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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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EL4.G8 cells show polarized morphology with a clear uropod that is regulated by Ser/Thr kinases and the actin cytoskeleton. (A) Phase-contrast view showing the hand mirror–shaped morphology of EL4.G8 cells with clear uropods (left, arrows). Aggregation of EL4.G8 cells by their uropods (left). (B–E) Selective localization of the uropod markers (CD44 and CD43) and p-ERM in the EL4.G8 cell uropod, whereas F-actin and LFA-1 are enriched in the leading edge. Fixed, permeabilized cells were stained with fluorescence-labeled probes for cell surface CD44, CD43, or LFA-1, and for intracellular ezrin, p-ERM, or F-actin. (F) Staurosporine and cytochalasin D disrupt uropod structure. Cells were treated with DMSO (cont.), staurosporine (stauro.), or cytochalasin D (cyt.D) and stained for p-ERM and CD44. (G) Staurosporine abolishes ERM phosphorylation and the interaction between CD44 and ezrin in EL4.G8 cells. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or staurosporine-treated cells (top and middle). CD44 ezrin interaction was detected by the immunoprecipitation of CD44 and Western blotting for ezrin (bottom). Bars (A–F), 10 μm.
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fig1: EL4.G8 cells show polarized morphology with a clear uropod that is regulated by Ser/Thr kinases and the actin cytoskeleton. (A) Phase-contrast view showing the hand mirror–shaped morphology of EL4.G8 cells with clear uropods (left, arrows). Aggregation of EL4.G8 cells by their uropods (left). (B–E) Selective localization of the uropod markers (CD44 and CD43) and p-ERM in the EL4.G8 cell uropod, whereas F-actin and LFA-1 are enriched in the leading edge. Fixed, permeabilized cells were stained with fluorescence-labeled probes for cell surface CD44, CD43, or LFA-1, and for intracellular ezrin, p-ERM, or F-actin. (F) Staurosporine and cytochalasin D disrupt uropod structure. Cells were treated with DMSO (cont.), staurosporine (stauro.), or cytochalasin D (cyt.D) and stained for p-ERM and CD44. (G) Staurosporine abolishes ERM phosphorylation and the interaction between CD44 and ezrin in EL4.G8 cells. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or staurosporine-treated cells (top and middle). CD44 ezrin interaction was detected by the immunoprecipitation of CD44 and Western blotting for ezrin (bottom). Bars (A–F), 10 μm.

Mentions: In general, the uropod is induced in a chemoattractant- or adhesion-dependent fashion (Sánchez-Madrid and del Pozo, 1999); thus, it seems relatively unstable. We took advantage of the mouse T lymphoma cell line EL4, a well-known cell line that is often used for investigating T cell functions, because it spontaneously constructs a uropod when grown in nonadhesive culture dishes and is therefore suitable for uropod studies. To obtain cells with clearer and more frequent uropod formation, we isolated a subclone, G8, with these features (∼80% of the G8 cells construct a uropod) from the parental line by limiting dilution (Fig. 1 A, arrows). EL4.G8 cells exhibited a hand mirror–shaped morphology, with a narrow neck segregating the cell body from the spherical uropod protrusion. We often observed that these cells attached to each other by their uropods to form aggregates, an observation that indicated that this structure is somewhat stickier than other parts of the cell (Fig. 1 A, right). Confocal microscopic analysis clearly demonstrated that transmembrane proteins such as CD44 and CD43 were selectively localized at the protruding part (Fig. 1, B and C). In addition, ezrin, one of the ERM proteins, also accumulated in this part (Fig. 1 B). A polyclonal antibody that recognizes the phosphorylated CT Thr residues of all three ERM proteins clearly stained the protruding structure (Fig. 1, C and D), suggesting that the ezrin accumulated in this compartment is the Thr567-phosphorylated form. In contrast, a large fraction of F-actin is accumulated at the cell margin opposite to the protruding structure; thus, this part is likely the leading edge, although a faint signal for actin filaments was also detected at the uropod protrusion (Fig. 1 D). A leukocyte integrin, LFA-1 (αLβ2), was also enriched in the leading edge (Fig. 1 E). Therefore, EL4.G8 cells show typical features of lymphocyte polarization (Sánchez-Madrid and del Pozo, 1999).


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)

EL4.G8 cells show polarized morphology with a clear uropod that is regulated by Ser/Thr kinases and the actin cytoskeleton. (A) Phase-contrast view showing the hand mirror–shaped morphology of EL4.G8 cells with clear uropods (left, arrows). Aggregation of EL4.G8 cells by their uropods (left). (B–E) Selective localization of the uropod markers (CD44 and CD43) and p-ERM in the EL4.G8 cell uropod, whereas F-actin and LFA-1 are enriched in the leading edge. Fixed, permeabilized cells were stained with fluorescence-labeled probes for cell surface CD44, CD43, or LFA-1, and for intracellular ezrin, p-ERM, or F-actin. (F) Staurosporine and cytochalasin D disrupt uropod structure. Cells were treated with DMSO (cont.), staurosporine (stauro.), or cytochalasin D (cyt.D) and stained for p-ERM and CD44. (G) Staurosporine abolishes ERM phosphorylation and the interaction between CD44 and ezrin in EL4.G8 cells. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or staurosporine-treated cells (top and middle). CD44 ezrin interaction was detected by the immunoprecipitation of CD44 and Western blotting for ezrin (bottom). Bars (A–F), 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172551&req=5

fig1: EL4.G8 cells show polarized morphology with a clear uropod that is regulated by Ser/Thr kinases and the actin cytoskeleton. (A) Phase-contrast view showing the hand mirror–shaped morphology of EL4.G8 cells with clear uropods (left, arrows). Aggregation of EL4.G8 cells by their uropods (left). (B–E) Selective localization of the uropod markers (CD44 and CD43) and p-ERM in the EL4.G8 cell uropod, whereas F-actin and LFA-1 are enriched in the leading edge. Fixed, permeabilized cells were stained with fluorescence-labeled probes for cell surface CD44, CD43, or LFA-1, and for intracellular ezrin, p-ERM, or F-actin. (F) Staurosporine and cytochalasin D disrupt uropod structure. Cells were treated with DMSO (cont.), staurosporine (stauro.), or cytochalasin D (cyt.D) and stained for p-ERM and CD44. (G) Staurosporine abolishes ERM phosphorylation and the interaction between CD44 and ezrin in EL4.G8 cells. The total amount of ezrin and the p-ERM level were determined by Western blotting using lysates from control or staurosporine-treated cells (top and middle). CD44 ezrin interaction was detected by the immunoprecipitation of CD44 and Western blotting for ezrin (bottom). Bars (A–F), 10 μm.
Mentions: In general, the uropod is induced in a chemoattractant- or adhesion-dependent fashion (Sánchez-Madrid and del Pozo, 1999); thus, it seems relatively unstable. We took advantage of the mouse T lymphoma cell line EL4, a well-known cell line that is often used for investigating T cell functions, because it spontaneously constructs a uropod when grown in nonadhesive culture dishes and is therefore suitable for uropod studies. To obtain cells with clearer and more frequent uropod formation, we isolated a subclone, G8, with these features (∼80% of the G8 cells construct a uropod) from the parental line by limiting dilution (Fig. 1 A, arrows). EL4.G8 cells exhibited a hand mirror–shaped morphology, with a narrow neck segregating the cell body from the spherical uropod protrusion. We often observed that these cells attached to each other by their uropods to form aggregates, an observation that indicated that this structure is somewhat stickier than other parts of the cell (Fig. 1 A, right). Confocal microscopic analysis clearly demonstrated that transmembrane proteins such as CD44 and CD43 were selectively localized at the protruding part (Fig. 1, B and C). In addition, ezrin, one of the ERM proteins, also accumulated in this part (Fig. 1 B). A polyclonal antibody that recognizes the phosphorylated CT Thr residues of all three ERM proteins clearly stained the protruding structure (Fig. 1, C and D), suggesting that the ezrin accumulated in this compartment is the Thr567-phosphorylated form. In contrast, a large fraction of F-actin is accumulated at the cell margin opposite to the protruding structure; thus, this part is likely the leading edge, although a faint signal for actin filaments was also detected at the uropod protrusion (Fig. 1 D). A leukocyte integrin, LFA-1 (αLβ2), was also enriched in the leading edge (Fig. 1 E). Therefore, EL4.G8 cells show typical features of lymphocyte polarization (Sánchez-Madrid and del Pozo, 1999).

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