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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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The C-terminal PBM is necessary for p114RhoGEF to be localized at apical cell–cell boundaries. (top schematic) p114RhoGEF possesses a potential PBM (VIFF) in its C-terminal tail. DLD-1 cells transfected with EGFP-tagged full-length p114RhoGEF (FL), N-terminal portion of p114RhoGEF (N), C-terminal portion of p114RhoGEF (C), or p114RhoGEFΔPBM (FLΔPBM), in which its last six amino acid residues were deleted, were doubly immunostained for EGFP and ZO-1. Fluorescence intensity of the EGFP or ZO-1 signal was scanned across cell–cell boundaries between control and EGFP-expressing cells (left and right of dotted lines, respectively). Five different cell–cell boundaries were measured (shown in different colors). (right) Vertical images are also shown. Although full-length and C-terminal p114RhoGEF accumulate along apical cell–cell boundaries, overlapping ZO-1, N-terminal p114RhoGEF, and FLΔPBM do not. Arrows and arrowheads show cell–cell boundaries marked by ZO-1. Bars, 10 µm.
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fig5: The C-terminal PBM is necessary for p114RhoGEF to be localized at apical cell–cell boundaries. (top schematic) p114RhoGEF possesses a potential PBM (VIFF) in its C-terminal tail. DLD-1 cells transfected with EGFP-tagged full-length p114RhoGEF (FL), N-terminal portion of p114RhoGEF (N), C-terminal portion of p114RhoGEF (C), or p114RhoGEFΔPBM (FLΔPBM), in which its last six amino acid residues were deleted, were doubly immunostained for EGFP and ZO-1. Fluorescence intensity of the EGFP or ZO-1 signal was scanned across cell–cell boundaries between control and EGFP-expressing cells (left and right of dotted lines, respectively). Five different cell–cell boundaries were measured (shown in different colors). (right) Vertical images are also shown. Although full-length and C-terminal p114RhoGEF accumulate along apical cell–cell boundaries, overlapping ZO-1, N-terminal p114RhoGEF, and FLΔPBM do not. Arrows and arrowheads show cell–cell boundaries marked by ZO-1. Bars, 10 µm.

Mentions: Because Lulu2 does not recruit p114RhoGEF to apical cell–cell boundaries, we next investigated the targeting mechanism of p114RhoGEF there. We first roughly mapped the region of p114RhoGEF required for its localization at apical cell–cell boundaries in DLD-1 cells. The full-length and C-terminal portion were recruited to apical cell–cell boundaries marked by ZO-1 staining, whereas the N-terminal portion was not (Fig. 5). We thus considered that a potential PBM in its C-terminal tail might be responsible for its targeting and tested this possibility. It was found that the mutant form of p114RhoGEF that lacks the potential PBM was not recruited to apical cell–cell boundaries (Fig. 5). Therefore, p114RhoGEF might be targeted to apical cell–cell boundaries via PDZ domain–mediated interaction.


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

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

The C-terminal PBM is necessary for p114RhoGEF to be localized at apical cell–cell boundaries. (top schematic) p114RhoGEF possesses a potential PBM (VIFF) in its C-terminal tail. DLD-1 cells transfected with EGFP-tagged full-length p114RhoGEF (FL), N-terminal portion of p114RhoGEF (N), C-terminal portion of p114RhoGEF (C), or p114RhoGEFΔPBM (FLΔPBM), in which its last six amino acid residues were deleted, were doubly immunostained for EGFP and ZO-1. Fluorescence intensity of the EGFP or ZO-1 signal was scanned across cell–cell boundaries between control and EGFP-expressing cells (left and right of dotted lines, respectively). Five different cell–cell boundaries were measured (shown in different colors). (right) Vertical images are also shown. Although full-length and C-terminal p114RhoGEF accumulate along apical cell–cell boundaries, overlapping ZO-1, N-terminal p114RhoGEF, and FLΔPBM do not. Arrows and arrowheads show cell–cell boundaries marked by ZO-1. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: The C-terminal PBM is necessary for p114RhoGEF to be localized at apical cell–cell boundaries. (top schematic) p114RhoGEF possesses a potential PBM (VIFF) in its C-terminal tail. DLD-1 cells transfected with EGFP-tagged full-length p114RhoGEF (FL), N-terminal portion of p114RhoGEF (N), C-terminal portion of p114RhoGEF (C), or p114RhoGEFΔPBM (FLΔPBM), in which its last six amino acid residues were deleted, were doubly immunostained for EGFP and ZO-1. Fluorescence intensity of the EGFP or ZO-1 signal was scanned across cell–cell boundaries between control and EGFP-expressing cells (left and right of dotted lines, respectively). Five different cell–cell boundaries were measured (shown in different colors). (right) Vertical images are also shown. Although full-length and C-terminal p114RhoGEF accumulate along apical cell–cell boundaries, overlapping ZO-1, N-terminal p114RhoGEF, and FLΔPBM do not. Arrows and arrowheads show cell–cell boundaries marked by ZO-1. Bars, 10 µm.
Mentions: Because Lulu2 does not recruit p114RhoGEF to apical cell–cell boundaries, we next investigated the targeting mechanism of p114RhoGEF there. We first roughly mapped the region of p114RhoGEF required for its localization at apical cell–cell boundaries in DLD-1 cells. The full-length and C-terminal portion were recruited to apical cell–cell boundaries marked by ZO-1 staining, whereas the N-terminal portion was not (Fig. 5). We thus considered that a potential PBM in its C-terminal tail might be responsible for its targeting and tested this possibility. It was found that the mutant form of p114RhoGEF that lacks the potential PBM was not recruited to apical cell–cell boundaries (Fig. 5). Therefore, p114RhoGEF might be targeted to apical cell–cell boundaries via PDZ domain–mediated interaction.

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