Regulation of the Rho GEF Pebble in Fibroblast Growth Factor dependent cell migration

The capability of cells to move is a highly regulated process in the human body. Cells can send and received signals that enable them to change position in an organism in a controlled fashion. These signals act upon a machine within the cell called the cytoskeleton, which allows cells to propel themselves forward in an organism or upon a substrate. Defects in the regulation of cell migration are the cause of many severe human diseases and a plethora of congenital disorders. Therefore to investigate the molecular detail of the signals and their action in cell migration is of utmost importance for our understanding of human diseases and to develop treatments against them.

Research on human cellular disease pathways is often hampered by the fact that we cannot do experiments with humans for obvious reasons. One possibility is to utilise diseased cells or tissues, but these are no longer in contact with the entire organism and thus only provided limited insight. Another possibility is the use of so called model organisms. As the principal genetic repertoire of all multicellular animals is surprisingly similar, we can use simple organisms to investigate human disease pathways. Our laboratory is using the fruit fly Drosophila melanogaster, which has been employed for over 100 year in biomedical research. The power of Drosophila lies in its ease to manipulate its genes by mutations and other modifications. Our previous research has identified a signalling pathway triggered by a Fibroblast Growth Factor that is responsible for the onset and the directionality of cell migration in the fly embryo. As very similar molecules exerts similar functions in human embryos, our research results are transferable to humans.

To identify the molecules that are involved in the regulation of cell migration through Fibroblast Growth Factor signalling we have conducted large scale genetic experiments. These genetic screens found mutations in other genes, which we then showed to be important for the regulation of the cytoskeleton during cell migration. In this research project we plan to determine how this regulation occurs on the molecular level and how the growth factor can modify the activity and the localisation of an important regulator of the cytoskeleton. We expect that the results from this research will provide new opportunities for research in drug discovery projects to establish assays that can detect small compounds leading to novel treatments of diseases in which cell migration is abnormal.

The induction of cell movements is an important cellular response elicited by Fibroblast growth factor (FGF) receptor signalling. Despite its relevance in human disease pathways, the molecular basis of FGF-dependent cell movements is only poorly understood. Our previous work has established that the fly homolog of the human proto-oncogene Ect2, called Pebble (Pbl) is required for FGF-dependent cell migration. These molecules are conserved in humans and are involved in controlling cell migration in normal and cancer cells. We discovered that the conserved carboxy-terminal tail of Pbl is important for the control of its substrate specificity and its localisation to the cell cortex in FGF-dependent cell migration, but it is unknown how FGF-signalling controls Pbl activity. To address these questions we propose to investigate the molecular requirement for the Pbl protein in cell migration and to identify and characterise regulators of Pbl using biochemical, molecular and genetic approaches. We will determine the specific molecular requirements of Pbl in FGF-dependent cell migration using a mutagenesis screen. We will identify posttranslational modifications of Pbl in response to FGF signalling and analyse their biological significance in transgenic animals. We will use proteomics and analyse novel mutants to identify proteins and genes that are involved in the regulation of Pbl in cell migration. Together these approaches will allow us to define the protein domains and amino acid motifs that are important for the regulation of Pbl in FGF-dependent cell migration and regulatory interaction partners of Pbl. Identifying those domains and their regulators will help to design assays that can be used to inhibit deregulated FGF-signalling and hyper-activated Ect2 in abnormal cell migration in diseases including cancer.

It is the nature of basic research that the economic and societal impact of the research results are rarely immediate, but that the advances in academic knowledge by basic research data are fundamental to the economic growth in moderns societies. For academic impact please see section on academic beneficiaries.

The immediate economic impact that this research project will produce is the training and employment of a postgraduate researcher and a bio-technological technical assistant, respectively. The training aspect for a postdoctoral scientist in this project is particularly high as it will encompass various disciplines, from molecular biology, high resolution imaging, genetics through modern quantitative proteomics. The major non-academic beneficiaries of this research are members of the public, including S2 pupils, teachers and any other interested individuals. This research uses the invertebrate model organism Drosophila, a classic genetic model used for research and education. In our research project we employ modern imaging techniques and state-of-the-art molecular and genetic methods. As outlined in 'pathways to impact' we plan a number of activities during the duration of the grant that are tailored towards specific interest groups.

An increasingly important potential impact of this particular research are the exploitation of the research data by drug discovery programmes. Our previous research has already informed research in mammalian systems, where a similar requirement of Ect2 and FGF signalling was found for cell migration in the mouse embryo or in cancer cells. The possibility of drug discovery approaches based on mutational, functional analyses of conserved pathways in Drosophila has now been realised in a small number of laboratories. More precisely, fruit flies can be used in whole animal drug discovery approaches once a clinically relevant pathway can be reconstituted as an adult phenotype that exhibits a high dynamic range - for example in the compound eye. In the present research project, we have made use of a phenotype in the adult compound eye to identify genetic interactors of oncogenic mimic gain-of-function forms of Pbl; in principle this assay could also be used to screen for small compounds that suppress this phenotype and thus the abnormal activity of Pbl. In the past such approaches have been successful and Drosophila-based screens may provide a unique opportunity to screen for bioactive compounds in a whole animal and on a large scale.