Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an In Vitro Cell System and Rat Tissues

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature

CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner

<div><p>Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein’s CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.</p></div

CX3CR1 in differentiated HAE cells interacts with RSV.

<p>Differentiated HAE cells, grown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130517#pone.0130517.g001" target="_blank">Fig 1</a>, were incubated with RSV and imaged by immunofluorescence and confocal microscopy. (<b>a</b>) Binding experiment meant to visualize viral particles in association with HAE cells. Cultures were incubated 2h with RSV, then fixed and processed. RSV virions appear in green, β-tubulin is shown in red, and CX3CR1 is colored purple. Two regions of interest, representative of other RSV-bound cells in this image, are outlined with white squares. The top one is shown in expanded views in <b>b</b>, and the other one is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130517#pone.0130517.s002" target="_blank">S2 Fig</a>. Red, green, and purple images corresponding to β-tubulin, RSV F, and CX3CR1 respectively, are shown individually. Also shown is a merged image combining the fluorescence channels for RSV F, CX3CR1, and motile cilia. (<b>c</b>) Infected HAE cells were incubated for 3 days after infection and imaged using the same antibodies and fluorophores as in <b>a</b> and <b>b</b> but pseudo-colored differently: β-tubulin is in blue, RSV F in green, and CX3CR1 is shown in red. The two images are xy planes of the same sample separated along the z axis by 3.7 μm. The bottom image shows cilia and apical cell body, including some purple color indicative of colocalized tubulin and CX3CR1 immunofluorescence. The top image crosses the plane of the nuclei, below the cilia. Only infected cells (green) are surrounded by red-colored CX3CR1-positive circular features. Uninfected cells in the same sample and cells from uninfected control samples do not show these CX3CR1-containing structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130517#pone.0130517.g001" target="_blank">Fig 1C</a>). (<b>d</b>) Confocal immunofluorescence of HAE cells grown, differentiated, and infected for 3 days with RSV strain MSA1. Both <i>en face</i> (xy) and side (xz and yz) views are shown in this image. The xy plane of the <i>en face</i> view mostly cuts through the cilia of the cells, above the cell body. Nuclei are shown in blue using DAPI and, as in panel <b>a</b>, anti-RSV F protein is in green, anti-β-tubulin in red, and anti-CX3CR1 in purple. As in panel <b>c</b>, large ovoid structures positive for CX3CR1 immunofluorescence are seen in the side views and located near the nuclei of infected cells.</p

CX3CR1 is expressed in human airway epithelial cells and is localized to the motile cilia.

<p>HAE cells were grown in an air-liquid interface (ALI) culture system until differentiation was achieved, and imaged by confocal immunofluorescence. (<b>a</b>) Anti-β-tubulin (green) and anti-CX3CR1 (red) immunodetection of the cells seen <i>en face</i> are shown individually and merged (scale bar 10 μm). An xz plane image (XZ Merge) further illustrates the localization of CX3CR1 in motile cilia (scale bar 5 μm). Nuclei, stained with DAPI, are rendered in blue. (<b>b</b>) Immunostaining with isotype control antibodies confirms the specificity of the immunostaining. The merge image and XZ merge image are in planes that cross nuclei to confirm the presence of cells and the absence of non-specific immunostaining. (<b>c</b>) Confocal immunofluorescence images of differentiated HAE cells. Cells were either infected with RSV strain MSA1 (top and bottom rows), or mock infected (middle row). Images were acquired either two hours (left two columns), 1 day (middle two columns), or 3 days (right two columns) after infection or mock infection. CX3CR1 immunofluorescence is shown in (purple) (top two rows), or using an isotype negative control (bottom row). The cells were also stained for nuclei using DAPI (blue). Two representative images are shown for each condition tested. The 2-hour time point images are of an xy plane above the nuclei, intersecting the motile cilia. CX3CR1 immunofluorescence is clearly detected in the cilia of both infected and uninfected cells but is absent in the isotype control samples. The 1-day and 3-day time points are in an xy plane crossing the nuclei, 4.3 μm below the motile cilia.</p

Antibody to G protein inhibits RSV binding to HAE cells.

<p>Wild type RSV was incubated with a negative control antibody or with 131-2G for 1h, and added to differentiated HAE cells for 2 h. (<b>a</b>) Fluorescence micrograph of cells fixed and immunostained against RSV F incubated with the indicated antibody. (<b>b</b>) Quantification of immunofluorescence intensity in a by counting pixels in moderate (40–100) or high (101–255) brightness ranges. Incubation with 131-2G significantly reduces immunofluorescence detection of RSV (<i>P</i> = 0.0001 by two-tailed t-test).</p