TβRIII inhibits cell migration in epithelial and cancer cells. (A) Cancer cell lines stably expressing TβRIII with Neo as controls (Ovca429 and MDA-MB231) and transiently expressing TβRIII with GFP as control (Ovca433 and Ovca3) were allowed to migrate for 18–24 h toward 10% FBS (see Materials and Methods). Fold migration relative to controls is presented and data represent the mean ± SE of 3 independent experiments performed in triplicate. The number of cells migrated is presented in Fig. S1A. (B) Ovca429 cells transiently expressing either TβRIII, TβRIIIΔGAG, TβRIIIΔCYTO, or GFP were allowed to migrate for 18–24 h toward 10% FBS. Data represent the mean ± SE of 3 independent experiments performed in triplicate. The number of cells migrated is presented in Fig. S1B. (C) NOSE007 cells in SFM in the top chamber of a transwell were allowed to migrate for 18–24 h toward 10% FBS 72 h after infection with adenovirus-expressing shRNA NTCs, human shRNA-TβRIII alone, human shRNA-TβRIII after infection with rat TβRIII (rTβRIII), or 48 h after infection with TβRIII, TβRIIIΔGAG, TβRIIIΔCYTO and GFP as controls. Fold migration relative to controls (shRNA NTCs for shRNA-TβRIII-infected cells or GFP control) is presented. Data represent the mean ± SE of 3 independent experiments performed in duplicate. *, P = 0.0036, relative to control. The number of cells migrated is presented in Fig. S1 C and D.

TβRIII alters the actin cytoskeletal organization of epithelial and cancer cells. (A–L) We plated 5–8 × 10⁴ of the indicated cell lines expressing the indicated proteins on coverslips and stained for actin by using rhodamine phalloidin. For transiently expressing TβRIII-IRES-GFP and control-GFP, cells were fixed and stained 48 h after infection and GFP-expressing cells were examined. Closed arrows indicate distinct lamellipodia in control cells and open arrows indicate rounded cells with multiple microspikes in TβRIII-expressing cells. Scale bar = 8.6 μm. (M and N) Subconfluent NOSE007 cells were infected with adenovirus expressing control shRNA NTCs (M) or shRNA-TβRIII (N). Cells were fixed and stained with actin-phalloidin-conjugated Alexa-488 72 h after infection and dsRed-expressing cells were examined. Open arrows indicate lamellipodia formation in shRNA-TβRIII cells. Scale bar = 21.6 μm.

TβRIII inhibits directional persistence, motility, and mediates alterations in the actin cytoskeleton through constitutive activation of Cdc42. (A and B) Migratory behavior of Ovca429Neo (A) (Movie S1) and Ovca429TβRIII (B) (Movie S2) cells tracked over a 120-min period. The origins of migration were superimposed at 0, 0. The tracks of representative individual cells during the 120-min period are presented. (C and D) Ovca429Neo and Ovca429TβRIII cell lines were serum-starved for 16 h followed by stimulation with TGF-β1 (200 pM) for the times indicated, and pull-down assays for active Cdc42 (C) and Rac1 (D) were performed. Five percent of lysate was assessed for effects on total Cdc42 and Rac1 levels. A representative experiment of 4 replicates is presented. Quantification of active Cdc42 or Rac1 is presented in Fig. S7 A and B. (E and F) The tracks of representative individual Ovca429TβRIII cells 24 h after infection with adenovirus-expressing GFP-tagged N17Cdc42 (E; see Movie S3) or GFP-tagged N17Rac1 (F) are presented. (G) Directional persistence was calculated under the indicated conditions from data acquired during live cell imaging. Data represent the mean ± SE for 3 independent experiments, n = 20. (H) Transwell migration toward a 10% FBS gradient was assessed 48 h after infecting Ovca429TβRIII cells with adenovirus-expressing GFP-tagged N17Cdc42 or GFP-tagged N17Rac1. Fold migration relative to Ovca429Neo control is presented. Data represent the mean ± SE for 5 independent experiments.

TβRIII-mediated Cdc42 activation, inhibition of migration, and alterations in the actin cytoskeleton are mediated through the interaction of TβRIII with β-arrestin2. (A) Transwell migration toward a 10% FBS gradient was assessed 48 h after infecting Ovca429 or Ovca433 cells with adenovirus-expressing GFP, full-length TβRIII (TβRIII), TβRIII unable to bind β-arrestin2 (TβRIII-T841A), or TβRIII unable to bind GIPC (TβRIII-DEL). Fold migration relative to GFP control is presented. Data represent the mean ± SE for 4 independent experiments for Ovca429 and 2 independent experiments for Ovca433, each done in triplicate. (#, P = 0.0189 and *, P = 0.0440). (B–D) The indicated cells were fixed and stained for actin using rhodamine phalloidin. GFP-expressing cells were examined and representative examples are presented. Scale bar = 6.6 μm. (E and G) Cells were serum starved and stimulated with TGF-β1 (200 pM) for the indicated times and pull-down assays for active Cdc42 conducted. Quantification is presented in Fig. S7C andFig. S7D, respectively. (F) Ovca429TβRIII cells were subjected to siRNA-mediated silencing of β-arrestin2 (Inset) and transwell migration performed toward 10% FBS. Fold migration relative to Neo controls are presented. Data represent the mean ± SE of 2 independent experiments.