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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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Lulu2 interacts with p114RhoGEF. (A, top schematic) Lulu2 has a FERM and a FERM adjacent (FA) domain. Amino acid numbers of mouse Lulu2 are indicated. Lysates of DLD-1 cells were examined for GST pull-down assays using GST or GST-FERM-FA. Endogenous p114RhoGEF was detected by immunoblotting. N, N terminus; C, C terminus. (B) Lysates of MDCK cells transfected with Flag-p114RhoGEF were examined for GST pull-down assays using GST, GST-FERM-FA, GST-FERM, or GST-FA (Fig. S4 B). (C) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top) Coprecipitated Flag-p114RhoGEF was detected by immunoblotting with anti-Flag antibody. Comparable amounts of Flag-p114RhoGEF were expressed (Input). (D) Lysates of DLD-1 cells were immunoprecipitated with control rabbit IgG or rabbit anti-Lulu2 antibody. Coprecipitated endogenous p114RhoGEF was detected by immunoblotting with anti-p114RhoGEF antibody. Characterization of the rabbit anti-Lulu2 antibody used is described in Fig. S2 (E and F). (E and F) DLD-1 cells doubly immunostained for Lulu2 and p114RhoGEF. Vertical images are shown in F. p114RhoGEF overlaps Lulu2 (arrows). (G) The in situ proximity ligation assay in DLD-1 cells. The assay was performed using goat anti-Lulu2 and rabbit anti-p114RhoGEF antibodies (Ab). ZO-1 was also stained using mouse anti–ZO-1 antibody to identify cell–cell boundaries. The ligation signals (red) were detected as dots at cell–cell boundaries in the samples incubated with anti-Lulu2 and anti-p114RhoGEF antibodies but scarcely detected in those incubated with anti-Lulu2 antibody and control rabbit IgG or anti-p114RhoGEF antibody and control goat IgG, suggesting that Lulu2 interacts with p114RhoGEF at cell–cell boundaries. Cytoplasmic dots are nonspecific signals in DLD-1 cells. Quantification of numbers of ligation dots at 100 cell–cell boundaries is shown in the right graph. n = 3 independent experiments, in each of which 100 cell–cell boundaries were examined. Error bars indicate SD. **, P < 0.01 by Student’s t test. Bars: (E) 20 µm; (F) 2 µm; (G) 10 µm.
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fig2: Lulu2 interacts with p114RhoGEF. (A, top schematic) Lulu2 has a FERM and a FERM adjacent (FA) domain. Amino acid numbers of mouse Lulu2 are indicated. Lysates of DLD-1 cells were examined for GST pull-down assays using GST or GST-FERM-FA. Endogenous p114RhoGEF was detected by immunoblotting. N, N terminus; C, C terminus. (B) Lysates of MDCK cells transfected with Flag-p114RhoGEF were examined for GST pull-down assays using GST, GST-FERM-FA, GST-FERM, or GST-FA (Fig. S4 B). (C) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top) Coprecipitated Flag-p114RhoGEF was detected by immunoblotting with anti-Flag antibody. Comparable amounts of Flag-p114RhoGEF were expressed (Input). (D) Lysates of DLD-1 cells were immunoprecipitated with control rabbit IgG or rabbit anti-Lulu2 antibody. Coprecipitated endogenous p114RhoGEF was detected by immunoblotting with anti-p114RhoGEF antibody. Characterization of the rabbit anti-Lulu2 antibody used is described in Fig. S2 (E and F). (E and F) DLD-1 cells doubly immunostained for Lulu2 and p114RhoGEF. Vertical images are shown in F. p114RhoGEF overlaps Lulu2 (arrows). (G) The in situ proximity ligation assay in DLD-1 cells. The assay was performed using goat anti-Lulu2 and rabbit anti-p114RhoGEF antibodies (Ab). ZO-1 was also stained using mouse anti–ZO-1 antibody to identify cell–cell boundaries. The ligation signals (red) were detected as dots at cell–cell boundaries in the samples incubated with anti-Lulu2 and anti-p114RhoGEF antibodies but scarcely detected in those incubated with anti-Lulu2 antibody and control rabbit IgG or anti-p114RhoGEF antibody and control goat IgG, suggesting that Lulu2 interacts with p114RhoGEF at cell–cell boundaries. Cytoplasmic dots are nonspecific signals in DLD-1 cells. Quantification of numbers of ligation dots at 100 cell–cell boundaries is shown in the right graph. n = 3 independent experiments, in each of which 100 cell–cell boundaries were examined. Error bars indicate SD. **, P < 0.01 by Student’s t test. Bars: (E) 20 µm; (F) 2 µm; (G) 10 µm.

Mentions: To understand the molecular mechanism downstream of Lulu2, we screened for interacting molecules by a GST pull-down assay using a GST-fused Lulu2 FERM-FA followed by liquid chromatography (LC)-mass spectrometry (MS)/MS analyses (Fig. 2 A, FERM-FA). Adaptin δ, HSP70, ribosomal protein L4, CWC22 splicing factor, heterogeneous nuclear riboprotein U, and p114RhoGEF were identified by MS/MS (unpublished data). Among them, we focused on p114RhoGEF, as it is a Rho-specific Dbl family RhoGEF (Niu et al., 2003; Nagata and Inagaki, 2005; Tsuji et al., 2010; Terry et al., 2011), a good candidate molecule downstream of Lulu2. We first confirmed this MS/MS result by immunoblotting using a specific antibody for p114RhoGEF (Fig. 2 A and Fig. S3, A–D, for the specificity of the antibody). By narrowing down the region of Lulu2 for binding to p114RhoGEF, we identified the FERM domain to be necessary and sufficient for binding (Fig. 2 B and Fig. S4 B). In this study, we mainly used MDCK cells to examine interactions of exogenously expressed molecules because MDCK cells are more efficiently transfected with plasmids than DLD-1 cells, and MDCK cells are expected to have components for Lulu2 to function because Lulu2 induces strong apical constriction in MDCK cells (Nakajima and Tanoue, 2010). The interaction between Myc-tagged full-length Lulu2 and Flag-tagged full-length p114RhoGEF was also detected by a coexpression and coimmunoprecipitation assay (Fig. 2 C). Furthermore, endogenous p114RhoGEF was coimmunoprecipitated with endogenous Lulu2 in DLD-1 cells (Fig. 2 D and Fig. S2, E and F). Recently, it was reported that p114RhoGEF regulates RhoA activity at apical cell–cell junctions in epithelial cells (Terry et al., 2011). We then examined the localization of p114RhoGEF in DLD-1 cells and found it to be well colocalized with Lulu2 along apical cell–cell boundaries (Fig. 2, E and F). We further tested the interaction between endogenous Lulu2 and p114RhoGEF along cell–cell boundaries by an in situ proximity ligation assay. The ligation signals were detected at cell–cell boundaries overlapping ZO-1, suggesting that Lulu2 might interact with p114RhoGEF there (Fig. 2 G). These results combined indicate that Lulu2 might interact with p114RhoGEF along apical cell–cell boundaries in epithelial cells.


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

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

Lulu2 interacts with p114RhoGEF. (A, top schematic) Lulu2 has a FERM and a FERM adjacent (FA) domain. Amino acid numbers of mouse Lulu2 are indicated. Lysates of DLD-1 cells were examined for GST pull-down assays using GST or GST-FERM-FA. Endogenous p114RhoGEF was detected by immunoblotting. N, N terminus; C, C terminus. (B) Lysates of MDCK cells transfected with Flag-p114RhoGEF were examined for GST pull-down assays using GST, GST-FERM-FA, GST-FERM, or GST-FA (Fig. S4 B). (C) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top) Coprecipitated Flag-p114RhoGEF was detected by immunoblotting with anti-Flag antibody. Comparable amounts of Flag-p114RhoGEF were expressed (Input). (D) Lysates of DLD-1 cells were immunoprecipitated with control rabbit IgG or rabbit anti-Lulu2 antibody. Coprecipitated endogenous p114RhoGEF was detected by immunoblotting with anti-p114RhoGEF antibody. Characterization of the rabbit anti-Lulu2 antibody used is described in Fig. S2 (E and F). (E and F) DLD-1 cells doubly immunostained for Lulu2 and p114RhoGEF. Vertical images are shown in F. p114RhoGEF overlaps Lulu2 (arrows). (G) The in situ proximity ligation assay in DLD-1 cells. The assay was performed using goat anti-Lulu2 and rabbit anti-p114RhoGEF antibodies (Ab). ZO-1 was also stained using mouse anti–ZO-1 antibody to identify cell–cell boundaries. The ligation signals (red) were detected as dots at cell–cell boundaries in the samples incubated with anti-Lulu2 and anti-p114RhoGEF antibodies but scarcely detected in those incubated with anti-Lulu2 antibody and control rabbit IgG or anti-p114RhoGEF antibody and control goat IgG, suggesting that Lulu2 interacts with p114RhoGEF at cell–cell boundaries. Cytoplasmic dots are nonspecific signals in DLD-1 cells. Quantification of numbers of ligation dots at 100 cell–cell boundaries is shown in the right graph. n = 3 independent experiments, in each of which 100 cell–cell boundaries were examined. Error bars indicate SD. **, P < 0.01 by Student’s t test. Bars: (E) 20 µm; (F) 2 µm; (G) 10 µm.
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fig2: Lulu2 interacts with p114RhoGEF. (A, top schematic) Lulu2 has a FERM and a FERM adjacent (FA) domain. Amino acid numbers of mouse Lulu2 are indicated. Lysates of DLD-1 cells were examined for GST pull-down assays using GST or GST-FERM-FA. Endogenous p114RhoGEF was detected by immunoblotting. N, N terminus; C, C terminus. (B) Lysates of MDCK cells transfected with Flag-p114RhoGEF were examined for GST pull-down assays using GST, GST-FERM-FA, GST-FERM, or GST-FA (Fig. S4 B). (C) Lysates of MDCK cells cotransfected with the indicated combinations of constructs were immunoprecipitated (IP) with anti-Myc antibody. (top) Coprecipitated Flag-p114RhoGEF was detected by immunoblotting with anti-Flag antibody. Comparable amounts of Flag-p114RhoGEF were expressed (Input). (D) Lysates of DLD-1 cells were immunoprecipitated with control rabbit IgG or rabbit anti-Lulu2 antibody. Coprecipitated endogenous p114RhoGEF was detected by immunoblotting with anti-p114RhoGEF antibody. Characterization of the rabbit anti-Lulu2 antibody used is described in Fig. S2 (E and F). (E and F) DLD-1 cells doubly immunostained for Lulu2 and p114RhoGEF. Vertical images are shown in F. p114RhoGEF overlaps Lulu2 (arrows). (G) The in situ proximity ligation assay in DLD-1 cells. The assay was performed using goat anti-Lulu2 and rabbit anti-p114RhoGEF antibodies (Ab). ZO-1 was also stained using mouse anti–ZO-1 antibody to identify cell–cell boundaries. The ligation signals (red) were detected as dots at cell–cell boundaries in the samples incubated with anti-Lulu2 and anti-p114RhoGEF antibodies but scarcely detected in those incubated with anti-Lulu2 antibody and control rabbit IgG or anti-p114RhoGEF antibody and control goat IgG, suggesting that Lulu2 interacts with p114RhoGEF at cell–cell boundaries. Cytoplasmic dots are nonspecific signals in DLD-1 cells. Quantification of numbers of ligation dots at 100 cell–cell boundaries is shown in the right graph. n = 3 independent experiments, in each of which 100 cell–cell boundaries were examined. Error bars indicate SD. **, P < 0.01 by Student’s t test. Bars: (E) 20 µm; (F) 2 µm; (G) 10 µm.
Mentions: To understand the molecular mechanism downstream of Lulu2, we screened for interacting molecules by a GST pull-down assay using a GST-fused Lulu2 FERM-FA followed by liquid chromatography (LC)-mass spectrometry (MS)/MS analyses (Fig. 2 A, FERM-FA). Adaptin δ, HSP70, ribosomal protein L4, CWC22 splicing factor, heterogeneous nuclear riboprotein U, and p114RhoGEF were identified by MS/MS (unpublished data). Among them, we focused on p114RhoGEF, as it is a Rho-specific Dbl family RhoGEF (Niu et al., 2003; Nagata and Inagaki, 2005; Tsuji et al., 2010; Terry et al., 2011), a good candidate molecule downstream of Lulu2. We first confirmed this MS/MS result by immunoblotting using a specific antibody for p114RhoGEF (Fig. 2 A and Fig. S3, A–D, for the specificity of the antibody). By narrowing down the region of Lulu2 for binding to p114RhoGEF, we identified the FERM domain to be necessary and sufficient for binding (Fig. 2 B and Fig. S4 B). In this study, we mainly used MDCK cells to examine interactions of exogenously expressed molecules because MDCK cells are more efficiently transfected with plasmids than DLD-1 cells, and MDCK cells are expected to have components for Lulu2 to function because Lulu2 induces strong apical constriction in MDCK cells (Nakajima and Tanoue, 2010). The interaction between Myc-tagged full-length Lulu2 and Flag-tagged full-length p114RhoGEF was also detected by a coexpression and coimmunoprecipitation assay (Fig. 2 C). Furthermore, endogenous p114RhoGEF was coimmunoprecipitated with endogenous Lulu2 in DLD-1 cells (Fig. 2 D and Fig. S2, E and F). Recently, it was reported that p114RhoGEF regulates RhoA activity at apical cell–cell junctions in epithelial cells (Terry et al., 2011). We then examined the localization of p114RhoGEF in DLD-1 cells and found it to be well colocalized with Lulu2 along apical cell–cell boundaries (Fig. 2, E and F). We further tested the interaction between endogenous Lulu2 and p114RhoGEF along cell–cell boundaries by an in situ proximity ligation assay. The ligation signals were detected at cell–cell boundaries overlapping ZO-1, suggesting that Lulu2 might interact with p114RhoGEF there (Fig. 2 G). These results combined indicate that Lulu2 might interact with p114RhoGEF along apical cell–cell boundaries in epithelial cells.

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