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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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Patj is necessary for p114RhoGEF to be recruited to apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) GST pull-down was performed with GST, GST-p114RhoGEF C2, or GST-p114RhoGEF C2ΔPBM (Fig. 3 A, C2). Myc-tagged Patj and Par3 expressed in MDCK cells were examined. (B) DLD-1 cells treated with control siRNA, Patj, or Par3 siRNA-1 were doubly immunostained for p114RhoGEF and ZO-1. (C) DLD-1 cells treated with control siRNA or Patj siRNA-1 were doubly immunostained for Par3 and ZO-1. (D) DLD-1 cells treated with control siRNA or Par3 siRNA-1 were doubly immunostained for Patj and ZO-1. (E) DLD-1 cells treated with control siRNA or Patj siRNA-1 were analyzed by immunoblotting with anti-p114RhoGEF antibody. (F) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top, GFP) Coimmunoprecipitated EGFP-p114RhoGEF was detected. Comparable amounts of EGFP-p114RhoGEF or EGFP-p114RhoGEFΔPBM were expressed (Input). (G) Lysates of DLD-1 cells were immunoprecipitated with anti-Patj antibody. Endogenous p114RhoGEF was detected. (H and I) DLD-1 cells treated with control siRNA or Patj siRNA-1 were stained for cingulin (H), F-actin, or myosin IIA (I). (J) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or Patj siRNA-1. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. n = 3 independent experiments, in each of which >100 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars: (B, H, and I) 20 µm; (C and D) 10 µm.
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fig6: Patj is necessary for p114RhoGEF to be recruited to apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) GST pull-down was performed with GST, GST-p114RhoGEF C2, or GST-p114RhoGEF C2ΔPBM (Fig. 3 A, C2). Myc-tagged Patj and Par3 expressed in MDCK cells were examined. (B) DLD-1 cells treated with control siRNA, Patj, or Par3 siRNA-1 were doubly immunostained for p114RhoGEF and ZO-1. (C) DLD-1 cells treated with control siRNA or Patj siRNA-1 were doubly immunostained for Par3 and ZO-1. (D) DLD-1 cells treated with control siRNA or Par3 siRNA-1 were doubly immunostained for Patj and ZO-1. (E) DLD-1 cells treated with control siRNA or Patj siRNA-1 were analyzed by immunoblotting with anti-p114RhoGEF antibody. (F) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top, GFP) Coimmunoprecipitated EGFP-p114RhoGEF was detected. Comparable amounts of EGFP-p114RhoGEF or EGFP-p114RhoGEFΔPBM were expressed (Input). (G) Lysates of DLD-1 cells were immunoprecipitated with anti-Patj antibody. Endogenous p114RhoGEF was detected. (H and I) DLD-1 cells treated with control siRNA or Patj siRNA-1 were stained for cingulin (H), F-actin, or myosin IIA (I). (J) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or Patj siRNA-1. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. n = 3 independent experiments, in each of which >100 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars: (B, H, and I) 20 µm; (C and D) 10 µm.

Mentions: To identify the molecule targeting p114RhoGEF, we tested several PDZ domain–containing molecules known to be localized at cell–cell boundaries for binding to p114RhoGEF by GST pull-down assays using the C2 fragment of p114RhoGEF. Among them, it was found that Patj and Par3, well-known apical cell polarity regulators (Suzuki and Ohno, 2006; Goldstein and Macara, 2007; Assémat et al., 2008; Martin-Belmonte and Mostov, 2008; Pieczynski and Margolis, 2011), bound to the C2 fragment (Fig. S4 A). The former has 10 PDZ domains, and the latter has three PDZ domains. Both Myc-tagged Patj and Par3 expressed in the cells efficiently bound to the C2 fragment of p114RhoGEF, and these interactions depended on the putative PBM of p114RhoGEF (Fig. 6 A and Fig. S4 B). To know which molecule actually targets p114RhoGEF in the cells, we examined the relationship among p114RhoGEF, Patj, and Par3 in terms of their localization. As previously reported in other cell types (Suzuki et al., 2001; Lemmers et al., 2002; Shin et al., 2005; Adachi et al., 2009), in DLD-1 cells, both Patj and Par3 overlap ZO-1 (unpublished data). In Patj-depleted cells, continuous ZO-1 staining was not affected, whereas it became partially fragmented in Par3-depleted cells (Fig. 6 B and Fig. S3, F, G, I, and J, RNAi). In both Par3- and Patj-depleted cells, p114RhoGEF mostly disappeared from cell–cell boundaries marked by ZO-1 staining (Fig. 6 B). In Patj-depleted cells, Par3 localization was not altered: Par3 still overlaps ZO-1 (Fig. 6 C). However, in Par3-depleted cells, Patj disappeared from cell–cell boundaries where ZO-1 accumulated (Fig. 6 D). These results suggest that Par3 is upstream of Patj, and Patj is upstream of p114RhoGEF in terms of accumulation at cell–cell boundaries. In addition, the total expression level of p114RhoGEF protein was not affected by Patj depletion (Fig. 6 E), indicating that the delocalization of p114RhoGEF in Patj-depleted cells is not caused by down-regulation of the total protein level of p114RhoGEF. The interaction between GFP-tagged full-length p114RhoGEF and Myc-tagged full-length Patj was detected in a coimmunoprecipitation assay (Fig. 6 F). This interaction, as expected, required the C-terminal PBM of p114RhoGEF (Fig. 6 F). Furthermore, endogenous p114RhoGEF was coimmunoprecipitated with endogenous Patj (Fig. 6 G). In addition, cingulin, which was recently reported to bind to the PH domain of p114RhoGEF and to be necessary for p114RhoGEF localization along cell–cell boundaries (Terry et al., 2011), remained localized there in Patj-depleted cells (Fig. 6 H), suggesting that cingulin might not mainly serve as a targeting molecule of p114RhoGEF.


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

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

Patj is necessary for p114RhoGEF to be recruited to apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) GST pull-down was performed with GST, GST-p114RhoGEF C2, or GST-p114RhoGEF C2ΔPBM (Fig. 3 A, C2). Myc-tagged Patj and Par3 expressed in MDCK cells were examined. (B) DLD-1 cells treated with control siRNA, Patj, or Par3 siRNA-1 were doubly immunostained for p114RhoGEF and ZO-1. (C) DLD-1 cells treated with control siRNA or Patj siRNA-1 were doubly immunostained for Par3 and ZO-1. (D) DLD-1 cells treated with control siRNA or Par3 siRNA-1 were doubly immunostained for Patj and ZO-1. (E) DLD-1 cells treated with control siRNA or Patj siRNA-1 were analyzed by immunoblotting with anti-p114RhoGEF antibody. (F) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top, GFP) Coimmunoprecipitated EGFP-p114RhoGEF was detected. Comparable amounts of EGFP-p114RhoGEF or EGFP-p114RhoGEFΔPBM were expressed (Input). (G) Lysates of DLD-1 cells were immunoprecipitated with anti-Patj antibody. Endogenous p114RhoGEF was detected. (H and I) DLD-1 cells treated with control siRNA or Patj siRNA-1 were stained for cingulin (H), F-actin, or myosin IIA (I). (J) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or Patj siRNA-1. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. n = 3 independent experiments, in each of which >100 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars: (B, H, and I) 20 µm; (C and D) 10 µm.
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fig6: Patj is necessary for p114RhoGEF to be recruited to apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) GST pull-down was performed with GST, GST-p114RhoGEF C2, or GST-p114RhoGEF C2ΔPBM (Fig. 3 A, C2). Myc-tagged Patj and Par3 expressed in MDCK cells were examined. (B) DLD-1 cells treated with control siRNA, Patj, or Par3 siRNA-1 were doubly immunostained for p114RhoGEF and ZO-1. (C) DLD-1 cells treated with control siRNA or Patj siRNA-1 were doubly immunostained for Par3 and ZO-1. (D) DLD-1 cells treated with control siRNA or Par3 siRNA-1 were doubly immunostained for Patj and ZO-1. (E) DLD-1 cells treated with control siRNA or Patj siRNA-1 were analyzed by immunoblotting with anti-p114RhoGEF antibody. (F) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top, GFP) Coimmunoprecipitated EGFP-p114RhoGEF was detected. Comparable amounts of EGFP-p114RhoGEF or EGFP-p114RhoGEFΔPBM were expressed (Input). (G) Lysates of DLD-1 cells were immunoprecipitated with anti-Patj antibody. Endogenous p114RhoGEF was detected. (H and I) DLD-1 cells treated with control siRNA or Patj siRNA-1 were stained for cingulin (H), F-actin, or myosin IIA (I). (J) Mixed cell cultures of parental and Myc-Lulu2–expressing MDCK cells were treated with control siRNA or Patj siRNA-1. Relative apical areas in Lulu2-expressing cells normalized by those in neighboring cells were quantified as in Fig. 4 B. n = 3 independent experiments, in each of which >100 cells were measured. Error bars indicate SD. *, P < 0.05 by Student’s t test. Bars: (B, H, and I) 20 µm; (C and D) 10 µm.
Mentions: To identify the molecule targeting p114RhoGEF, we tested several PDZ domain–containing molecules known to be localized at cell–cell boundaries for binding to p114RhoGEF by GST pull-down assays using the C2 fragment of p114RhoGEF. Among them, it was found that Patj and Par3, well-known apical cell polarity regulators (Suzuki and Ohno, 2006; Goldstein and Macara, 2007; Assémat et al., 2008; Martin-Belmonte and Mostov, 2008; Pieczynski and Margolis, 2011), bound to the C2 fragment (Fig. S4 A). The former has 10 PDZ domains, and the latter has three PDZ domains. Both Myc-tagged Patj and Par3 expressed in the cells efficiently bound to the C2 fragment of p114RhoGEF, and these interactions depended on the putative PBM of p114RhoGEF (Fig. 6 A and Fig. S4 B). To know which molecule actually targets p114RhoGEF in the cells, we examined the relationship among p114RhoGEF, Patj, and Par3 in terms of their localization. As previously reported in other cell types (Suzuki et al., 2001; Lemmers et al., 2002; Shin et al., 2005; Adachi et al., 2009), in DLD-1 cells, both Patj and Par3 overlap ZO-1 (unpublished data). In Patj-depleted cells, continuous ZO-1 staining was not affected, whereas it became partially fragmented in Par3-depleted cells (Fig. 6 B and Fig. S3, F, G, I, and J, RNAi). In both Par3- and Patj-depleted cells, p114RhoGEF mostly disappeared from cell–cell boundaries marked by ZO-1 staining (Fig. 6 B). In Patj-depleted cells, Par3 localization was not altered: Par3 still overlaps ZO-1 (Fig. 6 C). However, in Par3-depleted cells, Patj disappeared from cell–cell boundaries where ZO-1 accumulated (Fig. 6 D). These results suggest that Par3 is upstream of Patj, and Patj is upstream of p114RhoGEF in terms of accumulation at cell–cell boundaries. In addition, the total expression level of p114RhoGEF protein was not affected by Patj depletion (Fig. 6 E), indicating that the delocalization of p114RhoGEF in Patj-depleted cells is not caused by down-regulation of the total protein level of p114RhoGEF. The interaction between GFP-tagged full-length p114RhoGEF and Myc-tagged full-length Patj was detected in a coimmunoprecipitation assay (Fig. 6 F). This interaction, as expected, required the C-terminal PBM of p114RhoGEF (Fig. 6 F). Furthermore, endogenous p114RhoGEF was coimmunoprecipitated with endogenous Patj (Fig. 6 G). In addition, cingulin, which was recently reported to bind to the PH domain of p114RhoGEF and to be necessary for p114RhoGEF localization along cell–cell boundaries (Terry et al., 2011), remained localized there in Patj-depleted cells (Fig. 6 H), suggesting that cingulin might not mainly serve as a targeting molecule of p114RhoGEF.

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