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Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair.

Hunter MV, Lee DM, Harris TJ, Fernandez-Gonzalez R - J. Cell Biol. (2015)

Bottom Line: We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility.Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair.Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin.

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Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.

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E-cadherin overexpression delays wound closure. (A and B) Epidermal cells in embryos expressing wild-type levels of E-cadherin (endo–E-cad:GFP) or overexpressing E-cadherin (UAS–E-cad:GFP). Time after wounding is shown. Red lines indicate wound sites. Anterior left, dorsal up. Bars, 5 µm. (C) Mean wound area over time in embryos expressing wild-type levels of E-cadherin (blue; n = 7) or overexpressing E-cadherin (red; n = 8). (D and E) Relative E-cadherin fluorescence in cell interfaces with respect to the counterstain (Dlg; D) and wound closure rate for the fast phase of wound repair (E) in stage 14–15 embryos expressing endo–E-cadherin:GFP (endo; n = 5), ubi–E-cadherin:GFP (ubi; n = 5), or UAS–E-cadherin:GFP driven by tubulin-Gal4 (UAS; n = 5). (F–H) Kymographs showing the redistribution of E-cadherin:GFP along cell interfaces at the wound margin in embryos expressing endo–E-cadherin:GFP (F), ubi–E-cadherin:GFP (G), or UAS–E-cadherin:GFP (H). Anterior left, dorsal up. Bars, 30 s. (I) Percentage of decrease in E-cadherin:GFP fluorescence from interfaces at the wound margin 15 min after wounding in embryos expressing endo–E-cadherin:GFP (n = 23 interfaces), ubi–E-cadherin:GFP (n = 32 interfaces), and UAS–E-cadherin:GFP (n = 20 interfaces). Error bars, SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig7: E-cadherin overexpression delays wound closure. (A and B) Epidermal cells in embryos expressing wild-type levels of E-cadherin (endo–E-cad:GFP) or overexpressing E-cadherin (UAS–E-cad:GFP). Time after wounding is shown. Red lines indicate wound sites. Anterior left, dorsal up. Bars, 5 µm. (C) Mean wound area over time in embryos expressing wild-type levels of E-cadherin (blue; n = 7) or overexpressing E-cadherin (red; n = 8). (D and E) Relative E-cadherin fluorescence in cell interfaces with respect to the counterstain (Dlg; D) and wound closure rate for the fast phase of wound repair (E) in stage 14–15 embryos expressing endo–E-cadherin:GFP (endo; n = 5), ubi–E-cadherin:GFP (ubi; n = 5), or UAS–E-cadherin:GFP driven by tubulin-Gal4 (UAS; n = 5). (F–H) Kymographs showing the redistribution of E-cadherin:GFP along cell interfaces at the wound margin in embryos expressing endo–E-cadherin:GFP (F), ubi–E-cadherin:GFP (G), or UAS–E-cadherin:GFP (H). Anterior left, dorsal up. Bars, 30 s. (I) Percentage of decrease in E-cadherin:GFP fluorescence from interfaces at the wound margin 15 min after wounding in embryos expressing endo–E-cadherin:GFP (n = 23 interfaces), ubi–E-cadherin:GFP (n = 32 interfaces), and UAS–E-cadherin:GFP (n = 20 interfaces). Error bars, SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: Our results suggest that E-cadherin endocytosis is required for rapid wound repair. To investigate if E-cadherin specifically needs to be removed from the wound margin, we overexpressed E-cadherin in the embryonic epidermis using the upstream activating sequence (UAS)–Gal4 system (Brand and Perrimon, 1993) and the strong driver tubulin-Gal4, which is expressed in every cell at every stage of development (Lee and Luo, 1999). We quantified wound-closure rates in embryos overexpressing UAS–E-cadherin:GFP and found that wound repair proceeded at a significantly slower rate (17.5 ± 1.4 µm2/min) than in embryos expressing wild-type levels of E-cadherin (34.4 ± 4.0 µm2/min; P = 4.5 × 10−3; Fig. 7, A–C). Notably, when we quantified the rate of wound closure in embryos expressing E-cadherin:GFP at endogenous levels (endo–E-cadherin:GFP; Huang et al., 2009), mildly increased levels (ubi–E-cadherin:GFP; Oda and Tsukita, 2001), or high levels (UAS–E-cadherin:GFP), we found that slower wound closure correlated with increasing E-cadherin expression (Fig. 7, D and E). To further test if increasing E-cadherin levels disrupted wound closure, we overexpressed p120-catenin using tubulin-Gal4. p120-catenin stabilizes E-cadherin at adherens junctions by preventing its endocytosis (Ishiyama et al., 2010; Nanes et al., 2012). Overexpression of p120-catenin resulted in significantly higher levels of junctional E-cadherin (61.7% increase; P = 0.02; Fig. S5 E). When we overexpressed p120-catenin, the rate of wound closure decreased significantly, from 34.4 ± 4.0 µm2/min in controls to 18.7 ± 5.1 µm2/min in p120-catenin–overexpressing embryos (P = 0.03; Fig. S5, A–D). Together, our data suggest that E-cadherin levels must be tightly regulated to promote rapid embryonic wound repair.


Polarized E-cadherin endocytosis directs actomyosin remodeling during embryonic wound repair.

Hunter MV, Lee DM, Harris TJ, Fernandez-Gonzalez R - J. Cell Biol. (2015)

E-cadherin overexpression delays wound closure. (A and B) Epidermal cells in embryos expressing wild-type levels of E-cadherin (endo–E-cad:GFP) or overexpressing E-cadherin (UAS–E-cad:GFP). Time after wounding is shown. Red lines indicate wound sites. Anterior left, dorsal up. Bars, 5 µm. (C) Mean wound area over time in embryos expressing wild-type levels of E-cadherin (blue; n = 7) or overexpressing E-cadherin (red; n = 8). (D and E) Relative E-cadherin fluorescence in cell interfaces with respect to the counterstain (Dlg; D) and wound closure rate for the fast phase of wound repair (E) in stage 14–15 embryos expressing endo–E-cadherin:GFP (endo; n = 5), ubi–E-cadherin:GFP (ubi; n = 5), or UAS–E-cadherin:GFP driven by tubulin-Gal4 (UAS; n = 5). (F–H) Kymographs showing the redistribution of E-cadherin:GFP along cell interfaces at the wound margin in embryos expressing endo–E-cadherin:GFP (F), ubi–E-cadherin:GFP (G), or UAS–E-cadherin:GFP (H). Anterior left, dorsal up. Bars, 30 s. (I) Percentage of decrease in E-cadherin:GFP fluorescence from interfaces at the wound margin 15 min after wounding in embryos expressing endo–E-cadherin:GFP (n = 23 interfaces), ubi–E-cadherin:GFP (n = 32 interfaces), and UAS–E-cadherin:GFP (n = 20 interfaces). Error bars, SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig7: E-cadherin overexpression delays wound closure. (A and B) Epidermal cells in embryos expressing wild-type levels of E-cadherin (endo–E-cad:GFP) or overexpressing E-cadherin (UAS–E-cad:GFP). Time after wounding is shown. Red lines indicate wound sites. Anterior left, dorsal up. Bars, 5 µm. (C) Mean wound area over time in embryos expressing wild-type levels of E-cadherin (blue; n = 7) or overexpressing E-cadherin (red; n = 8). (D and E) Relative E-cadherin fluorescence in cell interfaces with respect to the counterstain (Dlg; D) and wound closure rate for the fast phase of wound repair (E) in stage 14–15 embryos expressing endo–E-cadherin:GFP (endo; n = 5), ubi–E-cadherin:GFP (ubi; n = 5), or UAS–E-cadherin:GFP driven by tubulin-Gal4 (UAS; n = 5). (F–H) Kymographs showing the redistribution of E-cadherin:GFP along cell interfaces at the wound margin in embryos expressing endo–E-cadherin:GFP (F), ubi–E-cadherin:GFP (G), or UAS–E-cadherin:GFP (H). Anterior left, dorsal up. Bars, 30 s. (I) Percentage of decrease in E-cadherin:GFP fluorescence from interfaces at the wound margin 15 min after wounding in embryos expressing endo–E-cadherin:GFP (n = 23 interfaces), ubi–E-cadherin:GFP (n = 32 interfaces), and UAS–E-cadherin:GFP (n = 20 interfaces). Error bars, SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: Our results suggest that E-cadherin endocytosis is required for rapid wound repair. To investigate if E-cadherin specifically needs to be removed from the wound margin, we overexpressed E-cadherin in the embryonic epidermis using the upstream activating sequence (UAS)–Gal4 system (Brand and Perrimon, 1993) and the strong driver tubulin-Gal4, which is expressed in every cell at every stage of development (Lee and Luo, 1999). We quantified wound-closure rates in embryos overexpressing UAS–E-cadherin:GFP and found that wound repair proceeded at a significantly slower rate (17.5 ± 1.4 µm2/min) than in embryos expressing wild-type levels of E-cadherin (34.4 ± 4.0 µm2/min; P = 4.5 × 10−3; Fig. 7, A–C). Notably, when we quantified the rate of wound closure in embryos expressing E-cadherin:GFP at endogenous levels (endo–E-cadherin:GFP; Huang et al., 2009), mildly increased levels (ubi–E-cadherin:GFP; Oda and Tsukita, 2001), or high levels (UAS–E-cadherin:GFP), we found that slower wound closure correlated with increasing E-cadherin expression (Fig. 7, D and E). To further test if increasing E-cadherin levels disrupted wound closure, we overexpressed p120-catenin using tubulin-Gal4. p120-catenin stabilizes E-cadherin at adherens junctions by preventing its endocytosis (Ishiyama et al., 2010; Nanes et al., 2012). Overexpression of p120-catenin resulted in significantly higher levels of junctional E-cadherin (61.7% increase; P = 0.02; Fig. S5 E). When we overexpressed p120-catenin, the rate of wound closure decreased significantly, from 34.4 ± 4.0 µm2/min in controls to 18.7 ± 5.1 µm2/min in p120-catenin–overexpressing embryos (P = 0.03; Fig. S5, A–D). Together, our data suggest that E-cadherin levels must be tightly regulated to promote rapid embryonic wound repair.

Bottom Line: We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility.Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair.Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.

Show MeSH
Related in: MedlinePlus