Integrative study of Unc5 signalling

Context of the research:
How does the brain work? This is one of the most fundamental questions a human being can ask. There are many sides to this question, philosophical as well as biological. My project focuses on particular proteins, the so-called Unc5 receptors, that are located on the surface of cells. Much like a navigation system, these proteins direct brain cells along the right paths and help them connect to each other. This gives rise to the neuronal networks that underlie the functions of our brain. These proteins also direct other cells in our body. For example, Unc5 receptors control the development of blood vessels is. They do this by regulating those cells that are responsible for growing new blood vessels. Some aspects of how Unc5 receptors guide and direct neurons and other cells are understood. Just like a navigator would do, they recognise specific signals (in this case other compatible molecules, referred to as ligands) in their environment. The recognition event involves physical interaction of the Unc5 receptor and ligands. Most ligands of Unc5 receptors also interact with other molecules. The result is a network of interactions, with Unc5 receptors constituting major nodal points. How Unc5 receptors guide cells depends on the surrounding molecules present in its environment.

Aims and objectives:
How do Unc5 receptors interact with specific ligands, and how do these ligands interact with further molecules? I will study these interactions with high resolution methods to understand the molecular details that define them. Following this, I will use this information to understand the specific biological functions of each of the interactions. For example, I will test what happens when Unc5 no longer recognises one of its ligands. WIll this affect specific cell properties, leaving others unaffected? Will it affect the way that Unc5 moves around on the surface of cells? My experiments will answer these questions by zooming in to the cell surface, using advanced microscopy methods, and by looking at the behaviour of multiple cells, using cell biology assays.

Potential applications and benefits:
Unc5 receptors are involved in fundamental processes including brain development and blood vessel formation. My results will provide an essential piece to the puzzle of how these processes are regulated. Given this information, could potentially be used to design new drugs related to diseases of the vascular and neuronal systems.
Unc5 receptors also play important roles in human cancers. In most cases, cancer cells have a reduced ability to use Unc5 receptors for their guidance. By revealing how Unc5 receptors control cell behaviour, I will also generate knowledge for the specific treatment of cancers with Unc5 receptor-related defects.
All information generated by this project will be published and made available for further exploitation. During the course of the project, I will engage in public outreach activities to communicate aspects of my work to a broad community.

This study focuses on Unc5, a cell surface receptor that plays essential roles in neural/vascular tissues and in cancer.
I will reveal the molecular properties of Unc5-ligand interactions by solving crystal structures of the Unc5 ectodomain in complex with its ligands. To achieve this, I will express the proteins recombinantly in mammalian cell cultures and use automated methods to produce crystals. Using synchrotron beamlines to collect X-ray diffraction data from these crystals, I will calculate their structural models.
I will use Surface Plasmon Resonance (SPR) and Multi-Angle Light Scattering (MALS) experiments to characterise the binding properties of Unc5 and its ligands. Based on the structural data, I will design mutants with specific ligand-binding properties.
I will study the properties of these mutants, alongside the wild type proteins, using single molecule localisation microscopy and Stimulated Emission Depletion (STED) microscopy techniques. I will thereby reveal their localisation and diffusion patterns at the cell surface.
Using cell biology assays, I will reveal the effects of Unc5-ligand interactions on cell behaviour (adhesion, repulsion, migration and proliferation). I will use a range of assays to achieve this. For example, I will perform "stripe" assays where cells choose between growing on alternating surface stripes: one coated with the protein of interest, the other coated with a neutral control protein. I will also perform assays in which cells (e.g. neurons) expressing Unc5 receptor choose the direction in which they grow within in a gradient of ligand protein. In collaboration, I will also reveal the effects of Unc5-ligand interactions in model tissues.
As a result, the proposed work will constitute a necessary step towards understanding this medically important signalling system on the molecular, cellular and tissue levels and generate knowledge for the specific treatment of cancers with defects in the Unc5 signalling system.

The proposed project focuses on an important biological system, the Unc5 receptors, which lie at the centre of brain development, blood vessel formation and cancer development. By studying these receptors, I will generate medically important results that will strengthen the UK's position in this research field. I will promptly publish my results and make them available to the community to maximize impact.

With respect to the commercial private sector, the results will benefit particularly the pharmaceutical and biotechnology industries. For example, the information that I will generate could lead to the identification of new screens and biomarkers, new drug targets and drugs, and new protocols for treating patients. These developments will be important both for the medical industry and the benefitting patients.

I will be involved in the training of undergraduate and graduate students. Some of these students choose careers in the above mentioned sectors, thereby benefitting these directly. More generally, by providing students with specific training, I will contribute to their career prospects and increase the pool of skilled workers that underlies the UK economy.

The proposed project will also benefit the general public, through the presentation of aspects of my research at public outreach events. For example, I have recently joined STEMNET, an organisation through which I will present aspects of my work in local schools. I will also help organize the annual UNIQ summer school at the Department of Biochemistry, which is designed to show A-level students what undergraduate study at Oxford University would be like.

It is difficult to estimate the exact timescales on which outcomes of this fundamental science project can directly result in application. However, I will facilitate this process by making my results publicly available and actively pursuing take-up within the relevant industries.