Genetic analysis of actin dynamics at epithelial wound edges in vivo

The surfaces and cavities of our bodies are covered by protective outer layers called epithelia, which consist of sheets of cells tightly adhered to one another. For instance, the outer layer of our skin is an epithelium, as are the linings of our gut and lungs. Epithelia function as barriers that protect our bodies against infective agents, toxic substances and fluid loss, therefore it is essential that if damaged, epithelia are rapidly repaired. Repair of epithelia, for instance during healing of a wound or recovery from surgery, can take several weeks, during which time the wounded individual is at increased risk of infection. In some circumstances epithelial repair fails completely, leaving the individual at constant risk of infection. Therefore the development of therapies to accelerate or improve epithelial repair would be of great benefit to human health. In order to develop such therapies, it is necessary to understand how epithelial repair occurs at the molecular and cellular level. Previous research has shown that there are two methods by which epithelial wound repair can occur. In the first method, cells at the wound edge extent protrusions into the wound. Using these protrusions, the cells grab hold of fibres within the wound and gradually pull themselves forward across the wound. In the second method of epithelial repair, the cells at the edge of wound construct a contractile cable around the wound edge. This cable then constricts like a purse-string and pulls the wound closed. The protrusions required for the first method of closure, and the cable required for the second, are both assembled from a protein called actin. Therefore in either case, a possible way of accelerating epithelial wound healing would be to develop a drug that altered the assembly of actin by wound edge cells, for instance causing them to make more protrusions or a thicker cable. To do this we need to understand how actin assembly is controlled in wound edge cells, as this would reveal ways in which actin assembly could be manipulated using drugs. The mechanisms controlling actin assembly will differ between cells forming protrusions and cables, so we would need to understand how actin is controlled in both situations. It is probable that we would need different drugs to accelerate protrusion-based and cable-based wound closure, therefore we need to be able to identify which type of closure is occurring if we are to select the appropriate method of treatment for a particular wound. We currently do not understand why some wounds heal using protrusions while others heal using a purse-string. The aims of this project are to identify what determines whether a wound heals using protrusions or a cable, and to establish how control of actin assembly differs between cells forming cables and protrusions. Our experiments will be performed using embryos of the fruit fly Drosophila. The advantage of using Drosophila for this research is that we can easily observe epithelial wound healing occurring in live embryos and we can use genetic techniques to determine which genes are important in the two forms of epithelial wound healing. We will study wound healing in two different epithelial tissues in the embryo, one of which forms an actin cable at wound edges, while the other forms protrusions. We will compare wound healing in these two tissues, and investigate why actin assembly at wound edges differs between them. We will use genetic techniques to identify the molecules that control actin assembly in wound edge cells in these two tissues. Our findings will greatly improve our understanding of how epithelia heal and will therefore assist in the development of therapies to accelerate epithelial wound healing. Spreading of cancer cells around the body involves changes in actin assembly by epithelial cells, therefore this work could also aid the development of therapies to treat cancer.

An epithelium is a sheet of cells that functions as a barrier separating a tissue or organism from its surroundings. Epithelia provide essential protection against infection, toxins and fluid loss, therefore it is vital that wounded epithelia are rapidly repaired. Epithelial repair can be achieved by two mechanisms: 1. A crawling mechanism, in which wound edge cells migrate across the substratum by extension of actin protrusions into the wound. 2. A purse-string mechanism in which a contractile actin cable forms in wound edge cells, constriction of which pulls the wound closed. The method of closure is therefore determined by the mode of actin assembly at the wound edge. The development of therapies to accelerate epithelial repair would greatly benefit human health, and manipulation of signaling pathways controlling actin assembly in wound edge cells is a strategy by which this could be achieved. However, actin signaling will differ between wound edge cells assembling protrusions and cables, therefore if we are to exploit this therapeutic approach, we need to understand under what circumstances each of the two modes of closure is used, and how actin signaling differs between them. This grant will therefore address 2 questions: 1. What determines the mode of actin assembly adopted by wound edge cells? 2. How does signaling differ in wound edge cells exhibiting different modes of actin assembly? We will employ a novel experimental strategy in which we will use genetics and imaging to scrutinise wound healing in two epithelial tissues in the Drosophila embryo; the epidermis and the amnioserosa. These two tissues exhibit different modes of actin assembly at wound edges and this project will involve comparative analysis and manipulation of wound healing in these two tissues. We will determine the influence of tension, adhesion and gene expression on actin assembly at the wound edge, and investigate the roles of Rho GTPases and PI 3-kinase in regulating actin dynamics.

1. POTENTIAL IMPACT ON HUMAN HEALTH. Epithelia provide essential protection against infective agents, toxins and fluid loss, therefore it is vital that damaged epithelia are repaired as rapidly as possible. The development of therapies to improve or accelerate epithelial repair would therefore greatly benefit human health. This project will explore how epithelial repair is regulated and how the characteristics of the wound environment affect the mechanism of repair. Our findings will assist the development of therapies to improve epithelial healing in two ways: 1. they will elucidate the signalling mechanisms that regulate epithelial wound healing, which will help in the identification of drug targets. 2. They will increase our understanding of the factors that influence the mechanism of repair, which will help in predicting the appropriate treatment for a particular tissue or wound. Our findings may also impact on human health by increasing our understanding of tumour metastasis and developmental processes such as neural tube closure, which also involve motility of epithelial cells and are likely to involve similar signalling pathways to epithelial wound healing. Our experiments will be performed in Drosophila embryos, and will make use of powerful genetic and imaging techniques not possible in higher organisms, which allow us to explore the cellular, molecular and mechanical mechanisms that underlie epithelial repair. An additional benefit of studying tissue repair in Drosophila is that it is consistent with the 3R's. An important next step towards realising the potential impact of our research is to test whether our findings are relevant in more complex animals and ultimately in patients. Being based in The Healing Foundation Centre we are well placed to do this, as we work alongside a number of groups studying tissue repair in vertebrate systems (E. Amaya, K. Mace, M. Hardman). We meet weekly, providing an excellent forum for discussion and collaboration. We also have regular meetings with clinicians (including G. McGrowther, J. Temple) in which we discuss the clinical applications of our research. During the course of this project we will maintain a constant discourse with our scientific and clinical colleagues, and with their assistance, explore all options for moving the findings of this research project towards clinical application. As well as being an excellent system for studying the cell biology of epithelial repair, the Drosophila embryo is also a potentially useful system for testing drugs and exploring their pharmacology. We can readily treat embryos with drugs and analyse the effect on epithelial healing. As we can image epithelial repair live and at high resolution, we can derive detailed information about the effect of a drug. The genetically tractability of Drosophila makes it useful for exploring the drug pharmacology. We have contacts within the biotech company Renovo and we will discuss with them the possibility of use our system in this way. 2. PUBLIC ENGAGEMENT. Tissue repair is a subject of great interest to the public, making this research ideal for public engagement. Our research generates striking images and our time-lapse movies bring complicated biological processes to life for lay audiences. Our research is thus ideal for promoting cutting edge science to the public and we are active contributors to such activities. For instance, TM has presented his work in a public lecture at the Birmingham museum ThinkTank, and regularly provides school pupils with the opportunity to visit his lab. These public engagement activities will be continued and expanded during the project. The Faculty has a dedicated media advisor who has been very successful in disseminating our research to the national and international media. The University also houses a popular museum (The Manchester Museum), which provides a direct and permanent link between our research activities and the local public.