Figure 8.
Figure 8. SGEF regulates apical constriction in epithelial cells. (A) Gastrula-stage Xenopus embryos expressing mRFP-ZO-1 (TJ marker) with 3xGFP-SGEF overexpressed (OE) at high levels (bottom). Yellow arrows point to apically constricted cells. Scale bar, 20 µm. (B) Time lapse of CTRL and 3xGFP-SGEF overexpression of a single cell over a period of 24 min. Note that the SGEF OE cell constricts apically whereas CTRL cell retains the same apical area. Scale bars, 10 µm. (C) Time projection of ZO-1 signal over a 203-s interval shows that junctions in SGEF OE cells are more dynamic than in CTRLs. Scale bar, 20 µm. (D) Graph showing the average apical surface area of SGEF OE cells is significantly smaller than CTRL cells, and some SGEF OE cells exhibit severe apical constriction. CTRL, n = 132 cells, three embryos, two experiments; SGEF OE, n = 147 cells, three embryos, two experiments. (E) CTRL and SGEF OE gastrula-stage Xenopus embryos were fixed and stained with Alexa Fluor 568–phalloidin to reveal F-actin. Images in the top row were taken with lower laser power optimized for viewing cell–cell junctions, and images in the bottom row were taken with higher laser power optimized for viewing medial-apical actin. Scale bar, 10 µm. (E′) F-actin intensity at BCJ was quantified from fixed phalloidin stained embryos. n = control: 288 junctions, 11 embryos, three experiments; SGEF OE: 304 junctions, 13 embryos, two experiments. (E′′) Medial-apical F-actin intensity was quantified from fixed phalloidin stained embryos. n = control: 50 cells, seven embryos, three experiments; SGEF OE: 50 cells, eight embryos, three experiments. (F) CTRL and SGEF OE embryos expressing an F-actin probe (Lifeact-mRFP, magenta in merge) and a myosin II intrabody (SF9-mNeon, green in merge) were live imaged by confocal microscopy. The control image shown is from a control region of a mosaic SGEF OE embryo. Scale bar, 10 µm. (G) CTRL and SGEF OE embryos coexpressing mNeon-Vinculin, mCherry-α-catenin, and BFP-membrane. Scale bar, 10 µm. (H) Graph comparing junctional intensities of vinculin (normalized to membrane probe intensity) in CTRL and SGEF OE embryos. n = control: 63 junctions, eight embryos, three experiments; SGEF OE: control: 63 junctions, eight embryos, three experiments. Confocal images in A, B, and E–G are brightest point of apical sections. All graphs show mean ± SEM. ****, P < 0.00005 using the Mann–Whitney U test.

SGEF regulates apical constriction in epithelial cells. (A) Gastrula-stage Xenopus embryos expressing mRFP-ZO-1 (TJ marker) with 3xGFP-SGEF overexpressed (OE) at high levels (bottom). Yellow arrows point to apically constricted cells. Scale bar, 20 µm. (B) Time lapse of CTRL and 3xGFP-SGEF overexpression of a single cell over a period of 24 min. Note that the SGEF OE cell constricts apically whereas CTRL cell retains the same apical area. Scale bars, 10 µm. (C) Time projection of ZO-1 signal over a 203-s interval shows that junctions in SGEF OE cells are more dynamic than in CTRLs. Scale bar, 20 µm. (D) Graph showing the average apical surface area of SGEF OE cells is significantly smaller than CTRL cells, and some SGEF OE cells exhibit severe apical constriction. CTRL, n = 132 cells, three embryos, two experiments; SGEF OE, n = 147 cells, three embryos, two experiments. (E) CTRL and SGEF OE gastrula-stage Xenopus embryos were fixed and stained with Alexa Fluor 568–phalloidin to reveal F-actin. Images in the top row were taken with lower laser power optimized for viewing cell–cell junctions, and images in the bottom row were taken with higher laser power optimized for viewing medial-apical actin. Scale bar, 10 µm. (E′) F-actin intensity at BCJ was quantified from fixed phalloidin stained embryos. n = control: 288 junctions, 11 embryos, three experiments; SGEF OE: 304 junctions, 13 embryos, two experiments. (E′′) Medial-apical F-actin intensity was quantified from fixed phalloidin stained embryos. n = control: 50 cells, seven embryos, three experiments; SGEF OE: 50 cells, eight embryos, three experiments. (F) CTRL and SGEF OE embryos expressing an F-actin probe (Lifeact-mRFP, magenta in merge) and a myosin II intrabody (SF9-mNeon, green in merge) were live imaged by confocal microscopy. The control image shown is from a control region of a mosaic SGEF OE embryo. Scale bar, 10 µm. (G) CTRL and SGEF OE embryos coexpressing mNeon-Vinculin, mCherry-α-catenin, and BFP-membrane. Scale bar, 10 µm. (H) Graph comparing junctional intensities of vinculin (normalized to membrane probe intensity) in CTRL and SGEF OE embryos. n = control: 63 junctions, eight embryos, three experiments; SGEF OE: control: 63 junctions, eight embryos, three experiments. Confocal images in A, B, and E–G are brightest point of apical sections. All graphs show mean ± SEM. ****, P < 0.00005 using the Mann–Whitney U test.

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