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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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Chemotactic migration of EL4.G8 cells toward SDF-1 and augmentation of the activity in T567D ezrin transfectants. Chemotactic activity was determined by constant-period counting using a flow cytometer and is shown as mean ± SD (A–C). (A) EL4.G8 cells exhibit typical chemotaxis toward SDF-1 (10 ng/ml), and this chemotaxis is markedly inhibited by pretreatment of the cells with pertussis toxin (PTx), staurosporine, or cytochalasin D. (B) EL4.G8 cell chemotaxis is dependent on ROCK activity. Cells were pretreated with Y-27632 and the chemotaxis assay was performed in medium containing the drug (in) or in medium only (wash out [w/o]). (C) T567D ezrin enhances chemotaxis. Transfectants preincubated with or without PTx were examined by the chemotaxis assay. Numbers represent the fold increase of chemotaxis toward SDF-1 (specific activity) compared with that of cells incubated in medium alone. A result typical of at least three experiments is shown.
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fig7: Chemotactic migration of EL4.G8 cells toward SDF-1 and augmentation of the activity in T567D ezrin transfectants. Chemotactic activity was determined by constant-period counting using a flow cytometer and is shown as mean ± SD (A–C). (A) EL4.G8 cells exhibit typical chemotaxis toward SDF-1 (10 ng/ml), and this chemotaxis is markedly inhibited by pretreatment of the cells with pertussis toxin (PTx), staurosporine, or cytochalasin D. (B) EL4.G8 cell chemotaxis is dependent on ROCK activity. Cells were pretreated with Y-27632 and the chemotaxis assay was performed in medium containing the drug (in) or in medium only (wash out [w/o]). (C) T567D ezrin enhances chemotaxis. Transfectants preincubated with or without PTx were examined by the chemotaxis assay. Numbers represent the fold increase of chemotaxis toward SDF-1 (specific activity) compared with that of cells incubated in medium alone. A result typical of at least three experiments is shown.

Mentions: In general, the uropod is observed during lymphocyte migration (Sánchez-Madrid and del Pozo, 1999), suggesting that this structure might be important for efficient lymphocyte motility. EL4.G8 cells exhibited clear chemotactic migration toward a chemokine, stromal cell–derived factor-1 (SDF-1), and this chemotaxis was prevented by pertussis toxin, which inactivates Gαi-coupled chemokine receptor signaling, as well as by staurosporine and cytochalasin D (Fig. 7 A). Y-27632 also inhibited the chemotaxis, although withdrawal of the drug restored the activity (Fig. 7 B), indicating that the effects of Y-27632 are reversible. Therefore, ROCK activity is required for the proper chemotactic migration of EL4.G8 cells. Interestingly, among the transfectants of various ezrin mutants, only T567D ezrin transfectants showed higher chemotactic activity toward SDF-1, whereas other transfectants had activity comparable to that of the control cells (Fig. 7 C). We confirmed similar enhancement of chemotaxis in other transfectant clones for T567D ezrin. Together, these findings support the notion that ezrin phosphorylation and Rho–ROCK signaling regulate the efficiency of lymphocyte migration.


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)

Chemotactic migration of EL4.G8 cells toward SDF-1 and augmentation of the activity in T567D ezrin transfectants. Chemotactic activity was determined by constant-period counting using a flow cytometer and is shown as mean ± SD (A–C). (A) EL4.G8 cells exhibit typical chemotaxis toward SDF-1 (10 ng/ml), and this chemotaxis is markedly inhibited by pretreatment of the cells with pertussis toxin (PTx), staurosporine, or cytochalasin D. (B) EL4.G8 cell chemotaxis is dependent on ROCK activity. Cells were pretreated with Y-27632 and the chemotaxis assay was performed in medium containing the drug (in) or in medium only (wash out [w/o]). (C) T567D ezrin enhances chemotaxis. Transfectants preincubated with or without PTx were examined by the chemotaxis assay. Numbers represent the fold increase of chemotaxis toward SDF-1 (specific activity) compared with that of cells incubated in medium alone. A result typical of at least three experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172551&req=5

fig7: Chemotactic migration of EL4.G8 cells toward SDF-1 and augmentation of the activity in T567D ezrin transfectants. Chemotactic activity was determined by constant-period counting using a flow cytometer and is shown as mean ± SD (A–C). (A) EL4.G8 cells exhibit typical chemotaxis toward SDF-1 (10 ng/ml), and this chemotaxis is markedly inhibited by pretreatment of the cells with pertussis toxin (PTx), staurosporine, or cytochalasin D. (B) EL4.G8 cell chemotaxis is dependent on ROCK activity. Cells were pretreated with Y-27632 and the chemotaxis assay was performed in medium containing the drug (in) or in medium only (wash out [w/o]). (C) T567D ezrin enhances chemotaxis. Transfectants preincubated with or without PTx were examined by the chemotaxis assay. Numbers represent the fold increase of chemotaxis toward SDF-1 (specific activity) compared with that of cells incubated in medium alone. A result typical of at least three experiments is shown.
Mentions: In general, the uropod is observed during lymphocyte migration (Sánchez-Madrid and del Pozo, 1999), suggesting that this structure might be important for efficient lymphocyte motility. EL4.G8 cells exhibited clear chemotactic migration toward a chemokine, stromal cell–derived factor-1 (SDF-1), and this chemotaxis was prevented by pertussis toxin, which inactivates Gαi-coupled chemokine receptor signaling, as well as by staurosporine and cytochalasin D (Fig. 7 A). Y-27632 also inhibited the chemotaxis, although withdrawal of the drug restored the activity (Fig. 7 B), indicating that the effects of Y-27632 are reversible. Therefore, ROCK activity is required for the proper chemotactic migration of EL4.G8 cells. Interestingly, among the transfectants of various ezrin mutants, only T567D ezrin transfectants showed higher chemotactic activity toward SDF-1, whereas other transfectants had activity comparable to that of the control cells (Fig. 7 C). We confirmed similar enhancement of chemotaxis in other transfectant clones for T567D ezrin. Together, these findings support the notion that ezrin phosphorylation and Rho–ROCK signaling regulate the efficiency of lymphocyte migration.

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