Integrative study of Unc5 signalling
Lead Research Organisation:
University of Oxford
Department Name: Biochemistry
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
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.
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.
Organisations
- University of Oxford (Lead Research Organisation, Project Partner)
- UNIVERSITY OF OXFORD (Collaboration)
- Max Planck Society (Collaboration)
- Rutherford Appleton Laboratory (Collaboration)
- European University Viadrina Frankfurt (Oder) (Collaboration)
- Uppsala University (Collaboration)
- University of Würzburg (Collaboration)
- Max Planck Institutes (Project Partner)
People |
ORCID iD |
Elena Seiradake (Principal Investigator) |
Publications
Ando T
(2022)
Tumor-specific interendothelial adhesion mediated by FLRT2 facilitates cancer aggressiveness.
in The Journal of clinical investigation
Araç D
(2016)
Understanding the Structural Basis of Adhesion GPCR Functions.
in Handbook of experimental pharmacology
Chavent M
(2016)
Structures of the EphA2 Receptor at the Membrane: Role of Lipid Interactions.
in Structure (London, England : 1993)
Chavent M
(2018)
Interactions of the EphA2 Kinase Domain with PIPs in Membranes: Implications for Receptor Function.
in Structure (London, England : 1993)
Chu TY
(2022)
GPR97 triggers inflammatory processes in human neutrophils via a macromolecular complex upstream of PAR2 activation.
in Nature communications
Jackson VA
(2018)
Structures of Teneurin adhesion receptors reveal an ancient fold for cell-cell interaction.
in Nature communications
Jackson VA
(2015)
Structural basis of latrophilin-FLRT interaction.
in Structure (London, England : 1993)
Jackson VA
(2016)
Super-complexes of adhesion GPCRs and neural guidance receptors.
in Nature communications
Johnson E
(2015)
Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins.
in Scientific reports
Seiradake E
(2016)
Structural Perspectives on Axon Guidance.
in Annual review of cell and developmental biology
Description | Pump priming award for research on adhesion GPCRs |
Amount | £8,615 (GBP) |
Funding ID | MRF/TT2015/2154 |
Organisation | University of Oxford |
Sector | Academic/University |
Country | United Kingdom |
Start | 06/2015 |
End | 06/2016 |
Description | Pump priming award for the purchase of a microscope |
Amount | £7,400 (GBP) |
Funding ID | 0003567 |
Organisation | University of Oxford |
Department | John Fell Fund |
Sector | Academic/University |
Country | United Kingdom |
Start | 06/2015 |
End | 12/2015 |
Description | Senior Research Fellowship in Basic Biomedical Science |
Amount | £1,810,007 (GBP) |
Funding ID | 202827/Z/16/Z |
Organisation | Wellcome Trust |
Department | Wellcome Trust Bloomsbury Centre |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2017 |
End | 12/2021 |
Description | Prof. Betsholtz and Prof. Langenhan, experts on adhesion GPCRs |
Organisation | University of Wurzburg |
Country | Germany |
Sector | Academic/University |
PI Contribution | My research also focuses on neural and vascular adhesion G-protein coupled receptors (GPCRs) and their protein complexes with other proteins, such as Uncoordinated-5. Adhesion GPCRs were recently shown to act as mechanosensitive receptors by the Langenhan team and others. In this recently established collaboration, I provide crystallography data that enable the design of specific experiments to understand the structural mechanisms in adhesion GPCR mechanosensation. I also provide protein and DNA reagents for collaborative experiments in the Betsholtz team, which involve genetically modified mice and mouse tissues. |
Collaborator Contribution | The Langenhan lab provides access to a specialised experimental set-up that allows the characterisation of adhesion GPCR responses to mechanical force. I know Prof. Langenhan well from repeated visits and have hosted members of his team in my lab for previous collaborative work on fly Latrophilins (manuscript in preparation). The Betsholtz team recently generated a new adhesion GPCR (GPR116) knock-out mouse line and provides access to assays and in vivo experiments to study GPR116 in the lung and at the blood brain barrier. The team provides access to specific experiments aiming at understanding adhesion GPCR functions in these tissues. |
Impact | The collaboration is multidisciplinary, combining structural biology, biophysical analysis and cell biology (Seiradake team) with mouse genetics (Betsholtz team) and specialised set-ups for measuring mechanical force (Langenhan team). |
Start Year | 2015 |
Description | Prof. Betsholtz and Prof. Langenhan, experts on adhesion GPCRs |
Organisation | Uppsala University |
Country | Sweden |
Sector | Academic/University |
PI Contribution | My research also focuses on neural and vascular adhesion G-protein coupled receptors (GPCRs) and their protein complexes with other proteins, such as Uncoordinated-5. Adhesion GPCRs were recently shown to act as mechanosensitive receptors by the Langenhan team and others. In this recently established collaboration, I provide crystallography data that enable the design of specific experiments to understand the structural mechanisms in adhesion GPCR mechanosensation. I also provide protein and DNA reagents for collaborative experiments in the Betsholtz team, which involve genetically modified mice and mouse tissues. |
Collaborator Contribution | The Langenhan lab provides access to a specialised experimental set-up that allows the characterisation of adhesion GPCR responses to mechanical force. I know Prof. Langenhan well from repeated visits and have hosted members of his team in my lab for previous collaborative work on fly Latrophilins (manuscript in preparation). The Betsholtz team recently generated a new adhesion GPCR (GPR116) knock-out mouse line and provides access to assays and in vivo experiments to study GPR116 in the lung and at the blood brain barrier. The team provides access to specific experiments aiming at understanding adhesion GPCR functions in these tissues. |
Impact | The collaboration is multidisciplinary, combining structural biology, biophysical analysis and cell biology (Seiradake team) with mouse genetics (Betsholtz team) and specialised set-ups for measuring mechanical force (Langenhan team). |
Start Year | 2015 |
Description | Prof. Dame Carol Robinson, expert in mass spectrometry |
Organisation | University of Oxford |
Department | Department of Chemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | My team uses a multidisciplinary approach to study neural and vascular receptors such as Uncoodinated-5 (Unc5) at the molecular, super-resolution and cellular, and tissue levels. We reveal novel X-ray crystallography, cell biology, confocal and super resolution microscopy data. My team also produces the high quality protein samples required for the analysis by mass spectrometry. |
Collaborator Contribution | The prestigious Robinson lab provides access to world-class mass spectrometry facilities and pioneering expertise in studying the 3D structure of proteins using mass spectrometry. These tools contribute to the characterisation of protein-ligand complexes and infer conclusions about their composition. |
Impact | This recently established collaboration contributed to my most recent paper (Jackson et al. Nature Communications 2016 (in press)). The collaboration is multidisciplinary: the Robinson team specialises in mass spectrometry, while my lab focuses on structural biology, cell biology and additional biophysical techniques (multi-angle light scattering, surface plasmon resonance). |
Start Year | 2015 |
Description | Prof. Mark Sansom, expert in molecular dynamics simulations |
Organisation | University of Oxford |
Department | Department of Biochemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | My lab studies cell guidance receptors involved in brain development, blood vessel formation and cancer. I use a range of powerful and cutting-edge techniques, especially X-ray crystallography, advanced cell biology assays, confocal and super resolution microscopy. Combining multiple techniques allows me to understand how membrane proteins, especially cell guidance receptors, function on the molecular, cellular and tissue levels. |
Collaborator Contribution | The Sansom lab are world-leading experts in all areas of computational studies of membrane proteins and related systems, ranging from molecular simulations of channels and transporters, to computational bionanoscience, and membrane protein folding and stability. The team also provides insights using multi-level simulations of membrane proteins in the context of addressing the 'gap' between molecular and systems descriptions of membranes. In contributing to our collaborative projects, the Sansom lab use structural models generated in my lab to model the stability of complexes and the interaction of proteins with lipidic membranes. |
Impact | Collaborative results are summarised in: Chavent et al. Structure 2016 Jackson et al. Nature Communications 2016 (in press) The collaboration involves the following disciplines: structural biology, biophysical analysis, cell biology (Seiradake lab), molecular dynamics simulations of proteins and lipid membranes (Sansom lab) |
Start Year | 2015 |
Description | Prof. Ruediger Klein and Prof. Amparo Acker-Palmer, experts in cell biology and in vivo mouse tools |
Organisation | European University Viadrina Frankfurt (Oder) |
Country | Germany |
Sector | Academic/University |
PI Contribution | My lab uses cutting-edge structural biology, biophysical and cell biology techniques to study membrane receptors in the neural and vascular systems. These techniques are highly complementary to those used in the Klein and Acker-Palmer labs. Our collaboration focuses on proteins involved in cell guidance during cortical development and vascular development, especially Latrophilin/Unc5/Flrt and Teneurin. |
Collaborator Contribution | I have an excellent established collaboration with the Klein lab, which provides access to cutting-edge in vivo tools using wild type and knock-out mouse lines. Previously, we generated ground-breaking results summarised in Seiradake et al. 2013 and 2014, Jackson et al. 2015 and 2016. Most recently, we established a collaborative project to study the function of Latrophilin/Unc5/Flrt and Teneurin in brain development, for which the Klein lab provide access to in vivo tools and specific mouse lines. The Acker-Palmer lab provides access to specialised in vivo experiments to study the functions of Latrophilin, Unc5 and Flrt in vascular development and cancer. These experiments involve wild type and knock-out mouse lines and are ideal to complement the in vivo work planned with the Klein lab, with whom the Acker-Palmer lab also collaborate. |
Impact | The collaboration with Prof. Klein contributed to results summarised in the following publications: Seiradake et al. Nat. Struct. Mol. Biol. 2013 Seiradake et al. Neuron 2014 Jackson et al. Structure 2015 Jackson et al. Nat. Commun. 2016 (in press) The collaboration with Prof. Acker-Palmer contributed substantially to: Seiradake et al. Neuron 2014 The collaboration is multidisciplinary, combining structural biology, biophysical analysis and cell biology (Seiradake team) with mouse genetics and in vivo tools to study processes in the neural system (Klein team) and in the vascular system (Acker-Palmer team). |
Start Year | 2011 |
Description | Prof. Ruediger Klein and Prof. Amparo Acker-Palmer, experts in cell biology and in vivo mouse tools |
Organisation | Max Planck Society |
Country | Germany |
Sector | Charity/Non Profit |
PI Contribution | My lab uses cutting-edge structural biology, biophysical and cell biology techniques to study membrane receptors in the neural and vascular systems. These techniques are highly complementary to those used in the Klein and Acker-Palmer labs. Our collaboration focuses on proteins involved in cell guidance during cortical development and vascular development, especially Latrophilin/Unc5/Flrt and Teneurin. |
Collaborator Contribution | I have an excellent established collaboration with the Klein lab, which provides access to cutting-edge in vivo tools using wild type and knock-out mouse lines. Previously, we generated ground-breaking results summarised in Seiradake et al. 2013 and 2014, Jackson et al. 2015 and 2016. Most recently, we established a collaborative project to study the function of Latrophilin/Unc5/Flrt and Teneurin in brain development, for which the Klein lab provide access to in vivo tools and specific mouse lines. The Acker-Palmer lab provides access to specialised in vivo experiments to study the functions of Latrophilin, Unc5 and Flrt in vascular development and cancer. These experiments involve wild type and knock-out mouse lines and are ideal to complement the in vivo work planned with the Klein lab, with whom the Acker-Palmer lab also collaborate. |
Impact | The collaboration with Prof. Klein contributed to results summarised in the following publications: Seiradake et al. Nat. Struct. Mol. Biol. 2013 Seiradake et al. Neuron 2014 Jackson et al. Structure 2015 Jackson et al. Nat. Commun. 2016 (in press) The collaboration with Prof. Acker-Palmer contributed substantially to: Seiradake et al. Neuron 2014 The collaboration is multidisciplinary, combining structural biology, biophysical analysis and cell biology (Seiradake team) with mouse genetics and in vivo tools to study processes in the neural system (Klein team) and in the vascular system (Acker-Palmer team). |
Start Year | 2011 |
Description | Single molecule tracking of cell surface receptors |
Organisation | Rutherford Appleton Laboratory |
Department | Central Laser Facility |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We produce cellular samples for the study and conduct the experiments under expert supervision |
Collaborator Contribution | The Martin-Fernandez team provide expert supervision for the experiments and assist with data analysis. |
Impact | This is a multi-disciplinary collaboration, combing structural biology, computer simulations and single molecule tracking. The labs involved are Seiradake and Sansom (Oxford University) and Martin-Fernandez (STFC central Laser Facility). |
Start Year | 2016 |
Description | School Visit (Gosford Hill) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | I led discussions at an event aimed at encouraging girls to enter science careers (organised by cogent skills and the WISE campaign, at Gosford Hill School, Kidlington, Oxfordshire). The event was well-received by the students, who were interested and engaged in discussions. |
Year(s) Of Engagement Activity | 2015 |
Description | Work placements, A-level students |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | I hosted work placements of two A-level students, Anna Maria Beauclerk (St. Gregory the Great, Oxford) and Lok Yee Chow (Rye St. Anthony, Oxford), in my lab. The students were enthusiastic and their frequent questions sparked in-depth scientific discussion. They both reported that the experience was very useful and furthered their interest in pursuing science-related careers. |
Year(s) Of Engagement Activity | 2015 |
Description | work placements, A-level students |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | I hosted work placements of two A-level students, Rachel Jimminson (Watford Grammar School for Girls, Watford) and Imogen Dickens (Oxford High School, Oxford), in my lab. They were enthusiastic students and both reported that the experience was useful and furthered their interest in pursuing science-related careers. |
Year(s) Of Engagement Activity | 2016 |