Iron is localized within dopamine neurovesicles.

<p>Visible light microscopy of freeze-dried cells (A), and epifluorescence microscopy of the same freeze-dried cells (B) enable the identification of dopamine distribution, while synchrotron X-ray fluorescence nano-imaging reveals the distribution of iron (C, D). Panels C and D represent comparison of the same region imaged in a fluorescent mode to visualize dopamine and with X-ray fluorescence to localize iron. Dopamine and iron are co-located within 200 nm structures characteristic of dopamine neurovesicles as identified by epifluorescence microscopy. A large number of iron and dopamine neurovesicles are found in neurite outgrowths (C) and distal ends (D). Min-max range bar units are arbitrary. Scale bars = 1 µm.</p

Synchrotron X-ray chemical nano-imaging reveals iron sub-cellular distribution.

<p>The synchrotron X-ray fluorescence nanoprobe end-station installed at ESRF was designed to provide a high flux hard X-ray beam of less than 90 nm size (FWHM, full width at half maximum). The intensity distribution in the focal plane is shown in (A); dopamine producing cells were exposed <i>in vitro</i> to 300 µM FeSO₄ during 24 h (B). Chemical element distributions, here potassium and iron, were recorded on distinct cellular areas such as cell bodies (C), neurite outgrowths, and distal ends (D). Iron was found in 200 nm structures in the cytosol, neurite outgrowths, and distal ends, but not in the nucleus. Iron rich structures are not always resolved by the beam and clusters of larger dimension are also observed. Min-max range bar units are arbitrary. Scale bars = 1 µm.</p

Cellular iron and zinc concentrations (µg/g dry mass; mean±SD; n = 6), obtained by PIXE quantitative micro-analysis.

<p>The data are the mean of six independent analyses performed on areas containing several hundred of cells for each condition of culture. The inhibition of dopamine synthesis (AMT, and AMT+Fe) results in a decrease of total iron concentration, while zinc concentration is not changed, suggesting a specific role of dopamine in iron homeostasis.</p

Nano-imaging of potassium, iron, and zinc in distal ends.

<p>Each series of images are representative of the entire cell population for each condition (control, 1mM AMT and/or 300 µM FeSO₄). The scanned area (left images, red squares) is shown on a bright field microscopy view of the freeze dried cell. Iron is located within dopamine vesicles of 200 nm size or more (Control, and Fe conditions). Iron concentration is close to the limit of detection in distal ends of AMT, and AMT+Fe cells; only a basal level of Fe is observed. Min-max range bar units are arbitrary for potassium and zinc distributions. For iron distribution the maximum threshold values in micrograms per squared centimeter are shown for each color scale. Scale bars = 1 µm.</p

Image_3_Cellular Fractionation and Nanoscopic X-Ray Fluorescence Imaging Analyses Reveal Changes of Zinc Distribution in Leaf Cells of Iron-Deficient Plants.pdf

<p>Multilevel interactions among nutrients occur in the soil-plant system. Among them, Fe and Zn homeostasis in plants are of great relevance because of their importance for plant and human nutrition. However, the mechanisms underlying the interplay between Fe and Zn in plants are still poorly understood. In order to elucidate how Zn interacts with Fe homeostasis, it is crucial to assess Zn distribution either in the plant tissues or within the cells. In this study, we investigated the subcellular Zn distribution in Fe-deficient leaf cells of cucumber plants by using two different approaches: cellular fractionation coupled with inductively coupled plasma mass spectrometry (ICP/MS) and nanoscopic synchrotron X-ray fluorescence imaging. Fe-deficient leaves showed a strong accumulation of Zn as well as a strong alteration of the organelles’ ultrastructure at the cellular level. The cellular fractionation-ICP/MS approach revealed that Zn accumulates in both chloroplasts and mitochondria of Fe deficient leaves. Nano-XRF imaging revealed Zn accumulation in chloroplast and mitochondrial compartments, with a higher concentration in chloroplasts. Such results show that (i) both approaches are suitable to investigate Zn distribution at the subcellular level and (ii) cellular Fe and Zn interactions take place mainly in the organelles, especially in the chloroplasts.</p

Image_1_Cellular Fractionation and Nanoscopic X-Ray Fluorescence Imaging Analyses Reveal Changes of Zinc Distribution in Leaf Cells of Iron-Deficient Plants.TIF

<p>Multilevel interactions among nutrients occur in the soil-plant system. Among them, Fe and Zn homeostasis in plants are of great relevance because of their importance for plant and human nutrition. However, the mechanisms underlying the interplay between Fe and Zn in plants are still poorly understood. In order to elucidate how Zn interacts with Fe homeostasis, it is crucial to assess Zn distribution either in the plant tissues or within the cells. In this study, we investigated the subcellular Zn distribution in Fe-deficient leaf cells of cucumber plants by using two different approaches: cellular fractionation coupled with inductively coupled plasma mass spectrometry (ICP/MS) and nanoscopic synchrotron X-ray fluorescence imaging. Fe-deficient leaves showed a strong accumulation of Zn as well as a strong alteration of the organelles’ ultrastructure at the cellular level. The cellular fractionation-ICP/MS approach revealed that Zn accumulates in both chloroplasts and mitochondria of Fe deficient leaves. Nano-XRF imaging revealed Zn accumulation in chloroplast and mitochondrial compartments, with a higher concentration in chloroplasts. Such results show that (i) both approaches are suitable to investigate Zn distribution at the subcellular level and (ii) cellular Fe and Zn interactions take place mainly in the organelles, especially in the chloroplasts.</p

Deciphering the Resistance-Counteracting Functions of Ferroquine in <i>Plasmodium falciparum</i>-Infected Erythrocytes

The aminoquinoline chloroquine (CQ) has been widely used for treating malaria since World War II. Resistance to CQ began to spread around 1957 and is now found in all malarious areas of the world. CQ resistance is caused by multiple mutations in the <i>Plasmodium falciparum</i> chloroquine resistance transporter (PfCRT). These mutations result in an increased efflux of CQ from the acidic digestive vacuole (DV) to the cytosol of the parasite. This year, we proposed a strategy to locate and quantify the aminoquinolines in situ within infected red blood cells (iRBCs) using synchrotron based X-ray nanoprobe fluorescence. Direct measurements of unlabeled CQ and ferroquine (FQ) (a ferrocene-CQ conjugate, extremely active against CQ-resistant strains) enabled us to evidence fundamentally different transport mechanisms from the cytosol to the DV between CQ and FQ in the CQ-susceptible strain HB3. These results inspired the present study of the localization of CQ and FQ in the CQ-resistant strain W2. The introduction of the ferrocene core in the lateral side chain of CQ has an important consequence: the transporter is unable to efflux FQ from the DV. We also found that resistant parasites treated by FQ accumulate a sulfur-containing compound, credibly glutathion, in their DV

Potassium elemental distribution of (parts of) single fibroblast cells obtained from control patient ‘LR’ and Friedreich’s ataxia patient ‘SL’.

<p>For each case, 5 different fibroblasts were scanned. Boundaries of the fibroblasts are indicated, as well as their nuclei (if present). Scans were obtained in ‘Low Dose’ mode, with a step size of 100 nm and a dwell time of 50 ms. Maps are normalized to incoming intensity, 1s measuring time and are dead time corrected.</p

Potassium and iron elemental distribution within control fibroblast case ‘PN’ (upper row) and FRDA fibroblast case ‘DJS’ (lower row).

<p>Scale bar indicates the background-corrected mass fraction in ppm, calculated from a mean cell thickness of 10 μm. Pixel size in the images is 55 nm; acquisition time for each pixel is 50 ms. Elemental maps were measured in ‘High dose’ mode and based on detector no. 5 (XIA05) only. All element maps were normalized to dead time, ring current and quantified using the Fundamental Parameter method, taking into account the ice layer thickness determined using the K-Kα/Kβ ratio. Red striped circles indicate areas with iron hot-spots, circles h1-h3 indicate exogenous iron hot-spots, white arrows spherical structures and red arrows fibre-like structures in the fibroblast cells.</p

Elemental distribution of P, S, Cl, Ca, Zn and Compton scatter within control fibroblast case ‘PN’ (upper row) and FRDA fibroblast case ‘DJS’ (lower row).

<p>Experimental conditions: see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190495#pone.0190495.g002" target="_blank">Fig 2</a>. White dashed circle indicates the nucleus border. n1-2 indicate the presence of nucleoli.</p