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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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T567D ezrin induces a plasma membrane polar cap in uropod-less cells. (A) T567D ezrin can cause formation of the polar cap in staurosporine-treated (arrows) but not in cytochalasin D–treated EL4.G8 cells. WT, T567D, and NT ezrin transfectants were treated with inhibitors and stained for CD44. Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 130). (C) T567D-ΔAB ezrin lacks the ability to induce polar cap formation in staurosporine-treated EL4.G8 cells. Cells were transiently transfected with constructs for T567D or T567D-ΔAB ezrin, and after 24 h the cells were treated with staurosporine and stained for CD44. In contrast with the results for T567D-ΔAB, clear polar caps are observed in T567D ezrin–transfected cells (arrows). Bar, 10 μm. (D) Percentage of cells exhibiting the polar cap in C (n > 130). (E) T567D ezrin can induce organization of the polar cap in X63.653 myeloma cells (arrows), which are naturally unpolarized with regard to the plasma membrane. X63.653 cells were transiently transfected with constructs for WT, T567D, NT, or T567D-ΔAB ezrin and stained for CD44 24 h after the transfection. Bar, 10 μm. (F) Percentage of cells exhibiting the polar cap in C (n > 120).
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fig3: T567D ezrin induces a plasma membrane polar cap in uropod-less cells. (A) T567D ezrin can cause formation of the polar cap in staurosporine-treated (arrows) but not in cytochalasin D–treated EL4.G8 cells. WT, T567D, and NT ezrin transfectants were treated with inhibitors and stained for CD44. Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 130). (C) T567D-ΔAB ezrin lacks the ability to induce polar cap formation in staurosporine-treated EL4.G8 cells. Cells were transiently transfected with constructs for T567D or T567D-ΔAB ezrin, and after 24 h the cells were treated with staurosporine and stained for CD44. In contrast with the results for T567D-ΔAB, clear polar caps are observed in T567D ezrin–transfected cells (arrows). Bar, 10 μm. (D) Percentage of cells exhibiting the polar cap in C (n > 130). (E) T567D ezrin can induce organization of the polar cap in X63.653 myeloma cells (arrows), which are naturally unpolarized with regard to the plasma membrane. X63.653 cells were transiently transfected with constructs for WT, T567D, NT, or T567D-ΔAB ezrin and stained for CD44 24 h after the transfection. Bar, 10 μm. (F) Percentage of cells exhibiting the polar cap in C (n > 120).

Mentions: As expected, staurosporine treatment disrupted the uropod as well as the polarized distributions of GFP signal and CD44 in WT ezrin transfectants (Fig. 3 A). Surprisingly, in T567D ezrin transfectants, despite the fact that the typical uropod also disappeared upon treatment with staurosporine, the GFP signal and CD44 clearly accumulated at one pole of the cells to form a capped structure (Fig. 3, A [arrows] and B). A similar cap structure was constructed in BW5147 cells transfected with T567D ezrin and treated with staurosporine (unpublished data). These findings strongly suggest that the T567D ezrin can preserve the plasma membrane polarity even in the uropod-abolished cells. Because polar cap formation was not observed in NT ezrin transfectants, this phenomenon requires the phosphorylated CT domain. In addition, cytochalasin D canceled the induction of polar cap formation by T567D ezrin (Fig. 3, A and B), suggesting that F-actin is also required for this event.


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)

T567D ezrin induces a plasma membrane polar cap in uropod-less cells. (A) T567D ezrin can cause formation of the polar cap in staurosporine-treated (arrows) but not in cytochalasin D–treated EL4.G8 cells. WT, T567D, and NT ezrin transfectants were treated with inhibitors and stained for CD44. Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 130). (C) T567D-ΔAB ezrin lacks the ability to induce polar cap formation in staurosporine-treated EL4.G8 cells. Cells were transiently transfected with constructs for T567D or T567D-ΔAB ezrin, and after 24 h the cells were treated with staurosporine and stained for CD44. In contrast with the results for T567D-ΔAB, clear polar caps are observed in T567D ezrin–transfected cells (arrows). Bar, 10 μm. (D) Percentage of cells exhibiting the polar cap in C (n > 130). (E) T567D ezrin can induce organization of the polar cap in X63.653 myeloma cells (arrows), which are naturally unpolarized with regard to the plasma membrane. X63.653 cells were transiently transfected with constructs for WT, T567D, NT, or T567D-ΔAB ezrin and stained for CD44 24 h after the transfection. Bar, 10 μm. (F) Percentage of cells exhibiting the polar cap in C (n > 120).
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fig3: T567D ezrin induces a plasma membrane polar cap in uropod-less cells. (A) T567D ezrin can cause formation of the polar cap in staurosporine-treated (arrows) but not in cytochalasin D–treated EL4.G8 cells. WT, T567D, and NT ezrin transfectants were treated with inhibitors and stained for CD44. Bar, 10 μm. (B) Percentage of cells exhibiting the polar cap in A (n > 130). (C) T567D-ΔAB ezrin lacks the ability to induce polar cap formation in staurosporine-treated EL4.G8 cells. Cells were transiently transfected with constructs for T567D or T567D-ΔAB ezrin, and after 24 h the cells were treated with staurosporine and stained for CD44. In contrast with the results for T567D-ΔAB, clear polar caps are observed in T567D ezrin–transfected cells (arrows). Bar, 10 μm. (D) Percentage of cells exhibiting the polar cap in C (n > 130). (E) T567D ezrin can induce organization of the polar cap in X63.653 myeloma cells (arrows), which are naturally unpolarized with regard to the plasma membrane. X63.653 cells were transiently transfected with constructs for WT, T567D, NT, or T567D-ΔAB ezrin and stained for CD44 24 h after the transfection. Bar, 10 μm. (F) Percentage of cells exhibiting the polar cap in C (n > 120).
Mentions: As expected, staurosporine treatment disrupted the uropod as well as the polarized distributions of GFP signal and CD44 in WT ezrin transfectants (Fig. 3 A). Surprisingly, in T567D ezrin transfectants, despite the fact that the typical uropod also disappeared upon treatment with staurosporine, the GFP signal and CD44 clearly accumulated at one pole of the cells to form a capped structure (Fig. 3, A [arrows] and B). A similar cap structure was constructed in BW5147 cells transfected with T567D ezrin and treated with staurosporine (unpublished data). These findings strongly suggest that the T567D ezrin can preserve the plasma membrane polarity even in the uropod-abolished cells. Because polar cap formation was not observed in NT ezrin transfectants, this phenomenon requires the phosphorylated CT domain. In addition, cytochalasin D canceled the induction of polar cap formation by T567D ezrin (Fig. 3, A and B), suggesting that F-actin is also required for this event.

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