Dose-response lethality curves reveal a differential sensitivity to Mn exposure for <i>smf</i> mutants compared to wild type worms.

<p>Upon 30 min exposure to MnCl₂ as L1 larvae the lethal concentration 50 (LD₅₀) at which half of the worms were dead at 24 h, was 47 mM for wildtype worms (black, N = 12), 93 mM for <i>smf-1</i> mutants (blue, N = 6), 26 mM for <i>smf-2</i> mutants (green, N = 7), and 126 mM for <i>smf-3</i> mutants (orange, N = 5). Error bars represent SEM, *** p<0.001.</p

Mn exposure leads to severe osmoregulation defects and developmental delay.

<p>(A) Excretory canal in a control wild type L1 larva (solid white arrow heads). (B, C) Enlargement of the excretory canal in L1 larvae acutely exposed to 35 mM MnCl₂, after 24 h (solid white arrowheads) is associated with vacuolization (hollow arrowheads). Vacuoles are also observed in the sheath cells of the chemosensory organs (D) and in the epidermis (E). (F) Control larva 24 h after 0 mM MnCl₂ treatment. (G) Dying vacuolated (black arrowheads) larva 24 h after 35 mM MnCl₂ exposure. (H) worms exposed to 35 mM MnCl₂ (grey) are about 30% shorter than control animals (black). I, most larvae exposed to 35 mM MnCl₂ are still in L1 stage at 24 h post-treatment when control animals are L2. Error bars represent SEM, *** p<0.001, scale bars are 5 µm.</p

<i>C. elegans</i> SMF transporters rescue EGTA sensitivity of yeast Δ<i>Smf1</i>+Δ<i>Smf-2</i> mutant.

<p><i>S. cerevisiae</i> double-mutant <i>Smf1</i>Δ+<i>Smf-2</i>Δ is hypersensitive to exposure to the divalent cation chelator EGTA (red), when compared to wildtype (dotted black line). Transvections of <i>C. elegans smf-1</i> (blue), <i>smf-2</i> (green) or <i>smf-3</i> (orange) cDNA rescue the double-mutant hypersensitivity to EGTA.</p

Expression pattern analysis of <i>C. elegans smf</i> genes.

<p>A, SMF-1::GFP strongly localizes to the anterior and posterior intestine (solid white arrowheads), to the anchor cell (hollow arrowhead) and to head neurons (white arrows). <i>smf-1::</i>GFP and SMF-1::GFP reveal expression of <i>smf-1</i> gene in rectal gland cells (B,C, black asterisks), in the uterus (uv1, uv2, utse syncytium, D,E, solid white asterisks) as well as in the adult spermatheca (F) and the L1 hyp7 epidermis (G, white arrowheads). Dotted lines outline the cuticle of the worm. Hollow asterisks indicate position of fertilized embryos. Hollow arrowheads indicate position of the vulva. <i>smf-3</i> is mainly expressed in the intestine as revealed by <i>smf-3</i>::GFP (H) and SMF-3::GFP (I), in the major epidermis hyp7 (J, dotted line) and head epidermis hyp1-6 (K, dotted line), and in head (L) and tail neurons (M). An antero-posterior gradient of <i>smf-2</i> expression is noticeable in the 9 marginal epithelial cells of the pharynx (mc1, mc2, mc3) and the 6 vpi cells of the pharyngeo-intestinal valve (N and O). Fainter expression is consistently observed in the proximal gonad (P, Q). Scale bars are 5 µm.</p

Working model for Mn and Fe uptake by SMF transporters in <i>C. elegans</i>.

<p>A: Regulation of Mn, Fe contents and SMF transporters upon low Mn exposure (0.001 mM to 3 mM), which is believed to be beneficial for the worm physiology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007792#pone.0007792-Lin1" target="_blank">[92]</a>. B: Regulation of Mn, Fe contents and SMF transporters upon high Mn exposure (50 mM to 150 mM), which was shown to be toxic (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007792#pone-0007792-g003" target="_blank">Fig. 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007792#pone-0007792-g004" target="_blank">4</a>). We propose that SMF-3* is the main transporter responsible for Mn uptake (A), and that it is degraded upon exposure to high Mn concentrations (B). Since high Fe content limit Mn uptake, SMF-3 may be inhibited by intracellular Fe (A). SMF-1 would be involved in Mn uptake to a lesser extent, and together with SMF-2*, would be responsible for Fe uptake. Upon Low Mn exposure SMF-2 would be mostly required for Fe uptake (A), whereas upon high Mn exposure, SMF-2 would be inhibited and SMF-1 would partially compensate for Fe uptake (B). In the case of SMF-2 and SMF-1, metal uptake could essentially take place in acidified endosomal compartments, as SMF-2 is mainly cytoplasmic and SMF-1 is detected in sub-apical compartments. Gap-junction communications between pharyngeal epithelia, vpi cells and intestinal cells permit Mn²⁺ and Fe²⁺ to diffuse distant from their site of uptake, allowing metal-dependent regulation of <i>smf</i> mRNA stability or transcription. A prediction of our model is that the Fe gradient established by SMF-2 activity would be reversed upon high Mn exposure (B), and could constitute the signal for <i>smf</i> expression regulation. Since basal <i>smf</i> mRNA levels depend on the integrity of each <i>smf</i> genomic sequence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007792#pone-0007792-g009" target="_blank">Fig. 9</a>), transcriptions of <i>smf</i> genes are assumed to be interdependent, maybe because they require a common transcription factor. * SMF-2 might transport Mn and SMF-3 might transport Fe, but these possibilities are not explored in this model. The size of the text reflects the concentration of the species.</p

Variations in Mn and Fe content in <i>smf</i> mutant worms upon Mn exposure.

<p>(A) WT and <i>smf</i> mutants take up Mn in a dose-dependent manner. <i>smf-2(gk133)</i> (green) takes up significantly more Mn than WT (black) and other mutant worms following exposure to 35 (# p<0.05), 100 (## p<0.001) and 150 mM (### p<0.001) MnCl₂. <i>smf-3(ok1035)</i> (orange) mutants take up significantly less Mn than other worms at 100 (## p<0.01) and 150 mM (### p<0.001). (B) Fe content varies differentially in <i>smf</i> mutants and WT upon Mn exposure. <i>smf-2(gk133)</i> (green) display significantly lower Fe levels (# p<0.05), while <i>smf-1(eh5)</i> and <i>smf-3(ok1035)</i> mutants show higher Fe levels (# p<0.05) than WT in absence of Mn treatment (0 mM), and at very low Mn concentration for <i>smf-1(eh5)</i> (0.001 mM). Error bars represent SEM. While # designate significant differences between genetic backgrounds exposed to the same manganese dose, * indicate significant differences between exposure doses within the same <i>C. elegans</i> strain: #/* p<0.05. ##/** p<0.01, ###/*** p<0.001.</p

SMF-3::GFP is down-regulated upon Mn exposure.

<p>SMF-3::GFP signal is strongly detected at the apical plasma membrane prior Mn treatment (A). After 1 hour of exposure to 35 mM MnCl2, SMF-3-GFP localizes to sub-apical vesicular compartments (B). C, SMF-3::GFP signal is strongly decreased at 5 hours post-treatment. After a day of recovery, SMF-3::GFP expression returns to control levels and SMF-3::GFP relocates to the apical plasma membrane (D). Scale bars are 5 µm. (E) quantification of apical plasma-membrane SMF-3::GFP in the whole intestine after 5 and 30 h of exposure in control and treated animals. While # designate significant differences between genetic backgrounds exposed to the same manganese dose, * indicate significant differences between exposure doses within the same <i>C. elegans</i> strain: #/* p<0.05. ##/** p<0.01, ###/p<0.001.</p

Subcellular localization of SMF::GFP reporters.

<p>SMF-1::GFP mostly localizes to the apical plasma membrane of the intestine and sub-apical compartments (A, D, dotted line in A underlines the basolateral membrane of the intestine, dotted lines in D delimit its apical plasma membrane). SMF-2::GFP is seen in cytoplasmic organelles in mc and vpi cells (E, cell plasma membranes are marked by a dotted line, <i>N</i> indicate the position of the nuclei). SMF-3::GFP is mainly restricted to the apical plasma membrane of the intestine and apical vesicular organelles (C, F, dotted line in C underlines the basolateral membrane of the intestine, dotted lines in D delimit its apical plasma membrane). Scale bars are 5 µm.</p

RNA and protein expression of TMEM135 in mouse tissues.

<p>(<b>A</b>) Northern blot analysis of TMEM135 transcripts in newborn hearts of mice deficient in the very long chain ACAD gene. (<b>B</b>) <b>B</b> is representative of quantification by densitometry of Northern blot in <b>A</b>. (<b>C</b>) Protein expression of TMEM135 in different mouse tissues. (<b>D</b>) Quantification by densitometry of western blots in <b>C</b>. N =  3 per group for wild-type control mice labeled (+/+) for VLCAD +/+, heterozygous mice (+/−) for VLCAD+/−, and null mutant mice (−/−) for VLCAD−/−.</p

Nile Red staining and MitoTracker Red staining, survival differences and DAF-16 levels among the three <i>C. elegans</i> strains.

<p>Fig. <b>6A</b> is representative staining with Nile Red in the worm. (<b>B</b>) Represents semi-quantitative assessment of Nile Red staining intensity, N = 4 per group. (<b>C</b>) Represents survival analysis among the three <i>C. elegans</i> strains at 20°C. (<b>D</b>) Represents survival analysis among the three <i>C. elegans</i> strains at 15°C. Wild-type  =  controls shown in red, <i>tmem 135</i>(−<i>/</i>−) <i> =  tmem135</i>-deleted animals shown in black, TMEM135::GFP =  <i>C. elegans</i> animal overexpressing TMEM135 shown in green. (<b>E</b>) Quantitative assessment of MitoTracker Red fluorescence. (<b>F</b>) Western blot analysis and quantification of DAF-16 levels in the three <i>C. elegans</i> strains, N = 6 per group, values are mean ± SEM.</p