The transforming Rho family GTPase Wrch-1 disrupts epithelial cell tight junctions and epithelial morphogenesis

Abstract

Wrch-1, an atypical and transforming Rho GTPase, regulates cellular activities including proliferation and actin organization, but its functions and effectors remain poorly characterized. We show here that Wrch-1 distributes along the apical and basolateral membranes in MDCK cells and binds the cell polarity protein Par6 in a GTP-dependent manner. Activated Wrch-1 negatively regulates the kinetics of tight junction (TJ) assembly during epithelial cell polarization but has no detectable effect on overall cell polarity in confluent monolayers. It also causes a dramatic cytoskeletal reorganization and multilayering in cells grown in two-dimensional culture and disrupts cystogenesis of cells grown in three-dimensional (3D) culture. Similarly, short hairpin RNA-mediated knockdown of Wrch-1 perturbs cystogenesis in 3D culture, suggesting that tight regulation of Wrch-1 activity is necessary for normal epithelial morphogenesis. A weakly transforming effector domain mutant of activated Wrch-1 that inhibits Par6 binding abrogates the ability of Wrch-1 to disrupt TJ formation, actin organization, and epithelial morphogenesis. We hypothesize that Wrch-1-induced morphological and growth transformation may occur in part through Par6-mediated disruption of TJs and actin organization.

FIG. 1.

GTP-bound Wrch-1 interacts with Par6 in vitro and in vivo. (A) COS-7 cell lysates expressing empty vector or HA-tagged Wrch-1 constructs were incubated with GST alone or GST-Par6, and the presence of Wrch-1 in the pulldown was probed by Western blot (immunoblot [IB]) analysis with anti-HA antibody. Pulldown of GST-Par6 was confirmed with anti-GST antibody. (B) COS-7 cells were cotransfected with empty vector or HA-tagged Wrch-1 along with Myc-tagged Par6. Par6 was immunoprecipitated (IP) with anti-Myc antibody, and immunoprecipitates were probed for Wrch-1 by immunoblotting with anti-HA antibody.

FIG. 2.

Wrch-1 displays overlapping localization with the cell polarity protein Par3 in polarized MDCKII cells. Cells stably expressing empty vector or HA-tagged Wrch-1 were grown to confluence on 12-mm Transwell filters and then fixed and stained using primary antibodies against the HA epitope tag alone (cyan) (A) or either the HA epitope tag (cyan) or Par3 (magenta) (B). IF staining was visualized using a confocal microscope. Square panels, xy sections; rectangular panels, xz sections (top to bottom). Scale bars, 20 μm.

FIG. 3.

Wrch-1 localizes to cell junctions in polarized MDCK cells. Cells were grown to confluence as described above and then fixed and probed for the HA epitope tag (cyan) and either occludin (magenta) (A), ZO-1 (magenta) (B), β-catenin (magenta) (C), or E-cadherin (magenta) (D). Square panels, xy sections; rectangular panels, xz sections (top to bottom). Scale bars, 20 μm.

FIG. 4.

Activated Wrch-1 disrupts TJ formation and integrity during cell polarization. (A) Equivalent expression in MDCKII cells of indicated HA-tagged Wrch-1 constructs and H-Ras(Q61L) shown by immunoblot (IB) with anti-HA and anti-Ras antibodies. The loading control was anti-β-actin. (B) MDCKII cells stably expressing empty vector or HA-tagged Wrch-1 were grown on 12-mm Transwell filters and subjected to calcium switch. Cells were fixed at the indicated intervals and stained using primary antibodies against the HA epitope tag (cyan) and ZO-1 (magenta). Square panels, xy sections; rectangular panels, xz sections (top to bottom). Scale bars, 20 μm. (C) Cells were subjected to calcium switch as in panel B. At indicated time points after calcium readdition, TER (Ω × cm²) was measured using an epithelial volt-ohmmeter. (D) Cells were subjected to calcium switch as in panel B. Paracellular flux of 4,000-kDa FITC-dextran (FD-4) or 40,000-kDa FITC-dextran (FD-40) tracer was examined 6 h and 18 h after calcium switch. RFU, relative fluorescence units. Bar graphs represent an average of three independent experiments carried out in triplicate for each cell line ± standard error of the mean. Significant P values of <0.01 or <0.001 are indicated by * or **, respectively. Tukey's multiple comparison test was used to determine significance between cell lines.

FIG. 5.

Activated Wrch-1 disrupts actin organization and cell morphology of MDCKII cells. (A) MDCKII cells stably expressing empty vector or the indicated HA-tagged Wrch-1 constructs were subjected to calcium switch and treated as in Fig. 4B, including probing for the HA epitope tag (cyan) or with Texas Red-phalloidin (magenta). Asterisks represent actin contractile rings and sites of cell-cell contact. (B) Cells as in panel A were grown to confluence on 12-mm Transwell filters and then fixed, stained, probed for the HA epitope tag (cyan) or with Texas Red-phalloidin (red), and visualized by confocal microscopy. Square panels, xy sections; rectangular panels, xz sections (top to bottom). Scale bars, 20 μm.

FIG. 6.

An effector domain mutation in Wrch-1 abrogates Par6-binding and its ability to disrupt TJ formation and actin organization during cell polarization. (A) Lysates from COS-7 cells transiently expressing the indicated constructs including the EDM Wrch-1(Q107L F86C) were subjected to GST pulldowns as in Fig. 1A. IB, immublotting. (B) Lysates from COS-7 cells expressing the indicated constructs were subjected to coimmunoprecipitation and immunoblotting as in Fig. 1B. (C) Stable expression in MDCKII cells of HA-tagged Wrch-1(Q107L F86C) was detected by immunoblotting with anti-HA antibody. Anti-β-actin was used as a loading control. (D) MDCKII cells stably expressing empty vector or Wrch-1(Q107L F86C) were subjected to calcium switch, and the localization of Wrch-1 (cyan) and ZO-1 (magenta) was evaluated as in Fig. 4B. Square panels, xy sections; rectangular panels, xz sections (top to bottom). Scale bars, 20 μm. (E) TER (Ω × cm²) was measured in cells subjected to calcium switch, at indicated time points after calcium readdition. (F) Actin organization was evaluated in cells expressing vector only or EDM Wrch-1 and subjected to calcium switch. Cells were fixed at indicated intervals after readdition of calcium and stained using either anti-HA antibody (cyan) or Texas Red-phalloidin (magenta). Asterisks represent actin contractile rings and sites of cell-cell contact.

FIG. 7.

Activated Wrch-1 disrupts epithelial morphogenesis and promotes anchorage-independent growth of MDCKII cells. (A) MDCKII cells stably expressing empty vector or indicated HA-tagged Wrch-1 constructs were grown in collagen I gels. After 10 days, collagen gels were fixed and stained for ZO-1 (cyan) and E-cadherin (magenta) (top panels) to examine epithelial cell polarity or with Texas Red-phalloidin (magenta) (bottom panel) to examine epithelial morphogenesis and lumen formation. Scale bars, 20 μm. Cysts containing a single lumen were quantified by counting cysts with single luminal area as positive and cysts with no lumen as negative. Bar graphs represent an average of three independent experiments carried out in triplicate for each cell line ± the standard error of the mean (SEM). Tukey's multiple comparison test was used to determine significance between cell lines. Significant P values of <0.001 are indicated by **. (B) Anchorage-independent growth. MDCKII cells were seeded into soft agar and analyzed for their ability to induce colony formation. Colonies formed after 14 days were stained and scanned, and the numbers of small (6 to 15 cells across) and large (>15 cells across) colonies were quantified. Images and bar graphs are representative of three separate experiments carried out in triplicate; shown are averages ± SEM. Significant P values of <0.001, obtained by using Tukey's multiple comparison test, are indicated by **.

FIG. 8.

Effector domain mutation in Wrch-1 abrogates Wrch-1-mediated disruption of epithelial morphogenesis and promotion of anchorage-independent growth of MDCKII cells. (A) MDCKII cells stably expressing empty vector or HA-tagged Wrch-1(Q107L F86C) were treated and evaluated as in Fig. 7A. (B) MDCKII cells stably expressing empty vector, WT Wrch-1, Wrch-1(Q107L), or Wrch-1(Q107L F86C) were seeded into soft agar and analyzed for their ability to induce anchorage-independent growth as indicated in Fig. 7B. SEM, standard error of the mean.

FIG. 9.

Loss of Wrch-1 expression perturbs epithelial morphogenesis. (A) mRNA levels of Wrch-1 or actin isolated from MDCKII cells stably expressing pRS-GFP, pRS-shWrch-1 no. 1 or pRS-shWrch-1 no. 2. Quantification of Wrch-1 mRNA levels was normalized to actin using ImageJ. Bar graphs represent an average of three independent experiments for each cell line ± the standard error of the mean (SEM). Tukey's multiple comparison test was used to determine significance between cell lines. Significant P values of <0.001 are indicated by **. (B) MDCKII cells stably expressing pRS-GFP, pRS-shWrch-1 no. 1, or pRS-shWrch-1 no. 2 were treated and evaluated as in Fig. 7A.