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Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF.

Nakajima H, Tanoue T - J. Cell Biol. (2011)

Bottom Line: In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions.This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C.We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Global Centers of Excellence Program for Integrative Membrane Biology, Graduate School of Medicine, Kobe University, Chuo-ku, Kobe 650-0017, Japan.

ABSTRACT
Myosin II-driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell-cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. In this paper, we characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain-containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell-cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells.

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p114RhoGEF is necessary for Lulu2 activity in the cells and is up-regulated by Lulu2 in vitro. (A and B) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or p114RhoGEF siRNA-1. (A) Cells were doubly immunostained for Myc and ZO-1. Note that Lulu2-expressing cells have higher cell heights than parental cells, resulting in the out of focus images of the parental cells. (B) Quantification of relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells. The dotted line marks where the relative apical area is 1. n = 3 independent experiments, in each of which >100 cells were measured. **, P < 0.01 by Student’s t test. (C and D) DLD-1 cells treated with control siRNA or p114RhoGEF siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). (E) Linearity index was quantified as in Fig. 1 E. (F) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA was monitored as an increase in fluorescence, which is indicative of the binding of N-methylanthraniloyl–GTP to small GTPases. p114RhoGEF possesses GEF activity toward RhoA (yellow), which is up-regulated in the presence of Lulu2 (blue). Three independent experiments were quantified. (left) The proteins used are detected by Western blotting (WB) with anti-Flag or anti-Myc antibody or stained with CBB. See Materials and methods for details. Error bars indicate SD. Bars, 20 µm.
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fig4: p114RhoGEF is necessary for Lulu2 activity in the cells and is up-regulated by Lulu2 in vitro. (A and B) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or p114RhoGEF siRNA-1. (A) Cells were doubly immunostained for Myc and ZO-1. Note that Lulu2-expressing cells have higher cell heights than parental cells, resulting in the out of focus images of the parental cells. (B) Quantification of relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells. The dotted line marks where the relative apical area is 1. n = 3 independent experiments, in each of which >100 cells were measured. **, P < 0.01 by Student’s t test. (C and D) DLD-1 cells treated with control siRNA or p114RhoGEF siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). (E) Linearity index was quantified as in Fig. 1 E. (F) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA was monitored as an increase in fluorescence, which is indicative of the binding of N-methylanthraniloyl–GTP to small GTPases. p114RhoGEF possesses GEF activity toward RhoA (yellow), which is up-regulated in the presence of Lulu2 (blue). Three independent experiments were quantified. (left) The proteins used are detected by Western blotting (WB) with anti-Flag or anti-Myc antibody or stained with CBB. See Materials and methods for details. Error bars indicate SD. Bars, 20 µm.

Mentions: To examine whether p114RhoGEF was involved in myosin II regulation by Lulu2, we used Lulu2-expressing MDCK cells, which were shown in the previous study to exhibit strong myosin II–dependent apical constriction when mixed with nonexpressing parental cells (Nakajima and Tanoue, 2010). By RNAi-mediated p114RhoGEF depletion, apical constriction of Lulu2-expressing MDCK cells became impaired, indicating that p114RhoGEF is necessary for myosin II activation by Lulu2 (Fig. 4, A and B; and Fig. S3 E, RNAi). Incomplete inhibition of apical constriction by p114RhoGEF RNAi might be because of incomplete loss of p114RhoGEF in Lulu2-expressing cells by our RNAi. A trace signal of p114RhoGEF staining was still detected after RNAi treatment in Lulu2-expressing cells (unpublished data).


Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF.

Nakajima H, Tanoue T - J. Cell Biol. (2011)

p114RhoGEF is necessary for Lulu2 activity in the cells and is up-regulated by Lulu2 in vitro. (A and B) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or p114RhoGEF siRNA-1. (A) Cells were doubly immunostained for Myc and ZO-1. Note that Lulu2-expressing cells have higher cell heights than parental cells, resulting in the out of focus images of the parental cells. (B) Quantification of relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells. The dotted line marks where the relative apical area is 1. n = 3 independent experiments, in each of which >100 cells were measured. **, P < 0.01 by Student’s t test. (C and D) DLD-1 cells treated with control siRNA or p114RhoGEF siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). (E) Linearity index was quantified as in Fig. 1 E. (F) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA was monitored as an increase in fluorescence, which is indicative of the binding of N-methylanthraniloyl–GTP to small GTPases. p114RhoGEF possesses GEF activity toward RhoA (yellow), which is up-regulated in the presence of Lulu2 (blue). Three independent experiments were quantified. (left) The proteins used are detected by Western blotting (WB) with anti-Flag or anti-Myc antibody or stained with CBB. See Materials and methods for details. Error bars indicate SD. Bars, 20 µm.
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fig4: p114RhoGEF is necessary for Lulu2 activity in the cells and is up-regulated by Lulu2 in vitro. (A and B) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or p114RhoGEF siRNA-1. (A) Cells were doubly immunostained for Myc and ZO-1. Note that Lulu2-expressing cells have higher cell heights than parental cells, resulting in the out of focus images of the parental cells. (B) Quantification of relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells. The dotted line marks where the relative apical area is 1. n = 3 independent experiments, in each of which >100 cells were measured. **, P < 0.01 by Student’s t test. (C and D) DLD-1 cells treated with control siRNA or p114RhoGEF siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). (E) Linearity index was quantified as in Fig. 1 E. (F) In vitro guanine nucleotide exchange reaction of p114RhoGEF toward RhoA was monitored as an increase in fluorescence, which is indicative of the binding of N-methylanthraniloyl–GTP to small GTPases. p114RhoGEF possesses GEF activity toward RhoA (yellow), which is up-regulated in the presence of Lulu2 (blue). Three independent experiments were quantified. (left) The proteins used are detected by Western blotting (WB) with anti-Flag or anti-Myc antibody or stained with CBB. See Materials and methods for details. Error bars indicate SD. Bars, 20 µm.
Mentions: To examine whether p114RhoGEF was involved in myosin II regulation by Lulu2, we used Lulu2-expressing MDCK cells, which were shown in the previous study to exhibit strong myosin II–dependent apical constriction when mixed with nonexpressing parental cells (Nakajima and Tanoue, 2010). By RNAi-mediated p114RhoGEF depletion, apical constriction of Lulu2-expressing MDCK cells became impaired, indicating that p114RhoGEF is necessary for myosin II activation by Lulu2 (Fig. 4, A and B; and Fig. S3 E, RNAi). Incomplete inhibition of apical constriction by p114RhoGEF RNAi might be because of incomplete loss of p114RhoGEF in Lulu2-expressing cells by our RNAi. A trace signal of p114RhoGEF staining was still detected after RNAi treatment in Lulu2-expressing cells (unpublished data).

Bottom Line: In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions.This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C.We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Global Centers of Excellence Program for Integrative Membrane Biology, Graduate School of Medicine, Kobe University, Chuo-ku, Kobe 650-0017, Japan.

ABSTRACT
Myosin II-driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell-cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. In this paper, we characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain-containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell-cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell-cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell-cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain-mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells.

Show MeSH
Related in: MedlinePlus