Figure 9.
Figure 9. The guanine nucleotide exchange activity of SGEF is required for junctional maintenance, whereas scaffolding activity of SGEF is required for apical contractility. (A) Active RhoG was precipitated from total lysates of CTRL, SGEF KD, Rescue mNeon-SGEF WT, and Rescue CD mNeon-SGEF using GST–ELMO and immunoblotted with anti-RhoG antibodies. (B) For quantification, active RhoG levels were normalized to total RhoG levels. Data are mean ± SEM of three independent experiments. **, P < 0.005; ns, nonsignificant using Student’s t test (two-tailed, unpaired). (C) Lysates from CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD were probed for E-cadherin, β-catenin, ZO-1, and myosin IIB antibodies. Tubulin was used as a loading control. (D and E) Confluent MDCK CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD were stained for endogenous E-cadherin, β-catenin, and mNeon-SGEF (green). Confocal images are maximum projections of apical Z-planes. Scale bar, 5 µm. (F) XZ view of MDCK cells from CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD stained for F-actin (magenta), nucleus (Hoechst), and mNeon-SGEF (green). Scale bar, 10 µm. (G) Confluent MDCK CTRL, SGEF KD, and SGEF KD cells rescued with m-Neon-SGEF 1-227, mNeon-SGEF 1-400, or mNeon-SGEF CD were steined for endogenous ZO-1, myosin IIB, and mNeoen-SGEF (green). Confocal images are maximum projections of apical Z-planes. Scale bar, 5 μm. (H) Linescan (6-µm line drawn perpendicular to center of junctions) of IF images in D. At least two fields from two independent experiments (≥150 junctions) were used for quantification. The intensity profiles from were manually centered around the highest peak for each condition. (I) Quantification of height in CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD cells. n = 50 cells for each condition. Error bars represent min to max values with all points. Error bars represent SEM. ****, P < 0.00005, using the Mann–Whitney U test. (J) Quantification of zigzag index of CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD. At least two fields from two independent experiments (≥200 junctions) were used for quantification. ****, P < 0.00005; ns, nonsignificant using Student’s t test (two-tailed, unpaired).

The guanine nucleotide exchange activity of SGEF is required for junctional maintenance, whereas scaffolding activity of SGEF is required for apical contractility. (A) Active RhoG was precipitated from total lysates of CTRL, SGEF KD, Rescue mNeon-SGEF WT, and Rescue CD mNeon-SGEF using GST–ELMO and immunoblotted with anti-RhoG antibodies. (B) For quantification, active RhoG levels were normalized to total RhoG levels. Data are mean ± SEM of three independent experiments. **, P < 0.005; ns, nonsignificant using Student’s t test (two-tailed, unpaired). (C) Lysates from CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD were probed for E-cadherin, β-catenin, ZO-1, and myosin IIB antibodies. Tubulin was used as a loading control. (D and E) Confluent MDCK CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD were stained for endogenous E-cadherin, β-catenin, and mNeon-SGEF (green). Confocal images are maximum projections of apical Z-planes. Scale bar, 5 µm. (F) XZ view of MDCK cells from CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD stained for F-actin (magenta), nucleus (Hoechst), and mNeon-SGEF (green). Scale bar, 10 µm. (G) Confluent MDCK CTRL, SGEF KD, and SGEF KD cells rescued with m-Neon-SGEF 1-227, mNeon-SGEF 1-400, or mNeon-SGEF CD were steined for endogenous ZO-1, myosin IIB, and mNeoen-SGEF (green). Confocal images are maximum projections of apical Z-planes. Scale bar, 5 μm. (H) Linescan (6-µm line drawn perpendicular to center of junctions) of IF images in D. At least two fields from two independent experiments (≥150 junctions) were used for quantification. The intensity profiles from were manually centered around the highest peak for each condition. (I) Quantification of height in CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD cells. n = 50 cells for each condition. Error bars represent min to max values with all points. Error bars represent SEM. ****, P < 0.00005, using the Mann–Whitney U test. (J) Quantification of zigzag index of CTRL, SGEF KD, and SGEF KD cells rescued with mNeon-SGEF 1–227, mNeon-SGEF 1–400, or mNeon-SGEF CD. At least two fields from two independent experiments (≥200 junctions) were used for quantification. ****, P < 0.00005; ns, nonsignificant using Student’s t test (two-tailed, unpaired).

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