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. 2011 May;1(3):114-121.
doi: 10.4161/bioa.1.3.17091.

Single cell wound repair: Dealing with life's little traumas

Maria Teresa Abreu-Blanco  1 , Jeffrey M Verboon, Susan M Parkhurst
Affiliations

Single cell wound repair: Dealing with life's little traumas

Maria Teresa Abreu-Blanco et al. Bioarchitecture. 2011 May.

Abstract

Cell wounding is a common event in the life of many cell types, and the capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Cell wounding is most frequent in tissues exposed to high levels of stress. Survival of such plasma membrane disruptions requires rapid resealing to prevent the loss of cytosolic components, to block Ca(2+) influx and to avoid cell death. In addition to patching the torn membrane, plasma membrane and cortical cytoskeleton remodeling are required to restore cell function. Although a general understanding of the cell wound repair process is in place, the underlying mechanisms of each step of this response are not yet known. We have developed a model to study single cell wound repair using the early Drosophila embryo. Our system combines genetics and live imaging tools, allowing us to dissect in vivo the dynamics of the single cell wound response. We have shown that cell wound repair in Drosophila requires the coordinated activities of plasma membrane and cytoskeleton components. Furthermore, we identified an unexpected role for E-cadherin as a link between the contractile actomyosin ring and the newly formed plasma membrane plug.

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Figures

Figure 1
Figure 1
Schematic diagram of the single cell wound repair process. Upon plasma membrane disruption, a Ca2+ influx triggers internal vesicles to fuse with each other and form a membrane patch. This membrane “patch” fuses with the plasma membrane at specific sites along the periphery of the disruption. Membrane resealing is followed by a process of plasma membrane and cortical cytoskeleton remodeling (adapted from McNeil et al.).
Figure 2
Figure 2
Plasma membrane and cytoskeleton responses to single cell wounds. (A) Vesicle-vesicle fusions at the wound site. Scanning electron micrographs of the surface of a Sea Urchin egg sheared to produce plasma membrane disruptions. The egg was sheared directly into fixative that contained Ca2+ (10 mM). The disruption site is filled with a large population of vesicles, supporting the idea that Ca2+ induces the recruitment of vesicles to the disruption site. [Figure adapted and reprinted from McNeill and Baker. Cell Tissue Research 2001; 304:141–6, with permission from Springer]. (B and C) Cytoskeleton response to single cell wounds. Confocal image of a laser wounded Xenopus oocyte showing that a continuous actomyosin ring (actin, green; myosin II, blue) forms during the wound healing process, surrounded by a radial arrangement of microtubules (red). Significantly, the actin (FA) and myosin II (M2) rings are not completely superimposed (C).(Figure adapted and reprinted from Mandato and Bement. Curr Biol 2003; 13:1096–105, with permission from Elsevier). (D) An intact cytoskeleton is required for proper cell wound repair. Disruption of the actin and myosin cytoskeleton impairs wound closure. Xenopus oocytes treated with Cytochalasin D (Cyto D), Latrunculin B (Lat B) and BDM shows defects in actin (red) and myosin II (green) recruitment and ring assembly. (Figure adapted and reprinted from Bement et al. Curr Biol 1999; 9:579–87, with permission from Elsevier).
Figure 3
Figure 3
Single cell wound repair in the early Drosophila embryo. (A) Cartoon of the early Drosophila embryo (Nuclear cycle 4 to 8; NC). The early embryo is a syncytium wherein the nuclei divide in the interior of the embryo without cytokinesis through nuclear cycle 8, thereby forming a large multinucleate cell. (B) Orthogonal view of embryos expressing actin and nuclear markers. Nuclei remain away from the cell periphery until NC10, allowing for the study of wound repair before this time point without the complication of damage to the nuclei. (C) Wound repair curves (area vs. time) of small, medium and large wounds follow a stereotyped response composed of three steps and independent of wound size. Post wounding the area expands, followed by a period of rapid contraction of wound area, and then a slower phase of closure as the wounded area is remodeled. (D–D‴) Time-lapse series following wound repair in embryos expressing plasma membrane and actin markers. Plasma membrane is recruited from the area surrounding the wound and by the trafficking of vesicles to the wound site. By 90″ post wounding, the membrane has formed a plug over the wounded area. Actin is recruited from around the wound and accumulates to form a tight ring around the plasma membrane plug, which progressively contracts until the ring closes 5′ post wounding. Scale bars: XY are 20 µm and XZ 10 µm unless otherwise indicated. (Figure reprinted with permission from Abreu-Blanco et al.; J Cell Biol 2011; DOI:10.1083/jcb.201011018).
Figure 4
Figure 4
E-Cadherin mediates the interaction between the plasma membrane and the underlying cytoskeleton in single cell wound repair. (A) Cartoon depicting the actomyosin purse-string in single and multi-cellular repair. In both single and multi-cellular repair, actin and myosin II co-localize to form an actomyosin cable. E-Cadherin co-localizes with this cable in the single cell repair model to tether it to the plasma membrane. In contrast, in multi-cellular repair, E-Cadherin expression is found at cellular junctions along the leading edge to link the actin and myosin II machinery into an intracellular purse-string. (B–B‴) Time-lapse series of E-Cadherin and actin. Membrane-associated E-Cadherin is recruited from the area around the wound and from particles that traffic towards the wound. E-Cadherin co-localizes with the actin ring. (C and D) E-Cadherin mutant embryos fail to assemble an actin cable and show wound healing defects. (C) Surface projection of a wildtype embryo expressing actin. An actin ring is observed 60″ post wounding in wildtype embryos, whereas in E-Cadherin mutants actin forms a diffuse ring and wounds over-expand. E-Cadherin mutant embryos do manage to eventually repair the wound. Scale bars: XY are 20 µm and XZ 10 µm unless otherwise indicated. (Figure reprinted with permission from Abreu-Blanco et al.; J Cell Biol 2011; DOI:10.1083/jcb.201011018).
Figure 5
Figure 5
Relationship between single cell and multicellular wound repair. (A) Actin and Myosin II accumulate in a ring surrounding the wound in the early Drosophila embryo. Surface projection and orthogonal view of actin and myosin II show both proteins co-localizing to form an actomyosin ring. [©Abreu-Blanco et al. Originally published in Journal of Cell Biology 2011; DOI: 10.1083/jcb.201011018]. (B) An actomyosin purse string is observed at the leading edge of multicellular wounds in late Drosophila embryos. Surface projection and orthogonal view of actin and myosin II show both proteins co-localizing to form an actomyosin purse string. (C) Actin and myosin II accumulate at the leading edge of the wound edge as well as at cell junctions near the wound in cellularized Xenopus embryos. This accumulation at the cellular junction is distance dependant and only junctions proximal to the wound will form these secondary accumulations. (Scale bars: XY are 20 µm and XZ 10 µm unless otherwise indicated). (Figure adapted and reprinted from Clark. Curr Biol 2009; 19:1389–95 with permission from Elsevier.)
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