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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 uropod formation in response to β1, but not β2 integrin engagement.(A) Wild-type or ERM-deficient T cells were allowed to interact with fibronectin-coated glass coverslips, fixed and imaged using DIC optics. Uropods are marked with arrows. (B) Quantitation of uropod formation in cells analyzed in (A). (C) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG followed by rVCAM-1 Fc and imaged as in (A). (D) Quantitation of uropod formation in cells analyzed in (C). (E) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG alone (top panels) or together with rICAM-1 Fc (bottom panels), and imaged as in A. (F) Quantitation of uropod formation in cells analyzed in (E). Data in B, D and F represent mean ± StDev of at least 5 coverslips, with 50–100 cells each, from 2–7 independent experiments. *p<0.05, **p<0.005.
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pone-0052368-g004: ERM proteins are required for uropod formation in response to β1, but not β2 integrin engagement.(A) Wild-type or ERM-deficient T cells were allowed to interact with fibronectin-coated glass coverslips, fixed and imaged using DIC optics. Uropods are marked with arrows. (B) Quantitation of uropod formation in cells analyzed in (A). (C) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG followed by rVCAM-1 Fc and imaged as in (A). (D) Quantitation of uropod formation in cells analyzed in (C). (E) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG alone (top panels) or together with rICAM-1 Fc (bottom panels), and imaged as in A. (F) Quantitation of uropod formation in cells analyzed in (E). Data in B, D and F represent mean ± StDev of at least 5 coverslips, with 50–100 cells each, from 2–7 independent experiments. *p<0.05, **p<0.005.

Mentions: Outside-in signals from engaged integrins can induce T cell polarization to form a leading edge and trailing uropod, setting the stage for migration [42], [43]. ERM proteins are localized to the T cell uropod and have been implicated in maintenance of T cell polarity [24], [30], [40], [44]. We therefore asked if ERM-deficient T cells polarize appropriately in response to fibronectin binding. T cells were settled on fibronectin-coated glass coverslips, and uropod formation was assessed by DIC microscopy. As anticipated, wild-type T cells formed a characteristic “hand mirror” shape, with a rounded cell body and a constricted trailing uropod (Figure 4A, uropods indicated with arrows). In contrast, ERM-deficient T cells were rounded or elongated, but rarely exhibited a clear uropod. Quantitative analysis showed that uropod formation was significantly reduced in T cells lacking ezrin or moesin alone, and profoundly reduced in cells lacking both ERM proteins (Figure 4B). This defect was not specific to fibronectin, since inefficient uropod formation in ERM-deficient T cells was also observed in response to VCAM-1, another β1 integrin ligand (Figure 4C and D). Since β2-integrin mediated binding to ICAM-1 was intact, we asked whether ERM-deficient T cells form uropods efficiently in response to ICAM-1. T cells were settled on coverslips coated with anti-hIgG and ICAM-1 Fc or with anti-hIgG alone. In the absence of integrin ligand, wild-type and ERM-deficient T cells did not form uropods (Figure 4E and F). However, in the response to ICAM-1, both wild-type and ERM-deficient T cells formed uropods at comparable frequencies. Taken together, these studies show that ERM proteins are specifically required for adhesion and uropod formation in response to β1 integrin ligands. Though ERM proteins have been implicated as structural uropod components, our data showing that ERM-deficient T cells can generate a normal uropod in response to ICAM-1 suggest that the defects in uropod formation reflect abnormal outside-in signaling through β1 integrins rather than an inability to form a uropod per se. Consistent with this, we have observed that proliferating ERM-deficient T cells also readily form uropods in tissue culture (data not shown).


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 uropod formation in response to β1, but not β2 integrin engagement.(A) Wild-type or ERM-deficient T cells were allowed to interact with fibronectin-coated glass coverslips, fixed and imaged using DIC optics. Uropods are marked with arrows. (B) Quantitation of uropod formation in cells analyzed in (A). (C) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG followed by rVCAM-1 Fc and imaged as in (A). (D) Quantitation of uropod formation in cells analyzed in (C). (E) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG alone (top panels) or together with rICAM-1 Fc (bottom panels), and imaged as in A. (F) Quantitation of uropod formation in cells analyzed in (E). Data in B, D and F represent mean ± StDev of at least 5 coverslips, with 50–100 cells each, from 2–7 independent experiments. *p<0.05, **p<0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585410&req=5

pone-0052368-g004: ERM proteins are required for uropod formation in response to β1, but not β2 integrin engagement.(A) Wild-type or ERM-deficient T cells were allowed to interact with fibronectin-coated glass coverslips, fixed and imaged using DIC optics. Uropods are marked with arrows. (B) Quantitation of uropod formation in cells analyzed in (A). (C) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG followed by rVCAM-1 Fc and imaged as in (A). (D) Quantitation of uropod formation in cells analyzed in (C). (E) Wild-type or ERM-deficient T cells were allowed to interact with glass coverslips coated with anti-human IgG alone (top panels) or together with rICAM-1 Fc (bottom panels), and imaged as in A. (F) Quantitation of uropod formation in cells analyzed in (E). Data in B, D and F represent mean ± StDev of at least 5 coverslips, with 50–100 cells each, from 2–7 independent experiments. *p<0.05, **p<0.005.
Mentions: Outside-in signals from engaged integrins can induce T cell polarization to form a leading edge and trailing uropod, setting the stage for migration [42], [43]. ERM proteins are localized to the T cell uropod and have been implicated in maintenance of T cell polarity [24], [30], [40], [44]. We therefore asked if ERM-deficient T cells polarize appropriately in response to fibronectin binding. T cells were settled on fibronectin-coated glass coverslips, and uropod formation was assessed by DIC microscopy. As anticipated, wild-type T cells formed a characteristic “hand mirror” shape, with a rounded cell body and a constricted trailing uropod (Figure 4A, uropods indicated with arrows). In contrast, ERM-deficient T cells were rounded or elongated, but rarely exhibited a clear uropod. Quantitative analysis showed that uropod formation was significantly reduced in T cells lacking ezrin or moesin alone, and profoundly reduced in cells lacking both ERM proteins (Figure 4B). This defect was not specific to fibronectin, since inefficient uropod formation in ERM-deficient T cells was also observed in response to VCAM-1, another β1 integrin ligand (Figure 4C and D). Since β2-integrin mediated binding to ICAM-1 was intact, we asked whether ERM-deficient T cells form uropods efficiently in response to ICAM-1. T cells were settled on coverslips coated with anti-hIgG and ICAM-1 Fc or with anti-hIgG alone. In the absence of integrin ligand, wild-type and ERM-deficient T cells did not form uropods (Figure 4E and F). However, in the response to ICAM-1, both wild-type and ERM-deficient T cells formed uropods at comparable frequencies. Taken together, these studies show that ERM proteins are specifically required for adhesion and uropod formation in response to β1 integrin ligands. Though ERM proteins have been implicated as structural uropod components, our data showing that ERM-deficient T cells can generate a normal uropod in response to ICAM-1 suggest that the defects in uropod formation reflect abnormal outside-in signaling through β1 integrins rather than an inability to form a uropod per se. Consistent with this, we have observed that proliferating ERM-deficient T cells also readily form uropods in tissue culture (data not shown).

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