Ezrin Is Highly Expressed in Early Thymocytes, but Dispensable for T Cell Development in Mice

Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored.We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/-) mice likely arise as a consequence of nutritional stress.We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways

Ezrin and moesin are required for efficient T cell adhesion and homing to lymphoid organs.

T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the β1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for β1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs

Random migration in collagen gels.

a<p><i>Meandering index</i> is defined as net displacement divided by track length, such that migration in a straight line gives a value of 1, while greater meandering leads to values closer to zero.</p>b<p><i>Angle</i> measures migration along the Y-axis. Values range from 0° to 180°, such that random migration should give a value of 90°.</p>c<p>Bearing measures migration along the X-axis. Values range from 90°–270°, such that random migration should give a value of 180°.</p

ERM proteins are required for T cell homing to peripheral lymphoid organs.

<p>Wild-type and ERM-deficient T cells were differentially labeled with CFSE and CMTMR, mixed in a 1∶1 ratio and injected into the tail veins of C57Bl/6 hosts. (A) Blood, spleen, and peripheral and mesenteric lymph nodes were collected 1 hour after injection, and cell suspensions were analyzed by flow cytometry. The ratio of adoptively transferred ERM-deficient to wild type T cells is shown. Data represent mean ± StDev from 5 mice in one experiment, representative of four experiments. * p<0.05, **p<0.005. (B and C) Lymph nodes were harvested 1 hour after injection for multi-photon imaging, and cell migration was tracked. (B) The percentage of cells showing active migration, defined as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052368#s4" target="_blank">Materials and Methods</a>. (C) Tracks, average velocities and average track lengths from one representative lymph node. Data represent mean ± StDev of three experiments.</p

ERM-deficient T cells can migrate efficiently in confined spaces.

<p>A) Wild-type or ERM-deficient T cells were placed in a 5 µm pore collagen gel in the absence (top panels) or presence (bottom panels) of a CCL19 gradient, and cell migration was imaged for 4 hours at 37°C. Tracks of individual cells are presented with the same point of origin. Data are representative of three experiments. Quantitative analysis is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052368#pone-0052368-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052368#pone-0052368-t002" target="_blank">2</a>. B and C) Frequency of individual cell velocities in (A) in the absence (B) or presence (C) of chemokine. Data are representative of at least three collagen gels per condition in two independent experiments.</p

ERM-deficient T cells can chemotax efficiently <i>in vitro</i>.

<p>Wild-type T cells expressing both ezrin and moesin (Ez^+/+Mo^SiC), T cells lacking moesin (Ez^+/+Mo^SiM), T cells lacking ezrin (Ez^−/−Mo^SiC), or T cells lacking both ezrin and moesin (Ez^−/−Mo^SiM), were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052368#s4" target="_blank">Materials and Methods</a>. (A) Cells were stained for CCR7 and assessed by flow cytometry. Filled histogram, isotype control. (B–D) Wild-type or the indicated ERM-deficient T cells were placed in a transwell assay in the absence or presence of 40 nM CCL19 for 2 hours. Cells that migrated across a 5 µm (B) or 3 µm (C and D) pore membrane to the bottom well were quantified, and are presented as the percentage of input. Data are mean ± StDev of quadruplicate wells from one experiment, representative of three experiments. *p<0.05, **p<0.005.</p

ERM-deficient cells undergo efficient diapedesis <i>in vitro</i>.

<p>(A,B) Diapedesis under static conditions. Confluent monolayers of 3B-11 endothelial cells were pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces. Cells were imaged in an environmental chamber every 30 seconds for 2 hours. (A) DIC images of cells undergoing diapedesis. Arrowheads indicate leading edges of migrating T cells. White arrows indicate cells migrating along the apical surface of endothelial cells; the same cells are marked with red arrows after passing below the endothelial cell layer. (B) Quantitative analysis of assays carried out as in A. Cells undergoing diapedesis are quantified as a percentage of total moving cells. Data represent mean ± StDev from six experiments. (C) Diapedesis under shear flow conditions. Confluent monolayers of 3B-11 endothelial cells were grown in flow chambers, pre-treated with or without TNFα to upregulate VCAM-1, and wild-type or ERM-deficient T cells were added to the apical surfaces and allowed to interact with endothelial cells under shear stress of 0.5 dyne/cm². Cells were imaged every 30 seconds for 1 hour, and the percentage of moving cells that underwent diapedesis was determined. Data represent mean ± StDev from three experiments.</p