Epithelial wound repair shares many similarities with the tissue movements that occur during normal embryo epithelial morphogenesis: both require cell shape changes and cell migrations that are dependent on the actin cytoskeleton. We are investigating the cellular and molecular mechanisms of single cell and multicellular (tissue) wound repair and their ensuing biological manifestations, in the embryo. We are particularly interested in the regulation of the actin cytoskeleton and in the role of the Rho family of small GTPases in these processes.
Single Cell Wound Repair
Most cells of the body are subjected to physiological events during normal functions that can lead to disruption of the cell’s plasma membrane. The capacity of single cells to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Upon disruption of the plasma membrane, an influx of calcium signals the deployment of vesicles that fuse with each other and with the plasma membrane to plug the hole. After the membrane has been sealed, repair of the cell’s cortical cytoskeleton is necessary to reestablish a normal cyto-architecture.
As the first thirteen nuclear divisions in the Drosophila embryo are not accompanied by cytokinesis, the early fly embryo can be considered as a giant single cell. The early embryo’s multinucleate nature is not unlike that of muscle cells; one of the major mammalian cell types undergoing continuous membrane tearing and employing single cell repair mechanisms. We employ 4D in vivo microscopy and fluorescently-tagged reporters, along with pharmacological and genetic manipulations, to define the series of changes that occur in response to wounding using this early stage (NC4-6) Drosophila embryo as a model.
Analysis of single cell wound repair in NC4 staged embryos allowed us to divide single cell wound repair into three distinct steps: i) Expansion, ii) Contraction, and iii) Closure. Upon wounding the cortical actin disappears and the initial wound area rapidly expands. This Expansion phase occurs during the first 30-60s post-wounding. Once actin accumulates in a ring flanking the wound, the diameter starts to reduce progressively (Contraction). Initiation of Contraction occurs in parallel with the accumulation of actin at the leading edge of the hole. The Contraction phase is followed by a slower Closure phase.
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To examine the signals that trigger wound repair and control the cytoskeleton changes, we are currently investigating the role of the Rho family small GTPases in single cell wound repair.
In addition to repairing individually damaged cells, wounded epithelial sheets must be able to seal the hole breaching the surface layer to avoid excessive fluid loss and prevent microbial invasion. Therefore, repair of wounds in epithelial multicellular tissues entails both repair of damaged single cells at the leading edge of the injury, as well as migration and closure of the tissue edges. There are two major ways in which this tissue repair has been shown to occur: i) dynamic actin protrusions (lamellipodial crawling), and ii) the formation of an actin purse-string.
The fly embryo has recently emerged as an excellent model to study epithelial wound repair. We conduct our wound healing assays by laser ablating the ventral surface of stage 15 embryos where protein expression is mostly zygotic and the net force on this surface is minimal.
We find that epithelial wound repair can be divided into four distinct steps: i) Expansion, which occurs immediately upon wounding as cells fall away and the leading edge pulls back; ii) Coalescence, during which protrusions become visible and recruitment of components needed for assembly of the actomyosin cable in leading edge cells takes place; iii) Contraction, during which the wound area decreases; and iv) Closure, where the wound edges fuse and final cellular remodeling occurs.
Protrusions appear during the Coalescence phase, and contraction begins before a complete actin cable is assembled. Disrupting either the actomyosin cable or the actin rich protrusions delays, but does not prevent, wound repair. Disrupting both mechanisms simultaneously is needed to prevent wound repair. Thus, fly embryos have a robust epithelial repair response using both an actin cable and actin-rich protrusions simultaneously to drive contraction of the wound. Protrusions are also needed during Closure to knot the hole closed.
In addition to imaging studies, we are conducting genetic screens to identify genes involved in the response to wounds, with the aim of uncovering the players, mechanism(s), and regulatory pathways of the cell and tissue wound repair processes.