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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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A polar cap covers the posterior part of the cell. (A) EL4.G8 cells exhibit a unique cytoplasmic polarity. EL4.G8 cells were stained for CD44 and β-tubulin, F-actin and β-tubulin, β-tubulin and the posterior cytoplasm (C5-ceramide), or GM130 and the posterior cytoplasm (C5-ceramide). Arrowheads and an arrow indicate the positions of the MTOC and the Golgi apparatus, respectively, in each cell. (B) The polar cap is constructed over the posterior part of the cytoplasm (arrows). Y-27632– or staurosporine-treated EL4.G8 cells were stained for CD44 and the posterior cytoplasm. Composite images for β-tubulin/CD44/nucleus are shown (right) together with the MTOC position (arrowheads). (C) The T567D ezrin–associated polar cap covers the posterior cytoplasm in Y-27632– or staurosporine-treated transfectants (arrows). A similar situation is observed in Y-27632 but not in staurosporine treatment in WT ezrin transfectants (arrows). (D) WT and Q63L RhoA show different intracellular localization in the stable transfectants. WT RhoA is distributed diffusely in the cytoplasm, including the leading edge (arrowheads), whereas Q63L RhoA is localized in the posterior cytoplasm beneath the uropod, or beneath the polar cap when the transfectant is treated with Y-27632 (arrows). Similar but less clear localization of WT RhoA in the posterior cytoplasm is also observed in Y-27632-treated WT RhoA transfectants (arrows). (E) Schematic representation of a typical arrangement of cellular structures in an EL4.G8 cell. (F) The T567D ezrin–associated polar cap covers the posterior cytoplasm in X63.653 cells (arrows). Bars (A–D and F), 10 μm.
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fig6: A polar cap covers the posterior part of the cell. (A) EL4.G8 cells exhibit a unique cytoplasmic polarity. EL4.G8 cells were stained for CD44 and β-tubulin, F-actin and β-tubulin, β-tubulin and the posterior cytoplasm (C5-ceramide), or GM130 and the posterior cytoplasm (C5-ceramide). Arrowheads and an arrow indicate the positions of the MTOC and the Golgi apparatus, respectively, in each cell. (B) The polar cap is constructed over the posterior part of the cytoplasm (arrows). Y-27632– or staurosporine-treated EL4.G8 cells were stained for CD44 and the posterior cytoplasm. Composite images for β-tubulin/CD44/nucleus are shown (right) together with the MTOC position (arrowheads). (C) The T567D ezrin–associated polar cap covers the posterior cytoplasm in Y-27632– or staurosporine-treated transfectants (arrows). A similar situation is observed in Y-27632 but not in staurosporine treatment in WT ezrin transfectants (arrows). (D) WT and Q63L RhoA show different intracellular localization in the stable transfectants. WT RhoA is distributed diffusely in the cytoplasm, including the leading edge (arrowheads), whereas Q63L RhoA is localized in the posterior cytoplasm beneath the uropod, or beneath the polar cap when the transfectant is treated with Y-27632 (arrows). Similar but less clear localization of WT RhoA in the posterior cytoplasm is also observed in Y-27632-treated WT RhoA transfectants (arrows). (E) Schematic representation of a typical arrangement of cellular structures in an EL4.G8 cell. (F) The T567D ezrin–associated polar cap covers the posterior cytoplasm in X63.653 cells (arrows). Bars (A–D and F), 10 μm.

Mentions: It is well-established that migrating lymphocytes exhibit unique intracellular polarity, i.e., that the microtubule organizing center (MTOC) and the Golgi apparatus are arranged in the posterior part behind the nucleus, thus revealing a cytoplasmic front–rear axis (Sánchez-Madrid and del Pozo, 1999). MTOC positioning in EL4.G8 cells was rather flexible: 70.5% of cells showed the rear MTOC, whereas in some cells, the MTOC was positioned clearly in the front cytoplasm, near the leading edge (Fig. 6 A). The Golgi apparatus was located in the middle or front part of the cell (in 72.6% of cells; Fig. 6 A), as was revealed by staining with anti-GM130 antibody. These unstable locations of the MTOC or the Golgi apparatus might be caused by the rapid cell cycle in this cell line. Ceramide analogues have been reported to label the Golgi apparatus (Pagano et al., 1991). However, fluorescence-labeled C5-ceramide clearly stained the cytoplasmic components in the rear part of the cell body and the uropod, in addition to the Golgi apparatus, in EL4.G8 cells (Fig. 6 A). In comparison to the MTOC or Golgi positioning, the intracellular arrangement of the front nucleus and the C5-ceramide–labeled rear cytoplasm was obviously stable (93.3% of cells showed this pattern). A similar staining pattern was observed in BW5147 cells bearing a uropod, although the MTOC and the Golgi apparatus in these cells were mostly detected in the rear cytoplasm (>95%; unpublished data). Thus, C5-ceramide was considered to be a useful probe for visualizing the posterior cytoplasm in EL4.G8 cells. Therefore, although they exhibit a slightly different feature of cell polarity from that observed in migrating lymphocytes, EL4.G8 cells show a unique asymmetry along the front–rear axis (Fig. 6 E).


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)

A polar cap covers the posterior part of the cell. (A) EL4.G8 cells exhibit a unique cytoplasmic polarity. EL4.G8 cells were stained for CD44 and β-tubulin, F-actin and β-tubulin, β-tubulin and the posterior cytoplasm (C5-ceramide), or GM130 and the posterior cytoplasm (C5-ceramide). Arrowheads and an arrow indicate the positions of the MTOC and the Golgi apparatus, respectively, in each cell. (B) The polar cap is constructed over the posterior part of the cytoplasm (arrows). Y-27632– or staurosporine-treated EL4.G8 cells were stained for CD44 and the posterior cytoplasm. Composite images for β-tubulin/CD44/nucleus are shown (right) together with the MTOC position (arrowheads). (C) The T567D ezrin–associated polar cap covers the posterior cytoplasm in Y-27632– or staurosporine-treated transfectants (arrows). A similar situation is observed in Y-27632 but not in staurosporine treatment in WT ezrin transfectants (arrows). (D) WT and Q63L RhoA show different intracellular localization in the stable transfectants. WT RhoA is distributed diffusely in the cytoplasm, including the leading edge (arrowheads), whereas Q63L RhoA is localized in the posterior cytoplasm beneath the uropod, or beneath the polar cap when the transfectant is treated with Y-27632 (arrows). Similar but less clear localization of WT RhoA in the posterior cytoplasm is also observed in Y-27632-treated WT RhoA transfectants (arrows). (E) Schematic representation of a typical arrangement of cellular structures in an EL4.G8 cell. (F) The T567D ezrin–associated polar cap covers the posterior cytoplasm in X63.653 cells (arrows). Bars (A–D and F), 10 μm.
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fig6: A polar cap covers the posterior part of the cell. (A) EL4.G8 cells exhibit a unique cytoplasmic polarity. EL4.G8 cells were stained for CD44 and β-tubulin, F-actin and β-tubulin, β-tubulin and the posterior cytoplasm (C5-ceramide), or GM130 and the posterior cytoplasm (C5-ceramide). Arrowheads and an arrow indicate the positions of the MTOC and the Golgi apparatus, respectively, in each cell. (B) The polar cap is constructed over the posterior part of the cytoplasm (arrows). Y-27632– or staurosporine-treated EL4.G8 cells were stained for CD44 and the posterior cytoplasm. Composite images for β-tubulin/CD44/nucleus are shown (right) together with the MTOC position (arrowheads). (C) The T567D ezrin–associated polar cap covers the posterior cytoplasm in Y-27632– or staurosporine-treated transfectants (arrows). A similar situation is observed in Y-27632 but not in staurosporine treatment in WT ezrin transfectants (arrows). (D) WT and Q63L RhoA show different intracellular localization in the stable transfectants. WT RhoA is distributed diffusely in the cytoplasm, including the leading edge (arrowheads), whereas Q63L RhoA is localized in the posterior cytoplasm beneath the uropod, or beneath the polar cap when the transfectant is treated with Y-27632 (arrows). Similar but less clear localization of WT RhoA in the posterior cytoplasm is also observed in Y-27632-treated WT RhoA transfectants (arrows). (E) Schematic representation of a typical arrangement of cellular structures in an EL4.G8 cell. (F) The T567D ezrin–associated polar cap covers the posterior cytoplasm in X63.653 cells (arrows). Bars (A–D and F), 10 μm.
Mentions: It is well-established that migrating lymphocytes exhibit unique intracellular polarity, i.e., that the microtubule organizing center (MTOC) and the Golgi apparatus are arranged in the posterior part behind the nucleus, thus revealing a cytoplasmic front–rear axis (Sánchez-Madrid and del Pozo, 1999). MTOC positioning in EL4.G8 cells was rather flexible: 70.5% of cells showed the rear MTOC, whereas in some cells, the MTOC was positioned clearly in the front cytoplasm, near the leading edge (Fig. 6 A). The Golgi apparatus was located in the middle or front part of the cell (in 72.6% of cells; Fig. 6 A), as was revealed by staining with anti-GM130 antibody. These unstable locations of the MTOC or the Golgi apparatus might be caused by the rapid cell cycle in this cell line. Ceramide analogues have been reported to label the Golgi apparatus (Pagano et al., 1991). However, fluorescence-labeled C5-ceramide clearly stained the cytoplasmic components in the rear part of the cell body and the uropod, in addition to the Golgi apparatus, in EL4.G8 cells (Fig. 6 A). In comparison to the MTOC or Golgi positioning, the intracellular arrangement of the front nucleus and the C5-ceramide–labeled rear cytoplasm was obviously stable (93.3% of cells showed this pattern). A similar staining pattern was observed in BW5147 cells bearing a uropod, although the MTOC and the Golgi apparatus in these cells were mostly detected in the rear cytoplasm (>95%; unpublished data). Thus, C5-ceramide was considered to be a useful probe for visualizing the posterior cytoplasm in EL4.G8 cells. Therefore, although they exhibit a slightly different feature of cell polarity from that observed in migrating lymphocytes, EL4.G8 cells show a unique asymmetry along the front–rear axis (Fig. 6 E).

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