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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.

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ERM proteins are required for efficient adhesion to β1, but not β2 integrin ligands.(A and C) Wild-type or the indicated ERM-deficient T cells were stained with Calcein-AM and settled in 96-well plates coated with fibronectin (A) or rICAM-1 Fc (C). Cells were either left unstimulated or stimulated as indicated, non-adherent cells were washed off, and fluorescence was measured using a microplate reader. Adherent cells are shown as a percentage of input. Data shown are means ± StDev from triplicate wells in one experiment, representative of three individual experiments. * p<0.05, **p<0.005. (B and D) Cells were stained with either anti-CD29 (B) or anti-CD18 (D) and analyzed by flow cytometry to assess surface integrin levels.
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pone-0052368-g003: ERM proteins are required for efficient adhesion to β1, but not β2 integrin ligands.(A and C) Wild-type or the indicated ERM-deficient T cells were stained with Calcein-AM and settled in 96-well plates coated with fibronectin (A) or rICAM-1 Fc (C). Cells were either left unstimulated or stimulated as indicated, non-adherent cells were washed off, and fluorescence was measured using a microplate reader. Adherent cells are shown as a percentage of input. Data shown are means ± StDev from triplicate wells in one experiment, representative of three individual experiments. * p<0.05, **p<0.005. (B and D) Cells were stained with either anti-CD29 (B) or anti-CD18 (D) and analyzed by flow cytometry to assess surface integrin levels.

Mentions: Overexpression of constitutively active ERM proteins has been shown to enhance adhesion to integrin ligands [30], [40]. To ask if deficiency in ERM proteins enhances or inhibits adhesion, wild-type, single deficient and double deficient T cells were allowed to settle on surfaces coated with the β1 integrin ligand fibronectin, non-adherent cells were washed away and specific binding was assessed. As anticipated, wild-type T cell blasts showed basal adhesion in the absence of stimulation, and adhesion was enhanced by treatment with either PMA or anti-CD3 (Figure 3A). ERM-deficient T cells showed significantly lower basal binding to fibronectin than wild-type cells. Moreover, activation-dependent binding of ERM-deficient T cells was strikingly lower than that of wild-type cells. Intermediate, but still statistically significant, defects were observed in T cells lacking only ezrin or moesin, pointing to overlapping function of these proteins in promoting β1 integrin-dependent adhesion. The diminished adhesion we observed in ERM-deficient T cells was not due to changes in β1 integrin expression; all cell populations expressed comparable surface levels of the β1 chain CD29 (Figure 3B). Somewhat surprisingly, parallel studies using the β2 integrin ligand ICAM-1 did not reveal ERM-protein dependent binding. As shown in Figure 3C, both basal and activation-induced binding of ERM-deficient T cells to ICAM-1 were comparable to that of wild-type T cells. As expected, ERM-deficient T cells expressed normal levels of the β2 chain CD18 (Figure 3D).


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 proteins are required for efficient adhesion to β1, but not β2 integrin ligands.(A and C) Wild-type or the indicated ERM-deficient T cells were stained with Calcein-AM and settled in 96-well plates coated with fibronectin (A) or rICAM-1 Fc (C). Cells were either left unstimulated or stimulated as indicated, non-adherent cells were washed off, and fluorescence was measured using a microplate reader. Adherent cells are shown as a percentage of input. Data shown are means ± StDev from triplicate wells in one experiment, representative of three individual experiments. * p<0.05, **p<0.005. (B and D) Cells were stained with either anti-CD29 (B) or anti-CD18 (D) and analyzed by flow cytometry to assess surface integrin levels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585410&req=5

pone-0052368-g003: ERM proteins are required for efficient adhesion to β1, but not β2 integrin ligands.(A and C) Wild-type or the indicated ERM-deficient T cells were stained with Calcein-AM and settled in 96-well plates coated with fibronectin (A) or rICAM-1 Fc (C). Cells were either left unstimulated or stimulated as indicated, non-adherent cells were washed off, and fluorescence was measured using a microplate reader. Adherent cells are shown as a percentage of input. Data shown are means ± StDev from triplicate wells in one experiment, representative of three individual experiments. * p<0.05, **p<0.005. (B and D) Cells were stained with either anti-CD29 (B) or anti-CD18 (D) and analyzed by flow cytometry to assess surface integrin levels.
Mentions: Overexpression of constitutively active ERM proteins has been shown to enhance adhesion to integrin ligands [30], [40]. To ask if deficiency in ERM proteins enhances or inhibits adhesion, wild-type, single deficient and double deficient T cells were allowed to settle on surfaces coated with the β1 integrin ligand fibronectin, non-adherent cells were washed away and specific binding was assessed. As anticipated, wild-type T cell blasts showed basal adhesion in the absence of stimulation, and adhesion was enhanced by treatment with either PMA or anti-CD3 (Figure 3A). ERM-deficient T cells showed significantly lower basal binding to fibronectin than wild-type cells. Moreover, activation-dependent binding of ERM-deficient T cells was strikingly lower than that of wild-type cells. Intermediate, but still statistically significant, defects were observed in T cells lacking only ezrin or moesin, pointing to overlapping function of these proteins in promoting β1 integrin-dependent adhesion. The diminished adhesion we observed in ERM-deficient T cells was not due to changes in β1 integrin expression; all cell populations expressed comparable surface levels of the β1 chain CD29 (Figure 3B). Somewhat surprisingly, parallel studies using the β2 integrin ligand ICAM-1 did not reveal ERM-protein dependent binding. As shown in Figure 3C, both basal and activation-induced binding of ERM-deficient T cells to ICAM-1 were comparable to that of wild-type T cells. As expected, ERM-deficient T cells expressed normal levels of the β2 chain CD18 (Figure 3D).

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