Figure 5.
Figure 5. LPAR2 signaling controls N-cadherin internalization. (A) N-cadherin immunostaining in controls and LPAR2MO cells. Bar, 10 µm. (B) Mean normalized fluorescence intensity along the cell–cell contact area is plotted for controls (green curve) and LPAR2MO cells (magenta curve). Error bars indicate SEM. (C) Maximum fluorescence levels at the free edge (78 cells from 13 independent experiments; Student’s two-tailed t test: P < 0.001; error bars indicate SD). (D) Maximum fluorescence levels at the cell–cell junctions (48 contacts from 13 independent experiments; Student’s two-tailed t test: P < 0.001; error bars indicate SD) are shown for controls (green dots) and LPAR2MO (magenta dots). (E) RT-PCR for Sox 9 (NC marker), β-catenin, and N-cadherin from embryos at premigratory (stage 19) and migratory (stage 24) NC injected with control MO (CMO) or LPAR2MO. (F) Western blot for N-cadherin and tubulin from dissected NC and eye tissue of the embryos injected with CMO or LPAR2MO. (G) Localization of p120-GFP expressed in controls and LPAR2MO cells. (H and I) Immunostaining for endogenous β-catenin in controls and LPAR2MO cells performed in vitro (H) or whole mount staining of dissected cranial tissues (I). Boxed regions are enlarged on the right. (J–L) Pulse-chase assay with NC cells injected with N-cadherin HaloTag (green) and membrane-mCherry (magenta) in controls (J), LPAR2MO cells (K), or cells coinjected with LPAR2MO and activated Rab5 (L). Boxed regions are enlarged in the “zoom” column. (M) Normalized fluorescence intensity levels for N-cadherin at the cell membrane in controls (green), LPAR2MO cells (magenta), and LPAR2MO-CA-Rab5 cells (cyan). n = 73 cells from three independent experiments. One-way ANOVA: P < 0.0001. Individual comparisons: **, P < 0.01; ***, P < 0.001. Error bars indicate SD. (N) Normalized number of intracellular vesicles per cells in controls (green), LPAR2MO cells (magenta), and LPAR2MO-CA-Rab5 cells (cyan). n = 291 cells from three independent experiments. One-way ANOVA: P < 0.0001. Individual comparisons: **, P < 0.01; ***, P < 0.001. Error bars indicate SD. Bars: (G–I) 50 µm; (G and H, high magnification) 10 µm; (J–K) 20 µm.

LPAR2 signaling controls N-cadherin internalization. (A) N-cadherin immunostaining in controls and LPAR2MO cells. Bar, 10 µm. (B) Mean normalized fluorescence intensity along the cell–cell contact area is plotted for controls (green curve) and LPAR2MO cells (magenta curve). Error bars indicate SEM. (C) Maximum fluorescence levels at the free edge (78 cells from 13 independent experiments; Student’s two-tailed t test: P < 0.001; error bars indicate SD). (D) Maximum fluorescence levels at the cell–cell junctions (48 contacts from 13 independent experiments; Student’s two-tailed t test: P < 0.001; error bars indicate SD) are shown for controls (green dots) and LPAR2MO (magenta dots). (E) RT-PCR for Sox 9 (NC marker), β-catenin, and N-cadherin from embryos at premigratory (stage 19) and migratory (stage 24) NC injected with control MO (CMO) or LPAR2MO. (F) Western blot for N-cadherin and tubulin from dissected NC and eye tissue of the embryos injected with CMO or LPAR2MO. (G) Localization of p120-GFP expressed in controls and LPAR2MO cells. (H and I) Immunostaining for endogenous β-catenin in controls and LPAR2MO cells performed in vitro (H) or whole mount staining of dissected cranial tissues (I). Boxed regions are enlarged on the right. (J–L) Pulse-chase assay with NC cells injected with N-cadherin HaloTag (green) and membrane-mCherry (magenta) in controls (J), LPAR2MO cells (K), or cells coinjected with LPAR2MO and activated Rab5 (L). Boxed regions are enlarged in the “zoom” column. (M) Normalized fluorescence intensity levels for N-cadherin at the cell membrane in controls (green), LPAR2MO cells (magenta), and LPAR2MO-CA-Rab5 cells (cyan). n = 73 cells from three independent experiments. One-way ANOVA: P < 0.0001. Individual comparisons: **, P < 0.01; ***, P < 0.001. Error bars indicate SD. (N) Normalized number of intracellular vesicles per cells in controls (green), LPAR2MO cells (magenta), and LPAR2MO-CA-Rab5 cells (cyan). n = 291 cells from three independent experiments. One-way ANOVA: P < 0.0001. Individual comparisons: **, P < 0.01; ***, P < 0.001. Error bars indicate SD. Bars: (G–I) 50 µm; (G and H, high magnification) 10 µm; (J–K) 20 µm.

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