrate that the most important characteristic was the shape and the size of the particles. The needle shaped particles induced the larger production of TNF-alpha, IL-6 and IL-10 by cells. To a less manner, the smallest particles induced an increase of the expression and production of the cytokines studied (TNF-alpha, IL-6 and IL-10). The sintering temperature appeared to be a less important characteristic even though it was involved in the dissolution/precipitation process.

Light elements quantification in stimulated cells cryosections studied by electron probe microanalysis.

The quantification of intracellular light elements such as carbon, nitrogen and oxygen may be useful in understanding the biological mechanisms, for example the generation of nitric oxide which is involved in apoptosis and inflammatory reaction. Electron energy loss spectroscopy (EELS) coupled with scanning transmission electron microscopy is a useful method to detect light elements in thin cryosections. Recent developments of X-ray detectors with ultra-thin protection windows allows the detection of such elements by energy dispersive X-ray spectroscopy. In the present study, we have demonstrated using both methods that the stimulation of BV-2 murine microglial cell line by gamma-interferon and lipopolysaccharide leads to the increase of intracellular oxygen concentration and no change in the intracellular nitrogen concentration. This indicates the use of exogeneous molecular oxygen in response to the stimulation. But the increase of oxygen concentration detected could not be only due to NO expression because the NO production was 1000 times less than the oxygen concentration increase observed.