Figure 4.
Figure 4. SGEF localizes at cell–cell junctions in epithelial cells. (A and B) IF of MDCK cells showing colocalization of mNeon-SGEF WT with Scribble and Dlg1, ZO-1 (TJ marker), and β-catenin (AJ marker). The right panels are single Z-planes along the length of the dotted yellow line. a, apical, b, basal. Scale bar, 10 µm, and XZ, 3 µm. (C) Gastrula-stage Xenopus embryos expressing mNeon-SGEF WT (green), TagBFP-ZO-1 (TJ marker), and PLEKHA7-mCherry (AJ marker) were live-imaged using confocal microscopy. En face views (left) are brightest point projections across multiple Z-planes. Side views (right) are average intensity projections along the length of the highlighted junction (see Materials and methods). Scale bars, XY, 10 µm, and XZ, 1 µm. (C′) Intensity profiles of SGEF (green solid line) relative to AJs (red dotted line) and TJs (blue dotted line) along the z axis in Xenopus gastrula-stage epithelial cells. Note that SGEF’s peak intensity is close to ZO-1’s, but it tapers away more slowly than ZO-1 along the lateral membrane. The graph shows normalized averaged intensities fitted with a smoothed curve; error bars indicate SD; n = 47 junctions, 18 embryos, five experiments. (D) Gastrula-stage Xenopus embryos expressing mNeon-RhoG (green), BFP-ZO-1 (TJ marker, blue), and PLEKHA7-mCherry (AJ marker, red) were live imaged using confocal microscopy. Brightest point projections of en face views and averaged side views of the highlighted junction (as in C) are shown. Scale bars, XY, 10 µm, and XZ, 1 µm. (D′) Intensity profiles of RhoG (green solid line) relative to AJs (red dotted line) and TJs (blue dotted line). Note that the RhoG signal is more basolateral compared with SGEF. The graph shows normalized averaged intensities fitted with a smoothed curve; error bars indicate SD. n = 19 junctions, 10 embryos, three experiments. (E) Gastrula-stage Xenopus embryos expressing mRFP-ZO-1 (TJ marker, magenta) and mNeon-SGEF WT (top, green), SGEF 1–227 (middle, green), or SGEF 228–871 (bottom, green) were live-imaged using confocal microscopy. En face views are brightest point projections across multiple Z-planes. Side views (right) are single Z-planes at the locations marked by yellow dotted lines. Note that WT SGEF and SGEF 1–227 appear junctional, whereas SGEF 228–871 appears diffusely localized. Scale bars, 20 µm, and XZ, 5 µm. (F) Quantification of the ratio of junctional to cytosolic intensities of mNeon-tagged SGEF WT, SGEF 1–227, and SGEF 228–871 in Xenopus embryos. n = SGEF WT: 234 junctions, 12 embryos, six experiments; SGEF 1–227: 214, 11, 5; SGEF 228–871: 180, 9, 4. Error bars represent SEM. ****, P < 0.00005 using the Mann–Whitney U test.

SGEF localizes at cell–cell junctions in epithelial cells. (A and B) IF of MDCK cells showing colocalization of mNeon-SGEF WT with Scribble and Dlg1, ZO-1 (TJ marker), and β-catenin (AJ marker). The right panels are single Z-planes along the length of the dotted yellow line. a, apical, b, basal. Scale bar, 10 µm, and XZ, 3 µm. (C) Gastrula-stage Xenopus embryos expressing mNeon-SGEF WT (green), TagBFP-ZO-1 (TJ marker), and PLEKHA7-mCherry (AJ marker) were live-imaged using confocal microscopy. En face views (left) are brightest point projections across multiple Z-planes. Side views (right) are average intensity projections along the length of the highlighted junction (see Materials and methods). Scale bars, XY, 10 µm, and XZ, 1 µm. (C′) Intensity profiles of SGEF (green solid line) relative to AJs (red dotted line) and TJs (blue dotted line) along the z axis in Xenopus gastrula-stage epithelial cells. Note that SGEF’s peak intensity is close to ZO-1’s, but it tapers away more slowly than ZO-1 along the lateral membrane. The graph shows normalized averaged intensities fitted with a smoothed curve; error bars indicate SD; n = 47 junctions, 18 embryos, five experiments. (D) Gastrula-stage Xenopus embryos expressing mNeon-RhoG (green), BFP-ZO-1 (TJ marker, blue), and PLEKHA7-mCherry (AJ marker, red) were live imaged using confocal microscopy. Brightest point projections of en face views and averaged side views of the highlighted junction (as in C) are shown. Scale bars, XY, 10 µm, and XZ, 1 µm. (D′) Intensity profiles of RhoG (green solid line) relative to AJs (red dotted line) and TJs (blue dotted line). Note that the RhoG signal is more basolateral compared with SGEF. The graph shows normalized averaged intensities fitted with a smoothed curve; error bars indicate SD. n = 19 junctions, 10 embryos, three experiments. (E) Gastrula-stage Xenopus embryos expressing mRFP-ZO-1 (TJ marker, magenta) and mNeon-SGEF WT (top, green), SGEF 1–227 (middle, green), or SGEF 228–871 (bottom, green) were live-imaged using confocal microscopy. En face views are brightest point projections across multiple Z-planes. Side views (right) are single Z-planes at the locations marked by yellow dotted lines. Note that WT SGEF and SGEF 1–227 appear junctional, whereas SGEF 228–871 appears diffusely localized. Scale bars, 20 µm, and XZ, 5 µm. (F) Quantification of the ratio of junctional to cytosolic intensities of mNeon-tagged SGEF WT, SGEF 1–227, and SGEF 228–871 in Xenopus embryos. n = SGEF WT: 234 junctions, 12 embryos, six experiments; SGEF 1–227: 214, 11, 5; SGEF 228–871: 180, 9, 4. Error bars represent SEM. ****, P < 0.00005 using the Mann–Whitney U test.

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