Figure 7. Tara-dependent maturation of the circumferential ring. (A–C) Confocal immunofluorescence micrographs of Tara-KD cells with or without transfection of HA-Tara or YFP–E-cadherin cultured with untransfected control MDCK cells. In co-cultures of control and Tara-KD cells, the Tara-KD cells showed a lower actin signal intensity at the zAJ/CR and cell–cell contacts than the control cells (A). In contrast, Tara-KD cells transfected with HA-Tara (B) or YFP-E-cadherin (C) exhibited actin signal intensities similar to the control cells. Bar, 10 µm. (A and B) HA, green; actin, red. (C) YFP, green; actin, red. (D) Scanning electron micrographs of Tara-KD cells with or without transfected HA-Tara or YFP–E-cadherin. The actin filaments of the CR were more loosely packed in the Tara-KD cells compared with the control cells. HA-Tara or YFP–E-cadherin partially restored the packing density of the CR actin filaments. Bar, 5 µm. (E) Electron micrographs of 3D cultures (cysts) of control cells and Tara-KD cells with or without transfection of HA-Tara or YFP–E-cadherin. Thin-section electron micrographs revealed that the spherical cell cysts were composed of a single-layered sheet of epithelial cells. Bar, 2 µm. The vertical-to-horizontal (Z/X) ratio of the diameters of control cell cysts was 0.92 (n = 10). The Tara-KD cell cysts were elliptical, and Tara-KD cells expressing exogenous HA-Tara or YFP–E-cadherin formed spherical cell cysts similar to the control cells. The Z/X ratios of the cell cysts made by Tara-KD cells, Tara-KD cells expressing HA-Tara, or Tara-KD cells expressing YFP–E-cadherin were 0.62, 0.87, and 0.91, respectively (n = 10). (F) Schematic drawing of the novel Tara/Trio RhoGEF/Rac1/p38MAPK/Tbx3/E-cadherin signaling cascade proposed here. This Tara/Trio/Rac1/p38/Tbx3 signaling cascade is the first E-cadherin–based AJ-initiated signal reported to regulate the transcription of E-cadherin in epithelial cell sheets. Here we found that Tara is enriched at AJs through its association with Trio RhoGEF, a binding partner of E-cadherin. By negatively regulating the RhoGEF activity of Trio in an EMT-independent manner, Tara down-regulates the activity of Tbx3, a transcriptional suppressor of E-cadherin. Furthermore, we found that Tara-KD remodeled the actin CR by decreasing the level of E-cadherin expression, although the protein level of cadherin-6 increased. We propose that Tara is a critical regulator of E-cadherin expression, and through this regulation Tara fine-tunes the cadherin-based actin CR and other properties of epithelial cell sheets.
Figure 7.

Tara-dependent maturation of the circumferential ring. (A–C) Confocal immunofluorescence micrographs of Tara-KD cells with or without transfection of HA-Tara or YFP–E-cadherin cultured with untransfected control MDCK cells. In co-cultures of control and Tara-KD cells, the Tara-KD cells showed a lower actin signal intensity at the zAJ/CR and cell–cell contacts than the control cells (A). In contrast, Tara-KD cells transfected with HA-Tara (B) or YFP-E-cadherin (C) exhibited actin signal intensities similar to the control cells. Bar, 10 µm. (A and B) HA, green; actin, red. (C) YFP, green; actin, red. (D) Scanning electron micrographs of Tara-KD cells with or without transfected HA-Tara or YFP–E-cadherin. The actin filaments of the CR were more loosely packed in the Tara-KD cells compared with the control cells. HA-Tara or YFP–E-cadherin partially restored the packing density of the CR actin filaments. Bar, 5 µm. (E) Electron micrographs of 3D cultures (cysts) of control cells and Tara-KD cells with or without transfection of HA-Tara or YFP–E-cadherin. Thin-section electron micrographs revealed that the spherical cell cysts were composed of a single-layered sheet of epithelial cells. Bar, 2 µm. The vertical-to-horizontal (Z/X) ratio of the diameters of control cell cysts was 0.92 (n = 10). The Tara-KD cell cysts were elliptical, and Tara-KD cells expressing exogenous HA-Tara or YFP–E-cadherin formed spherical cell cysts similar to the control cells. The Z/X ratios of the cell cysts made by Tara-KD cells, Tara-KD cells expressing HA-Tara, or Tara-KD cells expressing YFP–E-cadherin were 0.62, 0.87, and 0.91, respectively (n = 10). (F) Schematic drawing of the novel Tara/Trio RhoGEF/Rac1/p38MAPK/Tbx3/E-cadherin signaling cascade proposed here. This Tara/Trio/Rac1/p38/Tbx3 signaling cascade is the first E-cadherin–based AJ-initiated signal reported to regulate the transcription of E-cadherin in epithelial cell sheets. Here we found that Tara is enriched at AJs through its association with Trio RhoGEF, a binding partner of E-cadherin. By negatively regulating the RhoGEF activity of Trio in an EMT-independent manner, Tara down-regulates the activity of Tbx3, a transcriptional suppressor of E-cadherin. Furthermore, we found that Tara-KD remodeled the actin CR by decreasing the level of E-cadherin expression, although the protein level of cadherin-6 increased. We propose that Tara is a critical regulator of E-cadherin expression, and through this regulation Tara fine-tunes the cadherin-based actin CR and other properties of epithelial cell sheets.

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