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

Chen EJ, Shaffer MH, Williamson EK, Huang Y, Burkhardt JK - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Pennsylvania, USA.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

ERM-deficient T cells can chemotax efficiently in vitro.Wild-type T cells expressing both ezrin and moesin (Ez+/+MoSiC), T cells lacking moesin (Ez+/+MoSiM), T cells lacking ezrin (Ez−/−MoSiC), or T cells lacking both ezrin and moesin (Ez−/−MoSiM), were prepared as described in Materials and Methods. (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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585410&req=5

pone-0052368-g001: ERM-deficient T cells can chemotax efficiently in vitro.Wild-type T cells expressing both ezrin and moesin (Ez+/+MoSiC), T cells lacking moesin (Ez+/+MoSiM), T cells lacking ezrin (Ez−/−MoSiC), or T cells lacking both ezrin and moesin (Ez−/−MoSiM), were prepared as described in Materials and Methods. (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.

Mentions: As shown in Figure 1A, wild-type and ERM-deficient T cells expressed comparable surface levels of the chemokine receptor CCR7. We therefore asked if ERM protein deficiency affects chemotactic responses to CCR7 ligands using an in vitro transwell assay. In addition to testing cells deficient for both ezrin and moesin, cells expressing only ezrin or only moesin were also tested to address the possibility of redundancy. As shown in Figure 1B, in the absence of chemokine, a low percentage of wild-type, single deficient and double deficient T cells were able to cross a 5 µm pore membrane. In the presence of chemokine in the lower chamber, efficient chemotaxis was observed for all cell populations. This demonstrates that ERM-deficient T cells are able to respond to CCR7 ligands, and can chemotax efficiently. Since ERM proteins have been implicated in regulating T cell cortical rigidity and tension [29], [30], we reasoned that ERM-deficient T cells might show enhanced ability to cross a constricted barrier. To test this, we repeated the assay using transwell chambers with 3 µm pores. Surprisingly, T cells lacking ERM proteins migrated less efficiently than wild-type cells through the smaller pores (Figure 1C). In some experiments, T cells lacking both ezrin and moesin showed a more profound phenotype than T cells lacking only one ERM protein (Figure 1D), consistent with the idea that these proteins have partially overlapping functions in T cells [37].


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

Chen EJ, Shaffer MH, Williamson EK, Huang Y, Burkhardt JK - PLoS ONE (2013)

ERM-deficient T cells can chemotax efficiently in vitro.Wild-type T cells expressing both ezrin and moesin (Ez+/+MoSiC), T cells lacking moesin (Ez+/+MoSiM), T cells lacking ezrin (Ez−/−MoSiC), or T cells lacking both ezrin and moesin (Ez−/−MoSiM), were prepared as described in Materials and Methods. (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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585410&req=5

pone-0052368-g001: ERM-deficient T cells can chemotax efficiently in vitro.Wild-type T cells expressing both ezrin and moesin (Ez+/+MoSiC), T cells lacking moesin (Ez+/+MoSiM), T cells lacking ezrin (Ez−/−MoSiC), or T cells lacking both ezrin and moesin (Ez−/−MoSiM), were prepared as described in Materials and Methods. (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.
Mentions: As shown in Figure 1A, wild-type and ERM-deficient T cells expressed comparable surface levels of the chemokine receptor CCR7. We therefore asked if ERM protein deficiency affects chemotactic responses to CCR7 ligands using an in vitro transwell assay. In addition to testing cells deficient for both ezrin and moesin, cells expressing only ezrin or only moesin were also tested to address the possibility of redundancy. As shown in Figure 1B, in the absence of chemokine, a low percentage of wild-type, single deficient and double deficient T cells were able to cross a 5 µm pore membrane. In the presence of chemokine in the lower chamber, efficient chemotaxis was observed for all cell populations. This demonstrates that ERM-deficient T cells are able to respond to CCR7 ligands, and can chemotax efficiently. Since ERM proteins have been implicated in regulating T cell cortical rigidity and tension [29], [30], we reasoned that ERM-deficient T cells might show enhanced ability to cross a constricted barrier. To test this, we repeated the assay using transwell chambers with 3 µm pores. Surprisingly, T cells lacking ERM proteins migrated less efficiently than wild-type cells through the smaller pores (Figure 1C). In some experiments, T cells lacking both ezrin and moesin showed a more profound phenotype than T cells lacking only one ERM protein (Figure 1D), consistent with the idea that these proteins have partially overlapping functions in T cells [37].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Pennsylvania, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus