Effect of endocytosis inhibitors on endosomal sorting of cystinosin-LKG.

<p>HK-2 cells stably transfected with RFP-tagged cystinosin-LKG, after 48h serum starvation, were treated 30’ with 5 μg/ml chlorpromazine (CPZ) a clathrin-dependent endocytosis inhibitor or with 30 μg/ml methyl-β-cyclodextrin (MβCD) which affects clathrin-independent pathway. Qualitative analysis shows the presence of RFP-tagged cystinosin-LKG on the plasma membrane stained with WGA green, but the RFP signal accumulated more in CPZ treated cells (A). Scale bar = 20 μm. In the same experimental conditions, the uptake of Alexa Fluor® 488 transferrin (488-Tf) was assayed in order to confirm the inhibition of clathrin-dependent endocytosis (B). Scale bar = 20 μm. Quantitative analysis, achieved by protein surface biotinylation and SDS-PAGE, shows that CPZ treatment induces a significant increase of cystinosin-LKG presence on the plasma membrane (C). Means ± SEM of three experiments are shown.</p

Colocalization studies of cystinosin-LKG carrying the deletion of lysosomal sorting signal YFPQA.

<p>In HK-2 cells, transiently transfected with the pCTNS-LKG-RFP construct carrying the deletion of the (ΔFPQA), the deleted cystinosin-LKG-RFP could be seen on the plasma membrane as well as in lysosomes. The intensity profile, obtained with RGB Profiler, an ImageJ plugin, showed the RFP signal in lysosomes of ΔFPQA reduced compared to the wild type cystinosin-LKG. Scale bar = 20 μm.</p

Subcellular distribution of the cystinosin-LKG and ΔSSLKG mutant.

<p>HK-2 cells were stably transfected with RFP-tagged cystinosin-LKG or with its mutated form, deleted in C-terminal tail for the last five amino acids (ΔSSLKG). Cells were immunolabeled with LAMP-2 for lysosomes (A), PDI for endoplasmic reticulum (B), GM130 for Golgi (C). Scale bar = 10 μm. Analysis of ROIs (Regions of Interest) shows a Pearson’s Correlation (R_r) for RFP with LAMP-2 greater than with other organelle markers (<i>p</i> < 0.0005). In particular, the mutant ΔSSLKG shows an R_r for LAMP-2 significantly increased (<i>p</i> < 0.0005) compared to the wild type cystinosin-LKG. The presence of cystinosin-LKG and ΔSSLKG mutant on ER is low, moreover the analysis shows very low expression of cystinosin-LKG in the Golgi apparatus, whereas high R_r for GM130 in ΔSSLKG mutant suggests that it is accumulated significantly (<i>p</i> < 0.00001) on the Golgi apparatus (D). Means ± SEM of three experiments are shown.</p

Effect of silencing of AP-2 mu chain on endosomal sorting of cystinosin-LKG and ΔSSLKG mutant.

<p>siRNA to human AP-2 mu chain and a scrambled control were transfected into HK-2 cells overexpressing RFP tagged cystinosin-LKG and ΔSSLKG mutant. The analysis of the AP-2 expression by PCR and western blotting show an efficient silencing with a significant transcript reduction (p < 0.005) (A). After 72h silencing of AP-2 mu chain, expression of cystinosin-LKG on the plasma membrane is significantly reduced. Inhibition of the clathrin-independent pathway by treatment with 30 μg/ml methyl-β-cyclodextrin (MβCD) for 30’ combined to AP-2 silencing, permanently prevents endocytic sorting of cystinosin-LKG from the plasma membrane, triggering the accumulation of the protein on the plasma membrane (B). Scale bar = 20 μm. As previously showed, ΔSSLKG mutant is less expressed on the plasma membrane, and this condition is highlighted by AP-2 silencing. After AP-2 silencing, in fact, ΔSSLKG is very low on plasma membrane and the inhibition of the clathrin-independent pathway with MβCD does not affect significantly the distribution (C). Scale bar = 20 μm. Colocalization analysis between RFP and WGA green (plasma membrane) signals shows the Pearson’s Correlation significantly reduced in ΔSSLKG cells (<i>p</i> < 0.001) and in cystinosin-LKG with AP-2 silencing (<i>p</i> < 0.001). In the latter, after MβCD treatment, the Pearson’s Correlation increases about 24% (<i>p</i> < 0.001), while no significant effects is observed in ΔSSLKG mutant (D). Analysis of transferrin uptake indicates that cystinosin-LKG is expressed on the plasma membrane only in cells where AP-2 was not silenced (transferrin internalized) instead silencing of AP-2 associated to the accumulation of transferrin on the plasma membrane, affects negatively the presence of cystinosin-LKG on the plasma membrane (E). Scale bar = 20 μm. Means ± SEM of four (A, B, C, D) or three (E) experiments are shown.</p

Scheme of the cystinosin isoforms structure.

<p>Cystinosin (367 aa) on the left and the cystinosin-LKG isoform (400 aa) on the right, are the main known isoforms to date, for which has been described the transport of cystine. The open-source tool for visualization of proteoforms, PROTTER [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154805#pone.0154805.ref032" target="_blank">32</a>], displayed the hypothetical structure of the two isoforms. Red regions are two targeting motifs for the protein sorting to lysosomes: GYDQL located at the C-terminal end, and YFPQA located in the putative fifth inter-transmembrane loop. Cystinosin-LKG differs from the canonical cystinosin in the C-terminal region (orange) while the proposed motif critical for the protein sorting to the plasma membrane (SSLKG) is highlighted in green.</p

¹⁴C-cystine uptake from extracellular milieu in HK-2 transfected with different cystinosin isoforms.

<p>¹⁴C-cystine uptake assay performed in HK-2 cells transfected with vehicle, cystinosin, cystinosin-LKG, or ΔSSLKG mutant, shows low uptake of radiolabelled L-[¹⁴C]-cystine across plasma membrane in absence of a proton gradient (light grey columns). In the presence of a proton gradient (dark grey columns) HK-2 cells overexpressing cystinosin-LKG show a significant ¹⁴C-cystine uptake (p < 0.0005) compared to cells transfected with cystinosin; while HK-2 cells overexpressing ΔSSLKG mutant show lower ¹⁴C-cystine uptake (p < 0.004) compared to cells transfected with cystinosin-LKG. Means ± SEM of three experiments are shown.</p

Characterization of mesenchymal stem cells and <i>CTNS</i>(−/−) mutant target cells.

<p>(a) FACS analysis (left) of bmMSC surface markers (blue) and isotype controls (red) and bmMSC differentiation from baseline (upper right) to Alizarin Red S-stained calcium-containing osteogenic (middle right) and Oil-Red O-stained adipocyte (bottom right) phenotypes. (b) same FACS and differentiation analysis of amMSC cells. (c) schematic diagram depicting the common 57 Kb deletion removing the 5′ portion of the <i>CTNS</i> gene and PCR-proven genotype of control and mutant fibroblasts from a cystinosis patient. Intracellular cystine content of control and homozygous <i>CTNS</i> mutant fibroblasts is shown on the right bar-graph.</p

Analysis of stem cell microvesicles and their effects on cystine content of <i>CTNS</i>(−/−) mutant fibroblasts.

<p>(a) diameter of bmMSC microparticles released into conditioned medium. (b) effect of increasing amounts of bmMSC microvesicles on cystine in mutant fibroblasts after 24 hour incubation, compared to the effect of 1 mM cysteamine. (c) diameter of amMSC microparticles released into conditioned medium. (d) effect of increasing amounts of amMSC microvesicles on cystine in mutant fibroblasts after 24 hour incubation, compared to the effect of 1 mM cysteamine and in the presence of annexin V. Statistical significance is indicated by the corresponding p-value.</p

Effect of co-culture with CTNS-expressing cells on cystine content of mutant <i>CTNS</i> cells.

<p>(a) Cystine content of <i>CTNS</i>(−/−) fibroblasts co-cultured with increasing amounts of wildtype amMSC. (b) Cystine content of GFP-tagged <i>CTNS</i>(−/−) ciPTEC isolated by FACS after co-culture with unlabelled wildtype amMSC, mutant ciPTEC or <i>CTNS</i>-corrected ciPTEC as donors. Statistical significance is indicated by the corresponding p-value.</p

amMSC microvesicle transfer of CTNS^Red to acidic intracellular compartment of <i>CTNS</i>(−/−) mutant fibroblasts.

<p>(a) amMSC stably transfected with CTNS^Red immunofluorescent protein. (b) CTNS^Red in amMSC microvesicles. (c) single confocal plane showing CTNS^Red protein in amMSC (circles) and in co-cultured GFP-tagged <i>CTNS</i>(−/−) mutant fibroblasts (arrows). Dashed line indicates amMSC cluster also shown in brightfield insert. (d) fusion of CTNS^Red particle with LysoTracker stained endosome/lysosome (arrowhead), shown in insert at higher magnification.</p