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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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Calcium is required for E-cadherin redistribution at the wound margin, purse string formation, and rapid wound repair. (A, B, E, and F) Epidermal cells in embryos expressing endo–E-cadherin:GFP (A and B) or mCherry:moesin (E and F), injected with water (A and E) or BAPTA (B and F) before wounding. Bars, 5 µm. (C and G) Wound area over time for water-injected (blue; n = 8 in C and n = 7 in G) and BAPTA-injected (red; n = 10 in C and n = 7 in G) embryos. (D) Wound closure rate for the fast phase of tissue repair. (H) Maximum fold enrichment of mCherry:moesin at the wound margin. (I and J) Kymographs showing E-cadherin:GFP redistribution along cell interfaces at the wound margin. Anterior left, dorsal up. Bars, 30 s. (K) Percentage of decrease in E-cadherin:GFP fluorescence along wound margin interfaces 15 min after wounding in water-injected (n = 22 interfaces) and BAPTA-injected (n = 21 interfaces) embryos. (A–D) Time after wounding is shown. Red lines indicate wound sites. Yellow dotted lines outline the wounds. Anterior left, dorsal up. Error bars, SEM; *, P < 0.05; **, P < 0.01.
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fig6: Calcium is required for E-cadherin redistribution at the wound margin, purse string formation, and rapid wound repair. (A, B, E, and F) Epidermal cells in embryos expressing endo–E-cadherin:GFP (A and B) or mCherry:moesin (E and F), injected with water (A and E) or BAPTA (B and F) before wounding. Bars, 5 µm. (C and G) Wound area over time for water-injected (blue; n = 8 in C and n = 7 in G) and BAPTA-injected (red; n = 10 in C and n = 7 in G) embryos. (D) Wound closure rate for the fast phase of tissue repair. (H) Maximum fold enrichment of mCherry:moesin at the wound margin. (I and J) Kymographs showing E-cadherin:GFP redistribution along cell interfaces at the wound margin. Anterior left, dorsal up. Bars, 30 s. (K) Percentage of decrease in E-cadherin:GFP fluorescence along wound margin interfaces 15 min after wounding in water-injected (n = 22 interfaces) and BAPTA-injected (n = 21 interfaces) embryos. (A–D) Time after wounding is shown. Red lines indicate wound sites. Yellow dotted lines outline the wounds. Anterior left, dorsal up. Error bars, SEM; *, P < 0.05; **, P < 0.01.

Mentions: Our results showing that calcium is necessary for the polarization of the endocytic machinery to the wound margin predict that blocking calcium signaling should cause phenotypes similar to those resulting from blocking endocytosis. To investigate whether wound healing was delayed when we blocked calcium release, we measured the rate of wound closure in BAPTA-treated embryos (Fig. 6, A–D). Blocking calcium release led to wounds that closed at a rate of 11.9 ± 2.4 µm2/min, significantly slower than wounds in water-injected controls, which closed at a rate of 20.2 ± 3.4 µm2/min (P = 3.8 × 10−2; Fig. 6 D). We confirmed these results by treating embryos with 500 µM thapsigargin, a cell-permeable calcium chelator, which also delayed wound healing (Fig. S4, I and J). These data show that calcium release is necessary for rapid 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)

Calcium is required for E-cadherin redistribution at the wound margin, purse string formation, and rapid wound repair. (A, B, E, and F) Epidermal cells in embryos expressing endo–E-cadherin:GFP (A and B) or mCherry:moesin (E and F), injected with water (A and E) or BAPTA (B and F) before wounding. Bars, 5 µm. (C and G) Wound area over time for water-injected (blue; n = 8 in C and n = 7 in G) and BAPTA-injected (red; n = 10 in C and n = 7 in G) embryos. (D) Wound closure rate for the fast phase of tissue repair. (H) Maximum fold enrichment of mCherry:moesin at the wound margin. (I and J) Kymographs showing E-cadherin:GFP redistribution along cell interfaces at the wound margin. Anterior left, dorsal up. Bars, 30 s. (K) Percentage of decrease in E-cadherin:GFP fluorescence along wound margin interfaces 15 min after wounding in water-injected (n = 22 interfaces) and BAPTA-injected (n = 21 interfaces) embryos. (A–D) Time after wounding is shown. Red lines indicate wound sites. Yellow dotted lines outline the wounds. Anterior left, dorsal up. Error bars, SEM; *, P < 0.05; **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4555830&req=5

fig6: Calcium is required for E-cadherin redistribution at the wound margin, purse string formation, and rapid wound repair. (A, B, E, and F) Epidermal cells in embryos expressing endo–E-cadherin:GFP (A and B) or mCherry:moesin (E and F), injected with water (A and E) or BAPTA (B and F) before wounding. Bars, 5 µm. (C and G) Wound area over time for water-injected (blue; n = 8 in C and n = 7 in G) and BAPTA-injected (red; n = 10 in C and n = 7 in G) embryos. (D) Wound closure rate for the fast phase of tissue repair. (H) Maximum fold enrichment of mCherry:moesin at the wound margin. (I and J) Kymographs showing E-cadherin:GFP redistribution along cell interfaces at the wound margin. Anterior left, dorsal up. Bars, 30 s. (K) Percentage of decrease in E-cadherin:GFP fluorescence along wound margin interfaces 15 min after wounding in water-injected (n = 22 interfaces) and BAPTA-injected (n = 21 interfaces) embryos. (A–D) Time after wounding is shown. Red lines indicate wound sites. Yellow dotted lines outline the wounds. Anterior left, dorsal up. Error bars, SEM; *, P < 0.05; **, P < 0.01.
Mentions: Our results showing that calcium is necessary for the polarization of the endocytic machinery to the wound margin predict that blocking calcium signaling should cause phenotypes similar to those resulting from blocking endocytosis. To investigate whether wound healing was delayed when we blocked calcium release, we measured the rate of wound closure in BAPTA-treated embryos (Fig. 6, A–D). Blocking calcium release led to wounds that closed at a rate of 11.9 ± 2.4 µm2/min, significantly slower than wounds in water-injected controls, which closed at a rate of 20.2 ± 3.4 µm2/min (P = 3.8 × 10−2; Fig. 6 D). We confirmed these results by treating embryos with 500 µM thapsigargin, a cell-permeable calcium chelator, which also delayed wound healing (Fig. S4, I and J). These data show that calcium release is necessary for rapid 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