Limits...
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.

Show MeSH

Related in: MedlinePlus

Lulu2 accumulates along apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) DLD-1 cells doubly immunostained for Lulu2 and ZO-1. Lulu2 accumulates along cell–cell boundaries overlapping ZO-1 (arrows). (B) Vertical images of DLD-1 cells doubly immunostained for Lulu2 and ZO-1. (C and D) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). Arrows show cell–cell boundaries. (E) Quantification of junction linearity. Junction length (blue) and the distance between vertices (red) were measured in Lulu2-depleted cells or control cells. Linearity index is defined by the ratio of junction length to the distance between vertices. Error bars indicate SD. n = 3 independent experiments, in each of which >50 junctions were measured. **, P < 0.001 by Student’s t test. (F) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for Par3 or Patj. (G) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were immunostained for β-catenin. Arrows show zonula adherens. (bottom) Close-up views are also shown. (H) Lulu2 loss results in attenuation of the circumferential actomyosin belt. Bars: (A) 10 µm; (B) 2 µm; (C, D, F, and G) 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3198159&req=5

fig1: Lulu2 accumulates along apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) DLD-1 cells doubly immunostained for Lulu2 and ZO-1. Lulu2 accumulates along cell–cell boundaries overlapping ZO-1 (arrows). (B) Vertical images of DLD-1 cells doubly immunostained for Lulu2 and ZO-1. (C and D) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). Arrows show cell–cell boundaries. (E) Quantification of junction linearity. Junction length (blue) and the distance between vertices (red) were measured in Lulu2-depleted cells or control cells. Linearity index is defined by the ratio of junction length to the distance between vertices. Error bars indicate SD. n = 3 independent experiments, in each of which >50 junctions were measured. **, P < 0.001 by Student’s t test. (F) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for Par3 or Patj. (G) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were immunostained for β-catenin. Arrows show zonula adherens. (bottom) Close-up views are also shown. (H) Lulu2 loss results in attenuation of the circumferential actomyosin belt. Bars: (A) 10 µm; (B) 2 µm; (C, D, F, and G) 20 µm.

Mentions: We began by examining the localization of endogenous Lulu2 in DLD-1 cells, which exhibit the characteristic morphology of polarized epithelial cells with a well-developed circumferential actomyosin belt (Fig. S1), and found that Lulu2 accumulated along apical cell–cell boundaries, overlapping ZO-1, a tight junctional marker (Fig. 1, A and B; and Fig. S2, A–D, for the specificity of the antibody). Lulu2 was also detected in the cytoplasm as dots (Fig. 1 A), which we did not characterize further in this study. In addition, as DLD-1 cells mainly express the short form of Lulu2 (unpublished data), it was used in this study.


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

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

Lulu2 accumulates along apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) DLD-1 cells doubly immunostained for Lulu2 and ZO-1. Lulu2 accumulates along cell–cell boundaries overlapping ZO-1 (arrows). (B) Vertical images of DLD-1 cells doubly immunostained for Lulu2 and ZO-1. (C and D) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). Arrows show cell–cell boundaries. (E) Quantification of junction linearity. Junction length (blue) and the distance between vertices (red) were measured in Lulu2-depleted cells or control cells. Linearity index is defined by the ratio of junction length to the distance between vertices. Error bars indicate SD. n = 3 independent experiments, in each of which >50 junctions were measured. **, P < 0.001 by Student’s t test. (F) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for Par3 or Patj. (G) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were immunostained for β-catenin. Arrows show zonula adherens. (bottom) Close-up views are also shown. (H) Lulu2 loss results in attenuation of the circumferential actomyosin belt. Bars: (A) 10 µm; (B) 2 µm; (C, D, F, and G) 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198159&req=5

fig1: Lulu2 accumulates along apical cell–cell boundaries and regulates the circumferential actomyosin belt. (A) DLD-1 cells doubly immunostained for Lulu2 and ZO-1. Lulu2 accumulates along cell–cell boundaries overlapping ZO-1 (arrows). (B) Vertical images of DLD-1 cells doubly immunostained for Lulu2 and ZO-1. (C and D) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for F-actin, myosin IIA, diphosphorylated MRLC (2P-MRLC; C), or ZO-1 (D). Arrows show cell–cell boundaries. (E) Quantification of junction linearity. Junction length (blue) and the distance between vertices (red) were measured in Lulu2-depleted cells or control cells. Linearity index is defined by the ratio of junction length to the distance between vertices. Error bars indicate SD. n = 3 independent experiments, in each of which >50 junctions were measured. **, P < 0.001 by Student’s t test. (F) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were stained for Par3 or Patj. (G) DLD-1 cells treated with control siRNA or Lulu2 siRNA-1 were immunostained for β-catenin. Arrows show zonula adherens. (bottom) Close-up views are also shown. (H) Lulu2 loss results in attenuation of the circumferential actomyosin belt. Bars: (A) 10 µm; (B) 2 µm; (C, D, F, and G) 20 µm.
Mentions: We began by examining the localization of endogenous Lulu2 in DLD-1 cells, which exhibit the characteristic morphology of polarized epithelial cells with a well-developed circumferential actomyosin belt (Fig. S1), and found that Lulu2 accumulated along apical cell–cell boundaries, overlapping ZO-1, a tight junctional marker (Fig. 1, A and B; and Fig. S2, A–D, for the specificity of the antibody). Lulu2 was also detected in the cytoplasm as dots (Fig. 1 A), which we did not characterize further in this study. In addition, as DLD-1 cells mainly express the short form of Lulu2 (unpublished data), it was used in this study.

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