Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity - PubMed (original) (raw)

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity

Wei Yu et al. EMBO Rep. 2008 Sep.

Abstract

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Loss of RhoA function rescues the phenotype caused by AIIB2 treatment. (A,B) Treatment with AIIB2 decreases Rac1 activity, measured by GST-Pak3-CRIB pull-down assay. (C,D) RhoA activity is assessed by a GST-RBD-Rhotekin pull-down assay. (E) Reduction of RhoA with shRNA in two clones shown by immunoblot. (F) Quantification of cysts with normal polarity in cells transfected with control shRNA or RhoA shRNA in the absence or presence of AIIB2. A total of 100 cysts per condition were counted from three experiments; *P<0.001. (G–L) Representative confocal images. The two selected clones were plated in COLI for 5 days. Normal polarity was determined by gp135 (green) at the luminal surface and by β-catenin (red) at the BL surface; nuclei are shown in blue. Scale bars 20 μm. (M–O) Activation of RhoA causes inverted cysts. (M) Activation of RhoA by CNFy treatment is assessed by GST-Rhotekin pull-down assay. CNFy results in cysts with inverted polarity, which was indicated by gp135 at the peripheral surface (green) and β-catenin (red) at the BL surface (O, compare normal cyst in N); nuclei are shown in blue. a.u., arbitrary units; BL, basolateral; COLI, collagen I; CNFy, Yersinia pseudotuberculosis cytotoxic necrotizing factor; CRIB, Cdc42/Rac interactive binding; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione _S_-transferase; Pak3, p21 activated kinase 3; RBD, Ras binding domain; shRNA, short hairpin RNA.

Figure 2

Figure 2

Depletion of ROCK I rescues AIIB2 polarity. (A) Quantification of cysts with normal polarity in cells treated with Y27632; *P<0.001. (B–E) Representative confocal images of cysts stained with gp135 (green) and β-catenin (red); nuclei are shown in blue. (F–K) Depletion of ROCK I rescues AIIB2-induced phenotype. (F) Reduction of ROCK I expression level by RNAi is confirmed by QRT–PCR. (G) Quantification of cysts with normal polarity in cells transfected with shRNA to ROCK I; _n_=3; *P<0.001. (H–J) Confocal images of representative cysts. Cells infected with lentivirus for control or ROCK I were plated in COLI for 5 days and stained with gp135 (green) and β-catenin (red); nuclei are shown in blue. Scale bars, 20 μm. COLI, collagen I; QRT–PCR, quantitative reverse transcription–PCR; RNAi, RNA interference; ROCK, Rho kinase; shRNA, short hairpin RNA.

Figure 3

Figure 3

Inhibition of myosin activity rescues the phenotype induced by AIIB2 treatment. (A,B) MLC activity was analysed by Western blot of cysts for p-MLC against rabbit p-MLC at Ser 19. GAPDH acts as a loading control. (C,D) Confocal micrographs of p-MLC. (C) In normal cysts, p-MLC is localized mainly at the basal plasma membrane, some p-MLC are in the region of TJ. (D) In AIIB2-treated cysts, p-MLC is mislocalized. (E–J) Inhibition of myosin activity by blebbistatin rescues AIIB2-induced phenotype, as indicated by the apical marker gp135 (green in H) located at the luminal surface, and β-catenin (red in H) located peripherally and at the cell–cell contact (compare F). Note that blebbistatin treatment results in normal distribution of p-MLC in the presence of AIIB2 (J). Scale bars, 20 μm. MLC, myosin light chain; p-MLC, phosphorylation of MLC; TJ, tight junction.

Figure 4

Figure 4

Inhibition of ROCK or myosin II reverts Rac1N17 cysts. MDCK cells were stably transfected with tetracycline-repressible Myc-Rac1N17, which is expressed by removal of doxycycline in (B,D,F,H). (A,B) Immunostaining of Myc (green) confirms expression of exogenous Rac1N17 in (B) compared with negative control in (A). Inverted polarity is indicated by gp135 at the periphery and β-catenin at the cell–cell contact when Rac1N17 is expressed (D), but not in control cysts (C). Inhibition of ROCK by Y27632 (F) or myosin activity by blebbistatin (H) reverts the inverted orientation of polarity. (C–H) gp135 is green, β-catenin is red and nuclei are blue. (I) Quantification of polarity in Rac1N17 cysts by treatment with Y27632 or blebbistatin; *P<0.001. Scale bar, 10 μm. MDCK, Madin–Darby canine kidney cells; ROCK, Rho kinase.

Similar articles

Cited by

References

    1. Arthur WT, Burridge K (2001) RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. Mol Biol Cell 12: 2711–2720 - PMC - PubMed
    1. Cowan CR, Hyman AA (2007) Acto-myosin reorganization and PAR polarity in C. elegans. Development 134: 1035–1043 - PubMed
    1. Durkin ME, Yuan BZ, Zhou X, Zimonjic DB, Lowy DR, Thorgeirsson SS, Popescu NC (2007) DLC-1: a Rho GTPase-activating protein and tumour suppressor. J Cell Mol Med 11: 1185–1207 - PMC - PubMed
    1. Gassama-Diagne A, Yu W, ter Beest M, Martin-Belmonte F, Kierbel A, Engel J, Mostov K (2006) Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells. Nat Cell Biol 8: 963–970 - PubMed
    1. Goldstein B, Macara IG (2007) The PAR proteins: fundamental players in animal cell polarization. Dev Cell 13: 609–622 - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources