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Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation.

Lee JH, Katakai T, Hara T, Gonda H, Sugai M, Shimizu A - J. Cell Biol. (2004)

Bottom Line: T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner.Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation.Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.

ABSTRACT
Front-rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner. Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho-ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane "posteriority" in the induction of the uropod in T lymphocytes.

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Intracellular localization of ezrin mutants and the enhancement of uropod size in T567D ezrin transfectants. (A) GFP-tagged ezrin constructs used in this study. In point mutant constructs T567A or T567D, a CT phosphorylation site, Thr567 (T) was replaced by Ala (A) or Asp (D), respectively. (B and C) Intracellular localization of ezrin mutants in EL4.G8 cells. Stable transfectants were examined for the localization of GFP as well as CD44 (B) or F-actin (C). Each set of panels corresponds to the construct shown to the left in A. T567D ezrin was exclusively localized at the uropod (asterisks) and colocalized with the enhanced F-actin signal (C, arrows). Bar, 10 μm. (D) GFP-tagged proteins expressed in stable transfectants were detected by Western blotting using antiezrin or anti-GFP antibody. Polyclonal antiezrin antibody recognizes an epitope in the CT domain and, thus, does not detect the NT fragment. The arrowhead indicates endogenous ezrin. (E) Interaction between CD44 and ezrin mutants was detected by the immunoprecipitation of CD44 and Western blotting for GFP. (F) T567D ezrin increases uropod length. The length of the uropod was measured and expressed as mean ± SD (n > 200). Each bar represents an individual stable clone of a different type of ezrin transfectant.
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fig2: Intracellular localization of ezrin mutants and the enhancement of uropod size in T567D ezrin transfectants. (A) GFP-tagged ezrin constructs used in this study. In point mutant constructs T567A or T567D, a CT phosphorylation site, Thr567 (T) was replaced by Ala (A) or Asp (D), respectively. (B and C) Intracellular localization of ezrin mutants in EL4.G8 cells. Stable transfectants were examined for the localization of GFP as well as CD44 (B) or F-actin (C). Each set of panels corresponds to the construct shown to the left in A. T567D ezrin was exclusively localized at the uropod (asterisks) and colocalized with the enhanced F-actin signal (C, arrows). Bar, 10 μm. (D) GFP-tagged proteins expressed in stable transfectants were detected by Western blotting using antiezrin or anti-GFP antibody. Polyclonal antiezrin antibody recognizes an epitope in the CT domain and, thus, does not detect the NT fragment. The arrowhead indicates endogenous ezrin. (E) Interaction between CD44 and ezrin mutants was detected by the immunoprecipitation of CD44 and Western blotting for GFP. (F) T567D ezrin increases uropod length. The length of the uropod was measured and expressed as mean ± SD (n > 200). Each bar represents an individual stable clone of a different type of ezrin transfectant.

Mentions: To address the details of ezrin localization in the uropod, we transfected plasmids expressing various GFP-tagged mutant forms of ezrin into EL4.G8 cells and obtained multiple stable lines (Fig. 2, A and D). Transfection with the control vector expressing GFP resulted in cytoplasmic and nuclear distribution of this reporter protein (Fig. 2, B and C). In contrast, wild-type (WT) ezrin-GFP was preferentially localized at the peripheral membrane, especially in the uropod rather than the cell body, although a weak signal was also detected in the cytoplasm. A mutant ezrin in which the CT Thr was replaced by Ala (T567A) was distributed diffusely in the cytoplasm. This mutant cannot be phosphorylated at this residue and, therefore, does not undergo the intramolecular NT and CT domain dissociation and mimics the dormant form of ezrin (Gautreau et al., 2000; Coscoy et al., 2002). In marked contrast, another mutant with replacement of this Thr by Asp, T567D, which does not undergo NT and CT domain association and thus mimics the phosphorylated active form with membrane–cytoskeleton linker activity (Gautreau et al., 2000; Coscoy et al., 2002), exclusively accumulated in the uropod colocalized with CD44 (Fig. 2 B, asterisks). The fluorescent signal of the NT domain fragment tagged with GFP was also detected at the uropod, as well as at the peripheral membrane of the cell body, confirming the requirement of the NT domain for ezrin to localize in the uropod. In contrast, the CT domain fragment of ezrin was accumulated at the leading edge and was well-colocalized with F-actin, but was not abundant in the uropod, indicating that this fragment freely associates with the actin cytoskeleton. Such intracellular localization patterns of ezrin mutants were also observed in another T cell lymphoma line, BW5147, although the cells bearing a uropod in this line are a minor population and the sizes of the uropods are much smaller than in EL4.G8 cells (unpublished data).


Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation.

Lee JH, Katakai T, Hara T, Gonda H, Sugai M, Shimizu A - J. Cell Biol. (2004)

Intracellular localization of ezrin mutants and the enhancement of uropod size in T567D ezrin transfectants. (A) GFP-tagged ezrin constructs used in this study. In point mutant constructs T567A or T567D, a CT phosphorylation site, Thr567 (T) was replaced by Ala (A) or Asp (D), respectively. (B and C) Intracellular localization of ezrin mutants in EL4.G8 cells. Stable transfectants were examined for the localization of GFP as well as CD44 (B) or F-actin (C). Each set of panels corresponds to the construct shown to the left in A. T567D ezrin was exclusively localized at the uropod (asterisks) and colocalized with the enhanced F-actin signal (C, arrows). Bar, 10 μm. (D) GFP-tagged proteins expressed in stable transfectants were detected by Western blotting using antiezrin or anti-GFP antibody. Polyclonal antiezrin antibody recognizes an epitope in the CT domain and, thus, does not detect the NT fragment. The arrowhead indicates endogenous ezrin. (E) Interaction between CD44 and ezrin mutants was detected by the immunoprecipitation of CD44 and Western blotting for GFP. (F) T567D ezrin increases uropod length. The length of the uropod was measured and expressed as mean ± SD (n > 200). Each bar represents an individual stable clone of a different type of ezrin transfectant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172551&req=5

fig2: Intracellular localization of ezrin mutants and the enhancement of uropod size in T567D ezrin transfectants. (A) GFP-tagged ezrin constructs used in this study. In point mutant constructs T567A or T567D, a CT phosphorylation site, Thr567 (T) was replaced by Ala (A) or Asp (D), respectively. (B and C) Intracellular localization of ezrin mutants in EL4.G8 cells. Stable transfectants were examined for the localization of GFP as well as CD44 (B) or F-actin (C). Each set of panels corresponds to the construct shown to the left in A. T567D ezrin was exclusively localized at the uropod (asterisks) and colocalized with the enhanced F-actin signal (C, arrows). Bar, 10 μm. (D) GFP-tagged proteins expressed in stable transfectants were detected by Western blotting using antiezrin or anti-GFP antibody. Polyclonal antiezrin antibody recognizes an epitope in the CT domain and, thus, does not detect the NT fragment. The arrowhead indicates endogenous ezrin. (E) Interaction between CD44 and ezrin mutants was detected by the immunoprecipitation of CD44 and Western blotting for GFP. (F) T567D ezrin increases uropod length. The length of the uropod was measured and expressed as mean ± SD (n > 200). Each bar represents an individual stable clone of a different type of ezrin transfectant.
Mentions: To address the details of ezrin localization in the uropod, we transfected plasmids expressing various GFP-tagged mutant forms of ezrin into EL4.G8 cells and obtained multiple stable lines (Fig. 2, A and D). Transfection with the control vector expressing GFP resulted in cytoplasmic and nuclear distribution of this reporter protein (Fig. 2, B and C). In contrast, wild-type (WT) ezrin-GFP was preferentially localized at the peripheral membrane, especially in the uropod rather than the cell body, although a weak signal was also detected in the cytoplasm. A mutant ezrin in which the CT Thr was replaced by Ala (T567A) was distributed diffusely in the cytoplasm. This mutant cannot be phosphorylated at this residue and, therefore, does not undergo the intramolecular NT and CT domain dissociation and mimics the dormant form of ezrin (Gautreau et al., 2000; Coscoy et al., 2002). In marked contrast, another mutant with replacement of this Thr by Asp, T567D, which does not undergo NT and CT domain association and thus mimics the phosphorylated active form with membrane–cytoskeleton linker activity (Gautreau et al., 2000; Coscoy et al., 2002), exclusively accumulated in the uropod colocalized with CD44 (Fig. 2 B, asterisks). The fluorescent signal of the NT domain fragment tagged with GFP was also detected at the uropod, as well as at the peripheral membrane of the cell body, confirming the requirement of the NT domain for ezrin to localize in the uropod. In contrast, the CT domain fragment of ezrin was accumulated at the leading edge and was well-colocalized with F-actin, but was not abundant in the uropod, indicating that this fragment freely associates with the actin cytoskeleton. Such intracellular localization patterns of ezrin mutants were also observed in another T cell lymphoma line, BW5147, although the cells bearing a uropod in this line are a minor population and the sizes of the uropods are much smaller than in EL4.G8 cells (unpublished data).

Bottom Line: T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner.Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation.Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.

ABSTRACT
Front-rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner. Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho-ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane "posteriority" in the induction of the uropod in T lymphocytes.

Show MeSH
Related in: MedlinePlus