Wound Healing
Wound Healing
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 and microtubule cytoskeleton. We are investigating the cellular and molecular mechanisms of single cell and multicellular wound repair and their ensuing biological manifestations, in the Drosophila 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. We anticipate that the information learned from Drosophila can be extrapolated to higher organisms, thereby contributing to our understanding of the single cell and tissue wound repair pathways in mammals.
SINGLE CELL WOUND REPAIR. Survival of individual cells upon injury depends on rapid repair of the disruption through a complex series of events highlighted by membrane fusion and cytoskeletal remodeling.
See: JCB Biosights: May 16, 2011
We are characterizing the components and pathways involved in single cell repair using embryos prior to cellularization, at which stage the embryos mimic large multinucleate single cells. After wounding, the first response observed is a fast reduction in the wound size. Following this initial step, the wound remains visible until the embryo undergoes cellularization . We have conducted a series of wounding assays using different lines that express GFP fusion proteins: moesinGFP, shows the disruption of the cortical cytoplasm at the wound site; Rho1GFP, accumulates at the wound site as rapidly as 2 minutes post wounding, which is consistent with Rho activity as a cytoskeleton regulator. Proteins associated with ER and plasma membrane are also seen to be rapidly recruited to the wound site.
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Early-stage Drosophila embryo expressing GFP-moesin, to visualize actin. After a cycle of mitotic division, the embryo was laser ablated and the wound repair response followed by time lapse microscopy. Upon ablation, the wound expands and soon afterward initiates closure. Actin does not accumulate at the wound edge, rather, we observe that the cortical cytoskeleton is damaged and that the actin cap associated with each nucleus is disrupted. (Full size movie) |
In addition to imaging studies, we are conducting a microarray screen to identify genes involved in the transcriptional response to wounds, with the aim of uncovering the start and stop signals to wound repair. Together, these assays will provide details of the components, mechanism(s), and regulatory pathways of the cell wound repair process.
MULTICELLULAR 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. This closure is achieved by lamellipodial crawling, by contraction of an actin purse-string, or by a combination of these mechanisms.
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| Tissue wound repair could be achieved by lamellipodial crawling or by contraction of an actin purse string. This diagram illustrates wound repair using an actin purse string. The individual actin filaments (green bars) anchor to adherens junctions (orange rectangles) formed between adjacent cells. Contraction of the actin cable in each cell leads to apical cell constriction and reduced wound circumference. As wound closure proceeds, some cells are squeezed out of the front row such that fewer epithelial cells remain in the front row. The remaining cells form new adherens junctions and apical actin cable contraction continues until the contralateral cells meet and fuse. Asterisks indicate cells that will be lost from the leading edge; nuclei are blue dots. | ||
Late-stage fly embryos seal these wounds through the formation of an actin-based circumferential ring around the hole. This ring appears shortly after wounding of the epithelial layer (within 10 minutes) and constricts to close the exposed hole within a time frame of about 2 hours. Our focus is to characterize the signaling pathways involved in initiating and terminating this process, and to determine the molecular mechanisms underlying wound repair machinery assembly and contraction. We had previously determined a requirement for the Rho1 small GTPase in wounding through in vivo imaging and genetic analysis. Embryos that lack Rho1 display a period of apparent inactivity following wounding, but after about 2 hours begin to heal as wounds start contracting. Despite impaired actin ring formation at the wound periphery, these holes eventually close. We are currently examining a series of fly lines containing specific Rho mutations, allowing us to dissect the specific Rho functions required for each stage of epithelial sheet repair. We are also identifying and characterizing the downstream effectors required for these repair pathways.
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Multiple laser ablations made to late-stage embryos expressing GFP-moesin to visualize actin. Following wounding, actin accumulates at the wound edge, forming an actin cable that contracts to close the hole. (Full size movie) |
Together with our studies on single cell wound repair, we aim to generate a more complete picture of how tissues repair themselves in the embryo.
Last updated 05/17/11
