ANNUAL RESEARCH SYMPOSIUM - Jo Berry€¦ · SYMPOSIUM FRIDAY 29th SEPTEMBER 2017 East Midlands Conference Centre, University Park, Nottingham, NG7 2RJ PROGRAMME. 2 CONTENTS General

ANNUAL RESEARCH SYMPOSIUM

FRIDAY 29th SEPTEMBER 2017

East Midlands Conference Centre, University Park, Nottingham, NG7 2RJ

PROGRAMME

2

CONTENTS

General Information 3

Programme 4

Invited Speakers 5

Jo Berry - Artist 6

ABSTRACTS - Poster presentations 8

PARTICIPANTS 54

COMPARE Directors and PIs 58

International Advisory Board 60

Strategic Oversight Group 60

Support Team 61

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General Information

Poster presentations

Posters should be set up between 9:00-9:45am and removed at 17:00 (please note that the last poster session is between 16:05-17:00 so posters should not be removed before this). Velcro strips will be provided for fastening posters to boards. Poster board format is A0 portrait. All poster presenters should be at their posters during poster sessions.

Poster sessions:

12:50 – 14:00

16:05 – 17:00

Posters are numbered P1-P38. Please check the abstracts at the back of the programme for the number of your poster.

Venue address

EMCC, Beeston Lane, Nottingham. For travel by car, use the following navigation coordinates (52.938863, -1.203153) and postcode (NG7 2RJ) to find the conference centre.

Parking

Guests are entitled to complimentary car parking.

Wi-Fi

Free, Wi-Fi with a maximum bandwidth of 1GB is available throughout the venue.

Wifi address - DeVere East Midlands Conf Centre, accept the Terms and Conditions and it will connect.

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Programme

Time Session Speaker

9:30 Registration and coffee Atrium

Session Chair: Steve Watson, University of Birmingham

09:50 Welcome and introduction Steve Hill University of Nottingham

10:00 RAMPs and Atypical Chemokine Receptors Kathleen Caron University of North Carolina, USA

Session Chair: Natalie Poulter, University of Birmingham

10:40 Single-molecule imaging of GPCR signalling Davide Calebiro University of Birmingham

11:05 GPCR signalling platforms for pain and analgesia Meri Canals Monash University, Australia

11:30 Imaging coronary microcirculation in the beating murine heart

Neena Kalia University of Birmingham

11:45 The configuration of GPVI drives the binding with fibrin and collagen and its role in thrombus formation

Marie-Blanche Onselaer University of Birmingham

12:00 Team Science Team Science Committee

12:05 Lunch Atrium

12:50 Poster session Atrium

Session Chair: Steve Hill, University of Nottingham

14:00 Endosomal Platforms for the Signaling Train to Pain

Nigel Bunnett Colombia University, USA

14:40 Dynamics in GPCR signalling Dmitry Veprintsev University of Nottingham

Session Chair: Jeanette Woolard, University of Nottingham

15:05 Expansion microscopy as a tool for 3D super-resolution imaging

Rob Neely University of Birmingham

15:20 Next generation approaches for understanding GPCR/ion channel function in diabetes

David Hodson University of Birmingham

15:35 Illuminating Receptor-Ligand Interactions with Fluorescent Ligands

Barrie Kellam University of Nottingham

15:50 Using BRET to quantify the molecular pharmacology of the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2).

Laura Kilpatrick University of Nottingham

16:05 Poster session Atrium, refreshments available

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Invited Speakers Steve Hill

Co-Director of COMPARE University of Nottingham

Kathleen Caron

Professor and Chair of Department of Cell Biology and Physiology University of North Carolina

Davide Calebiro

Professor of Molecular Endocrinology University of Birmingham

Meri Canals

Monash Fellow Monash University

Neena Kalia

Senior Lecturer in Microcirculation Research University of Birmingham

Marie-Blanche Onselaer Research Fellow University of Birmingham

Nigel Bunnett

6

Professor of Surgery Colombia University

Dmitry Veprintsev

Professor of Molecular and Cellular Pharmacology University of Nottingham

David Hodson

Professor of Cellular Metabolism University of Birmingham

Laura Kilpatrick

Research Fellow University of Nottingham

Jo Berry - Artist

Rob Neely

Senior Lecturer in Physical Chemistry University of Birmingham

Barrie Kellam

Professor of Medicinal Chemistry University of Nottingham

7

I exhibit regularly and widely throughout the Country and internationally with pieces in the Victoria & Albert Museum (V&A), Arts Council England (ACE) East Midland Collections, Nottingham University Medical School and Zeiss, Munich, Germany. Residencies include the Florence Trust Studios, London, the Natural History Museum, London, and the Lakeside Arts Centre, University of Nottingham. Public art commissions include Millfield Sculpture Commission, Derbyshire Moorlands, Sheffield Galleries and Museums Trust, New Shetland Museum & Archives and Blackpool Illuminations. My research involves collaborative ventures with scientists who specialise in advanced imaging, including;

Cell Signalling and Pharmacology Group (CS&PG) and Molecular and Cellular Biology Group (MCB), University of Nottingham.

Versatile Imaging as a three-dimensional sketch at the Natural History Museum (NMH), London.

Dermal drug delivery – How to increase bioavailability in viable skin. A multidisciplinary project, Centre for Cellular Imaging (CCI) Sahlgrenska Academy Gothenburg University, Chalmers and Malmo University.

It is generating new knowledge from scientific subject–matter through visual investigation and has opened up a ‘space’ for new engagement and interaction adding to knowledge of how art and science can connect. Links are being established further through a network of art and science organisations, funders, practitioners, academics and the media, including Zeiss, the Royal Microscopical Society and the British Pharmacological Society.

Confocal Spinal Chord

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ABSTRACTS - Poster presentations (12:50 & 16:05)

9

ABSTRACT TITLES

P1

Use of Fluorescence Correlation Spectroscopy to study the diffusion of μ-opioid receptor in the membranes of single cells.

Arisbel Batista-Gondin

P2

Zeb1 controls endothelial quiescence through metabolic reprogramming.

Andrew Benest

P3

Use of fluorescent ligands to determine ligand binding kinetics of the adenosine A3-receptor in living cells.

Monica Bouzo-Lorenzo

P4

Studying functional and binding properties of CXCR4 intracellular loop 1 pepducins by GloSensorTM and NanoBRET.

Birgit Caspar

P5

The use of NanoBRET to determine the binding affinity of A1 adenosine receptor agonists and antagonists in both rat and human receptor-subtypes.

Samantha Cooper

P6

Cac and ca-a1D VGCC subunits regulate tissue integrity and tumour progression in the Drosophila dorsal epithelium.

Africa Couto

P7

Fluorescence correlation spectroscopy for the analysis of cell surface receptors at very low levels of expression.

Joëlle Goulding

P8

Functional genetics of lung function associated gene GPR126.

Robert Hall

P9

Effect of C terminus Truncations on light induced signalling and internalisation of optogenetic Rhodopsin-α1A Adrenoceptor chimeras.

Nicholas Holliday

10

P10

Human multidrug transporter ABCG2 is purified for fluorescence based single-particle analysis via SMALP mediated extraction.

Aaron Horsey

P11

Modelling cytoskeletal gene mutations implicated in inherited thrombocytopenia using CRISPR edited iPSC derived megakaryocytes and super-resolution microscopy.

Abdullah Khan

P12

Characterisation of ADAM10-regulating tetraspanins Tspan14 and Tspan15 on platelets using new monoclonal antibodies.

Chek Ziu (Connie) Koo

P13

Vasculoprotective effects of haematopoietic stem cells in myocardial ischaemia/reperfusion (IR) injury

Adam Lokman

P14

Dissecting the spatio-temporal dynamics of GPCR during memory formation in Drosophila neurons.

Isabella Maiellaro

P15

Regulation of the platelet collagen receptor GPVI by ADAM10 and tetraspanins.

Alexandra Matthews

P16

Development of a fluorescent antagonist binding assay for the Gastrin Releasing Peptide BB2 Receptor.

Liciane Medeiros

P17

Novel fluorescent β-adrenoceptor ligands.

Sarah Mistry

P18

Expansion microscopy as a tool for 3D super-resolution imaging.

Robert Neely

11

P19

The configuration of GPVI drives the binding with fibrin and collagen and its role in thrombus formation.

Marie-Blanche Onselaer

P20

Receptor Clustering Prevents GPVI Shedding And Leads To Sustained Signalling.

Chiara Pallini

P21

Measuring binding of fluorescent variants of VEGF165a, VEGF165b and VEGF121a to VEGFR2 using NanoBRET.

Chloe Peach

P22

Persistent homology as a tool to classify topology in single molecule localization microscopy.

Jeremy Pike

P23

The podoplanin-CLEC-2 axis inhibits inflammation in sepsis.

Julie Rayes

P24

The use of Fluorescence Correlation Spectroscopy to investigate ligand-receptor interaction at the Neuropeptide Y Y1 receptor.

Rachel Richardson

P25

EP4 receptor agonists inhibit cellular responses in airway remodelling.

Elizabeth Rosethorne

P26

Unnatural amino acid mutagenesis and photoaffinity cross-linking to determine the binding site for CGRP at its receptor.

John Simms

P27

Structural insights into GPCR activation using essential dynamics and molecular dynamics simulations.

John Simms

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P28

Investigating the regulatory mechanisms of actin nodule formation and turnover in platelets.

Victoria Simms

P29

Platelet membrane receptor glycoprotein Ib (GP)Ib-IX complex analysed using dSTORM super-resolution light microscopy.

Alex Slater

P30

DNA origami: a tool for single molecule localisation microscopy.

Darren Smith

P31

Using NanoBRET to quantify antibody binding to a thermostabilised turkey β1-adrenoceptor.

Mark Soave

P32

Large-scale screening for membrane protein interactions involved in platelet-monocyte interactions

P33

Role of endothelial cell ADAM10 and TspanC8 tetraspains in angiogenesis.

Justyna Szyroka

P34

Tetraspanin Tspan18 promotes haemostasis and thrombo-inflammation through regulation of Orai1-dependent store-operated Ca2+ entry.

Michael G Tomlinson

P35

Production of antibodies against the SMALP-solubilised adenosine 2a receptor by phage display.

Romez Uddin

P36

The inner workings of a GPCR: Signalling-specific conformations of vasopressin V2 receptor.

Dmitry Veprintsev

13

P37

Utilising CRISPR/Cas9 and NanoBRET to monitor ligand binding at G protein-coupled receptors under endogenous promotion.

Carl White

P38

Light sheet microscopy: revealing platelet biology in 4 dimensions.

Malou Zuidscherwoude

P39

Expansion Microscopy (ExM) as a tool for investigating DNA repair

Emma Faulkner

P1

Use of Fluorescence Correlation Spectroscopy to study the diffusion of μ-opioid receptor in the membranes of single cells. Arisbel Batista-Gondin 1,2, Michelle Halls2, Meritxell Canals2, Stephen Briddon1

1 University of Nottingham, UK 2 Monash University, Australia.

The μ-opioid receptor (MOR) is a G protein-coupled receptor (GPCR) with physiological importance in mediating the analgesic effects of opioids such as morphine. The differential regulation of this receptor could contribute to the adverse effects of opioids, limiting their clinical use. Regulation of MOR occurs via multiple mechanisms including receptor desensitization, internalization, recycling and plasma membrane redistribution (Halls et al, 2016).

In this study, Fluorescence Correlation Spectroscopy (FCS) was used to investigate the diffusion properties and the molecular brightness of SNAP-MOR stably expressed in the human embryonic kidney (HEK) 293 cell line.

Our data revealed that stimulation of MOR with morphine, an agonist that causes low receptor internalization, does not change the basal diffusion properties of the receptor. In contrast, stimulation with DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin), an agonist that causes high receptor internalization, slows down the diffusion of the receptor within the plasma membrane.

The lateral diffusion of MOR within different microdomains of the plasma membrane upon activation by distinct opiate analgesics could contribute to the signalling elicited by agonists and ultimately to their clinical effects for the treatment of pain. Further studies will investigate what factors and signalling components contribute to the lateral diffusion of MOR and visualise such redistribution through high-resolution microscopy techniques.

Halls IP et al, (2016), Plasma membrane localization of the μ-opioid receptor controls spatiotemporal signalling. Sci Signal, 9:414.

P2

Zeb1 controls endothelial quiescence through metabolic reprogramming. Andrew Benest, Joshua Bourne, Pamela Collier, Spandan Kalra, Chris Denning, Anna Grabowska, David Bates University of Nottingham, UK

Blood vessel formation is a multistep process, involving the coordinated activity of multiple cell types (mainly endothelial cells (ECs) and pericytes) and involvement of multiple different signalling pathways. In addition to cytokine signalling in response to hypoxia, angiogenesis can be activated by changing the metabolic state of the endothelial cell.

Recent work has demonstrated the dynamic interplay between ‘quiescence’ factors (such as FOXO1 expression) which reduce the activity of glycolytic activity of PFKFB3 and c-MYC. Here we show that Zeb1 (a transcription factor with known functions in cancer progression) is expressed by quiescent (non-angiogenic) endothelial cells and is silenced in angiogenic endothelial cells in the developing murine retina.

In vitro analyses demonstrates that Zeb1 is expressed by confluent monolayers of Human Vein Umbilical Cord Endothelial cells (HUVECs) but is downregulated in actively growing cells. Zeb1 knockdown promotes an angiogenic EC phenotype: including enhanced PFKFB3, c-myc and VEGFR2 and reduced FOXO1, Tie2 and VE-cadherin expression. Consistently, Zeb1 knockdown EC are metabolic more active (both through oxidative and glycolytic pathways). Furthermore, loss of Zeb1 promotes EC migration and proliferation in vitro.

In conclusion, Zeb1 appears to be linked to key quiescence regulating signalling pathways and furthermore regulates aspects of these pathways. Zeb1 expression is a component of maintaining the quiescence endothelium.

P3 Use of fluorescent ligands to determine ligand binding kinetics of the adenosine A3-receptor in living cells. Monica Bouzo-Lorenzo1,

Lizi Xia 2, Leigh Stoddart 1, Laura Heitman 2, Steve Briddon1, Steve Hill1

1. University of Nottingham, UK 2. Leiden University, Netherlands The adenosine A3 receptor (A3AR) belongs to a family of four G-protein coupled receptors (A1, A2A, A2B and A3) that all respond to adenosine. A3AR couples mainly to Gi/o proteins and is involved in a variety of intracellular signalling pathways and physiological functions. Its peripheral location allows it to mediate a sustained cardio-protective function during cardiac ischemia, being involved in the inhibition of neutrophil degranulation in neutrophil-mediated tissue injury. It has also been implicated in neuroprotective and neurodegeneration. For all these reasons, A3AR has been extensively studied as a drug target.

Currently, the prediction of the in vivo efficacy of a drug is based mainly on steady-state metrics like affinity or potency values. However, the binding of a drug to its target is not static, is a dynamic and reversible process. For this reason, the usage of end point measurements has been leading during the last years to a very high attrition rates in drug discovery. In order to improve the prediction of the efficacy of a candidate drug is necessary to introduce in the studies the drug's binding kinetics which is the time it takes for a drug molecule to bind to and dissociate from its target protein (Guo et al, 2016).

One way to determine ligand-binding kinetics of receptors in their natural cellular environment is monitored in real time the interaction of fluorescently labelled agonists and antagonist with NLuc (nanoluciferase) tagged receptors using Bioluminescence Resonance Energy Transfer (BRET) (Stoddart et al, 2015). In this study, different fluorescent ligands for the adenosine receptors in combination with NLuc tagged A3 receptor were used to determine the kinetics of the ligand-receptor interactions in living cells using BRET.

Guo D et al, (2017), Kinetic Aspects of the Interaction between Ligand and G Protein Coupled Receptor: The Case of the Adenosine Receptors. Chem Rev, 117:38-66.

Stoddart LA, (2015), Application of BRET to monitor ligand binding to GPCRs. Nat Methods, 12:661-3.

P4

Studying functional and binding properties of CXCR4 intracellular loop 1 pepducins by GloSensorTM and NanoBRET.

Birgit Caspar, Joëlle Goulding, Leigh Stoddart, Stephen Briddon, Stephen Hill

University of Nottingham, UK

Lipidated peptides, known as pepducins (Covic et al, 2002), which are derived from the sequence of one of the internal loops of a GPCR, have been shown to act as either positive or negative allosteric modulators. Pepducins have been described for the chemokine receptor, CXCR4, which act as positive allosteric modulators and also show agonist behaviour in the absence of the endogenous ligand CXCL12. To date, their precise mode of action is unclear. In this study, we aimed to investigate the interaction of CXCR4 and intracellular loop 1 pepducins.

CXCL12 and pepducins showed the ability to inhibit forskolin mediated cAMP production in a GloSensorTM (Promega) cAMP assay in CXCR4-expressing HEK cells. Control pepducins without a lipid tail showed reduced potency. The agonist action of both CXCL12 and the pepducins was antagonised by the CXCR4 selective antagonist AMD3100 (0.1 to 10 µM). No significant effects were observed for CXCL12 or the pepducins in native HEK cells.

To further study the interaction of the pepducins with CXCR4, the binding of fluorescently labelled ligand was studied with a NanoBRET assay (Stoddart et al, 2015). Using HEK cells expressing CXCR4 tagged with NanoLuc on its N-terminus (NL-CXCR4), the affinity of fluorescent CXCL12 (CXCL12-red) was determined (pKd = 7.61 ± 0.10 nM, n = 5). The binding of CXCL12 to CXCR4 could be inhibited by pre-incubation with the small molecule antagonists AMD3100, IT1t as well as pepducins. Various control pepducins showed no ability to disrupt the binding between CXCL12 and CXCR4.

Our data shows that receptor-dependent signalling pathways can be activated by pepducins and that the lipid tail plays a crucial role in the binding of pepducins to CXCR4. The NanoBRET studies suggest that pepducins have a direct influence on the binding of CXCL12 to CXCR4. Further work will be aimed at understanding their exact mechanism of action.

Covic L et al, (2002), Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. Proc Natl Acad Sci, 99, 643–648.

Stoddart LA et al, (2015), Application of BRET to monitor ligand binding to GPCRs. Nat. Methods, 12: 661–663.

P5

The use of NanoBRET to determine the binding affinity of A1 adenosine receptor agonists and antagonists in both rat and human receptor-subtypes.

Samantha Cooper, Mark Soave, Stephen Hill, Jeanette Woolard

University of Nottingham, UK

The A1 adenosine receptor (A1AR) is a vital regulator of many biological functions and has been implicated as a therapeutic target for several disease indications (Borea et al, 2016), (Peleli et al, 2017). Here we have used a proximity assay to determine the binding affinities of A1AR agonists and antagonists to the human A1AR. Interestingly, compounds which are receptor-subtype specific in rats may not be as specific in humans (Klotz et al, 1997).

The specific binding of A1AR ligands was measured in both the rat and human A1AR using the previously characterised bioluminescence energy transfer (BRET) proximity assay (Stoddart et al, 2015). Here, the N-terminus of the human or rat A1AR was tagged with the NanoLuc luciferase and stably expressed in HEK293 cells. The xanthine amine congener (XAC)-based fluorescent ligand CA200645 was used to monitor the binding of unlabelled ligands in living cells using NanoBRET.

CA200645 specifically bound the A1AR in a concentration-dependant manner, resulting in a large specific NanoBRET signal, confirming CA200645 is a potent ligand of A1ARs with a log KD value of -7.51±0.41 in rat and -7.62±0.01 in human (n=5). We then carried out competition assays for the A1AR with CA200645 against the endogenous A1AR ligand adenosine, agonists (capadenoson and NECA) and antagonist (DPCPX). Competing ligands were added at increasing concentrations simultaneously with CA200645 (25nM) and incubated for 1 hour to determine ligand pKi values for A1ARs using the Cheng-Prussoff equation. Values obtained (mean + s.e.mean) were: for the rat Adenosine pKi=5.35 ± 0.09, capadenoson pKi=7.83 ± 0.09, NECA pKi=6.17 ± 0.08 and DPCPX pKi=9.28 ± 0.08. For the Human: Adenosine pKi=4.55±0.07, capadenoson pKi=6.86±0.14, NECA pKi=5.50±0.24 and DPCPX pKi=8.19±0.09 (n=5 in each case).

These data highlight the use of the NanoBRET proximity assay to study receptor-ligand interactions and have demonstrated species variation. Importantly, these data demonstrate the ability to measure agonist affinities with a high level of accuracy.

Borea PA et al, (2016), Adenosine as a Multi-Signalling Guardian Angel in Human Diseases: When, Where and how does it Exert its Protective Effects? Trends in Pharmacological Sciences, 37:419-434.

Peleli M et al, (2017), Pharmacological Targeting of Adenosine Receptor Signaling. Molecular Aspects of Medicine, 55:4-8.

Klotz KN et al, (1997), Comparative pharmacology of human adenosine receptor subtypes–characterization of stably transfected receptors in CHO cells. Naunyn-Schmiedeberg's archives of pharmacology, 357:1-9.

Stoddart LA et al, (2015), 'Application of BRET to Monitor Ligand Binding to GPCRs'. Nature

Methods, 12:661-663.

P6

Cac and ca-a1D VGCC subunits regulate tissue integrity and tumour progression in the Drosophila dorsal epithelium.

Africa Couto, Marios A. Georgiou

University of Nottingham, UK

Calcium is a ubiquitous second messenger that regulates a plethora of cellular behaviours, from cell division to cell death. As a result, misregulation of calcium signalling has devastating consequences, cancer being one of them. We use the Drosophila dorsal epithelium to model early stages of tumourigenesis and assess the contribution of calcium signalling to normal and pathologic epithelium behaviour. By combining sophisticated Drosophila genetic techniques with powerful confocal microscopy, we follow the morphology and behaviour of mutant clones of cells surrounded in high spatial and temporal resolution (Couto et al, 2017). Moreover, we have established a protocol to monitor spontaneous intracellular calcium dynamics in the living epithelium. Using these tools, we show here that cac and ca-a1D, pore forming subunits of the Voltage Gated Calcium Channels (VGCCs), cooperate with the tumour suppressor and cell polarity regulator lgl to regulate tumour progression in the Drosophila dorsal epithelium. cac and ca-mutant clones are larger than wild type clones, as a result of the greater fitness of the mutant tissue compared to that of the surrounding wild type tissue, which suggests a supercompetition mechanism (Baker, 2017). VGCC mutant cells also present significantly longer and larger calcium spikes compared to wild type cells. Thus, VGCCs regulate calcium dynamics in this epithelial tissue. Taken together, our data suggests that Drosophila VGCCs are important for regulating calcium dynamics in epithelial cells, and that this calcium signal is important for tissue growth and homeostasis. Mutations that transform cells into supercompetitors, allow the morphologically normal mutant tissue to unnoticeably expand at the expense of wild-type surrounding cells, a mechanism which has been paralleled to ‘field cancerisation’. This is an early step in tumourigenesis that creates a preneoplastic field where subsequent mutations can accumulate to develop cancer. Mutations that create supercompetition could offer markers of precancerous tissue expansion and targets for preventive therapy.

Couto A et al, (2017), An apicobasal gradient of Rac activity determines protrusion form and position. Nat Commun, 8:15385.

Baker NE, (2017), Mechanisms of cell competition emerging from Drosophila studies.

Curr Opin Cell Biol, 48:40.

P7

Fluorescence correlation spectroscopy for the analysis of cell surface receptors at very low levels of expression. Joëlle Goulding, Alexander Kondrashov, Ngoc Vothi, Chris Denning, Stephen Briddon, Stephen Hill

University of Nottingham, UK Fluorescence imaging is commonly employed in the study of fluorescently tagged membrane proteins however this normally relies on a high expression level to be detectable. More sensitive techniques such as fluorescence correlation spectroscopy (FCS) have the potential to be used to quantify receptor number and movement in systems where fluorescence imaging is not possible. FCS is the study of the fluctuations in fluorescence intensity measured in a defined confocal volume, and allows the quantification of concentration and diffusion coefficients of the fluorescent species of interest (Briddon et al, 2007).

We have used FCS to study the membrane organisation of a SNAP-tagged β2-adrenoceptor (β2AR) stably expressed in human stem cell (HUES7) derived fibroblasts (Cowan et al, 2004) which was not easily detected by standard confocal microscopy. Confocal imaging of the fibroblasts showed a large degree of constitutive internalisation but no clear membrane receptor localisation. Sequential FCS reads allowed distinct membrane and intracellular components of SNAP-β2AR diffusion to be measured, with differing characteristics.

In summary, FCS allows us to study receptor dynamics of the SNAP-labelled β2AR in fibroblasts which are not visible using standard fluorescent microscopy approaches. FCS could therefore be an important tool for characterisation of endogenous receptors expressed at low levels.

Briddon S et al, (2007), Pharmacology under the microscope: the use of fluorescence correlation spectroscopy to determine the properties of ligand-receptor complexes. Trends Pharmacol Sci, 28:637–645.

Cowan et al, (2004), Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med, 350:1353-6.

P8

Functional genetics of lung function associated gene GPR126. Robert Hall, Bo Liu, Ian Hall, Ian Sayers

University of Nottingham, UK

GPR126 lies on chromosome 6q24 and variants in this region have reproducibly been associated with lung function in genome wide association studies (GWAS) (Soler et al, 2015).

We aimed to further define i) potential causal variants in the haplotype, ii) the expression of GPR126 in lung and iii) the effects of genetic variants associated with lung function.

Q-PCR and immunohistochemistry were used to profile GPR126 expression in lung and airway relevant cells. Stable cell lines expressing the wild-type GPR126 and a potential causal SNP, Ser123Gly, have been produced to assess the effects of this variant on protein signalling via a cAMP luciferase reporter cell line. A range of ligands identified from the literature including collagen type IV (3µg/ml), short peptides derived from collagen type IV and the prion protein (up to 50µM), and the tethered agonist stachel (1mM) were used to treat cells overexpressing GPR126. cAMP was measured indirectly by measuring luminescence produced by luciferase.

Q-PCR showed that GPR126 mRNA is expressed in human bronchial epithelial cells and human airway smooth muscle cells. GPR126 protein expression is seen in lung tissue by immunohistochemistry with strongest staining seen in smooth muscle.

Incubation of cells overexpressing GPR126 with collagen type IV and peptides derived from collagen 4 and the prion protein did not result in an increase in luminescence. However treatment of cells with a 1mM concentration of the stachel peptide resulted in an approximate five-fold increase in luminescence in cells overexpressing both variants of GPR126 but not in empty vector and no vector controls (p=0.01, n=5).

This cell system will be useful in identifying potential agonists and antagonists for GPR126. The stachel peptide is a useful tool to help identify the biological function of GPR126 in airway relevant cells as well as identifying any additional signalling pathways.

Soler AM et al, (2015), Sixteen new lung function signals identified through 1000 Genomes Project reference panel imputation. Nature communications, 6:8658.

P9 Effect of C terminus Truncations on light induced signalling and internalisation of optogenetic Rhodopsin-α1A Adrenoceptor chimeras. Nicholas Holliday, Laura Humphrys, Tomas Bellamy University of Nottingham, UK Chimeric opsin-G protein coupled receptors (OptoXR), for dissecting signalling by optogenetics, include the α1A adrenoceptor-OptoXR, coupled to Gq and increases in intracellular Ca2+ (Airan et al, 2009). To generate OptoXRs with distinct spatiotemporal signalling profiles, we designed two truncated receptors targeting C tail phosphorylation sites of the α1A-OptoXR, and compared Ca2+ response profiles and internalisation in response to light stimulation.

SNAP-tagged-α1A-OptoXR cDNAs in pcDNA3.1 were truncated at Ser371 or Thr471 by PCR based mutagenesis, and stably expressed in HEK293T cells. Cells were kept in the dark prior to imaging calcium responses and receptor internalisation on a LSM710 Confocal microscope (Zeiss), using 488 nm laser excitation to also stimulate OptoXR activation. Calcium mobilisation was assessed after loading with 10µM Fura Red-AM (20 min, 1 fps), while receptor internalisation (30 min, 0.1 fps) was monitored after first labelling receptors with 0.5µM SNAPsurface488, as the change in plasma membrane fluorescence intensity. Where required media was supplemented with 1 µM 9-cis-retinal for 1 h prior to stimulation.

9-cis-retinal supplementation increased the proportion of SNAP-α1A-OptoXR cells eliciting calcium responses (45.5±4.1% compared to 24.0±5.5% in the absence of retinal, n=3; P<0.05 one way ANOVA / Tukey’s post-test), the magnitude of the peak response, and the number of Ca2+ transients within the 20 min stimulation period (10.1±0.9 compared to 5.0±0.7, n=18-56 cells, 3 experiments; P<0.05). S371 truncation (but not T471) further increased the percentage cell responders (76.7±5.9%) and the number of calcium transients (14.3±0.9; each P<0.05 compared to non-truncated). SNAP-α1A-OptoXR underwent light induced internalisation, but only with 9-cis-retinal present (48.5±2.1%, initial plasma membrane intensity at 30 min; n=3, 20 cells per experiment). Retinal-dependent responses were prevented by S371 truncation. We conclude that through limiting receptor desensitisation, the Ser371 truncation delivers an α1A-OptoXR with sustained response kinetics.

Supported by an AJ Clark studentship to LJH (British Pharmacological Society)

Airan RD et al, (2009), Temporally precise in vivo control of intracellular signalling. Nature, 458:1025–1029.

P10

Human multidrug transporter ABCG2 is purified for fluorescence based single-particle analysis via SMALP mediated extraction. Aaron Horsey, Stephen Briddon, Ian Kerr, Nicholas Holliday

University of Nottingham, UK

ABCG2 is a human ATP-binding cassette (ABC) transporter expressed in a number of barrier tissues (e.g. kidney epithelium). It influences pharmacokinetics and bioavailability of various drugs in addition to other physiological roles (e.g. uric acid transport) (Wong et al, 2016).

As with other membrane proteins, ABCG2 is technically challenging to study as it is poorly extracted from membranes. In this project styrene-maleic acid co-polymer lipid particles (SMALPs) are used to isolate ABCG2 in nanodiscs in the native membrane (Gulati et al, 2014). Current work aimed to assess oligomeric state of SMALP-ABCG2 particles by fluorescence correlation spectroscopy (FCS) to enable future study of substrate binding parameters (e.g. stoichiometry). Labelled and His-tagged ABCG2 constructs stably expressed in HEK293T cells exhibited appropriate membrane localisation and function by confocal microscopy and mitoxantrone efflux assays. SMALP-based extraction and downstream purification yielded sufficient ABCG2 (~10ug mL-1) for analysis by FCS and dynamic light scattering (DLS).

DLS analysis has shown expected particle sizes of 15-20nm and diffusion coefficients have been obtained by FCS, consistent with a homogenous particle distribution. Photon counting histogram analysis has revealed a molecular brightness of SMALP-ABCG2 that indicates the isolation of dimeric ABCG2. The current data can be built upon to further explore characteristics and functionality of ABCG2 in SMALPs.

Gulati S et al, (2014), Detergent-free purification of ABC (ATP-binding-cassette) transporters. Biochem J, 461:269-78.

Wong K et al, (2016), Plasma membrane dynamics and tetrameric organisation of ABCG2 transporters in mammalian cells revealed by single particle imaging techniques. BBA Mol Cell Res, 1863:19–29.

P11 Modelling cytoskeletal gene mutations implicated in inherited thrombocytopenia using CRISPR edited iPSC derived megakaryocytes and super-resolution microscopy. Abdullah Khan, Victoria Simms, Jeremy Pike, Stephen Thomas, Neil Morgan

University of Birmingham, UK

Single molecule localisation microscopy methods like dSTORM (direct Stochastic Optical Reconstruction Microscopy) and PALM (Photoactivated Localisation Microscopy) resolve structures at 10-20nm, and allow for unique insights into protein stoichiometry and spatial relationships (Bates et al, 2007), (Gordon et al 2004). However, key obstacles remain in developing highly accurate quantitative single molecule approaches (Nieuwenhuizen et al, 2015). The genomic tagging of PALM fluorophores through CRISPR-Cas9 (Clustered Regularly Interspaced Palindromic Repeats) offers an excellent opportunity for generating stable cell lines expressing a defined single molecule probe at endogenous levels, without the biological disruption and variability inherent to transfection. A fundamental question is whether these comparatively low levels of expression can successfully satisfy the stringent labelling demands of super-resolution SMLM (Single Molecule Localisation Microscopy). In our work we apply CRISPR-Cas9 gene editing to tag a cytoskeletal protein (α-tubulin) and demonstrate a relationship between expression level and the subsequent quality of PALM imaging, and that spatial resolutions comparable to dSTORM can be achieved with this CRISPR-PALM method. Our approach shows a relationship between choice of tag and the total expression of labelled protein, which has important implications for the development of future PALM tags. CRISPR-PALM allows for nanoscopic spatial resolution and the unique quantitative benefits of single molecule localization microscopy through endogenous expression, as well as the capacity for super-resolved live cell imaging. With the advent of induced pluripotent stem cell (iPSC) technology and cutting edge methods for the differentiation of megakaryocytes and platelets, CRISPR-PALM also offers a unique opportunity for the single molecule investigation of proteins of interest through the endogenous expression of photoswitchable tags (Khan et al, 2017). Future work will involve modelling patient mutations implicated in inherited thrombocytopenia, with a focus on investigating the effects of these novel genotypes through state-of-the art SMLM and live cell imaging.

Bates M et al, (2007), Multi-color super-resolution imaging with photo-switchable fluorescent probes. Science, 317:1749-1753.

Gordon MP et al, (2004), Single-molecule high-resolution imaging with photobleaching. Proc Natl Acad Sci USA, 102: 17565-17569.

Nieuwenhuizen RP et al, (2015), Quantitative localization microscopy: effects of photophysics and labeling stoichiometry. PLOS one, 10: e01278989.

Khan AO et al, (2017), CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution. Scientific Reports, 7: 8450.

P12

Characterisation of ADAM10-regulating tetraspanins Tspan14 and Tspan15 on platelets using new monoclonal antibodies. Chek Ziu (Connie) Koo, Peter Noy, Alexandra Matthews, Hanh Nguyen, Philip Morrison, Clara Apicella, Edward Davis, Murat Keles, Natalie S. Poulter, Michael Tomlinson

University of Birmingham, UK The platelet-specific collagen and fibrin receptor GPVI has emerged as a potential anti-thrombotic drug target. GPVI can be shed from the platelet surface by the ‘molecular scissor’ ADAM10, which is regulated by interaction with one of six tetraspanins (Tspan5, 10, 14, 15, 17 or 33) termed TspanC8s. Proteomic studies suggest that human platelets express three TspanC8s: Tspan14, 15 and 33. We hypothesise that ADAM10 exists as three different scissors on platelets with different substrate specificities, depending on the associated TspanC8. (Matthews et al, 2017), (Matthews et al, 2017). However, characterisation of Tspan14, 15 and 33 complexes with ADAM10 is hindered by the lack of effective TspanC8 monoclonal antibodies (mAbs), perhaps due to the small size of TspanC8s and their ‘masking’ from an antibody response by the larger ADAM10. The aim of this study was to devise a novel TspanC8 mAb generation strategy and to characterise platelet TspanC8s.

ADAM10-deficient mouse embryonic fibroblasts stably transfected with human Tspan14 or Tspan15 were used as immunogens for generation of mouse mAbs by the integrated life sciences company Abpro. Eight Tspan14 and four Tspan15 mAbs were generated. Using the Tspan15 mAbs, Tspan15 expression was demonstrated on human platelets by flow cytometry, western blotting and super-resolution microscopy, and its interaction with ADAM10 was confirmed by co-immunoprecipitation. Tspan15 was not detected on ADAM10 CRISPR/Cas9-knockout cell lines, suggesting that ADAM10 is required for Tspan15 stability. Similar experiments are in progress using the Tspan14 mAbs.

We have discovered a novel method to generate tetraspanin mAbs. The requirement of ADAM10 for Tspan15 expression suggests that the major function of Tspan15 is to regulate ADAM10. This strengthens the theory that ADAM10 should be regarded as six different scissors depending on the associated TspanC8, rather than a single scissor.

This work is funded by the British Heart Foundation, the Biotechnology and Biological Sciences Research Council, and COMPARE University of Birmingham and University of Nottingham Midlands.

Matthews AL et al, (2017), Regulation of A disintegrin and metalloproteinase (ADAM) family sheddases ADAM10 and ADAM17: The emerging role of tetraspanins and rhomboids. Platelets, 28:333-341.

Matthews AL et al, (2017), Scissor sisters: regulation of ADAM10 by the TspanC8 tetraspanins. Biochem Soc Trans, 45:719-730.

P13

Vasculoprotective effects of haematopoietic stem cells in myocardial ischaemia/reperfusion (IR) injury Adam Lokman, Dean Kavanagh, Abigail Colley, Neena Kalia University of Birmingham, UK

The clinical success of stem cell (SC) therapy for myocardial infarction is compromised by poor cardiac homing following systemic delivery. Given that such therapy may depend on beneficial paracrine effects, it is a further hindrance that little is known about the inflammatory response dynamics within myocardial microcirculation in vivo (Bulluck et al, 2016), (Kopp et al, 2005).

3D-printed stabilisers were bonded to the anaesthetised (ketamine/medetomidine) mouse beating heart to enable confocal intravital imaging of ventricular microcirculation. Antibodies were injected to label neutrophils and platelet receptors respectively with blood flow visualisation achieved through vascular staining. In some mice, haematopoietic SCs (HSCs; HPC-7s) were introduced intra-arterially, some dyed/labelled as required. IR injury was induced by 45 minutes (reversible) ligation of the left anterior descending (LAD) artery followed by 150 minutes reperfusion.

Neutrophil adhesion and platelet accumulation were both significantly (p<0.05) and rapidly increased in injured microvessels. This response was significantly reduced in the presence of exogenous HSCs (p<0.05). A significant (p<0.05) decrease in functional capillary density was also observed in injured hearts. Although HSC adhesion was not significantly enhanced as a result of injury, a time-dependent increase in adhesion was observed in sham and injured hearts. No significant change in number or velocity of free-flowing HSCs was observed following injury either. Interestingly, despite reduced capillary perfusion, approximately 10-20 HSCs were observed trafficking through the heart at each time point throughout reperfusion.

Intravital microscopy has allowed successful visualisation of the microvascular inflammatory response and HSC homing events in the beating mouse heart post-ischaemia-reperfusion injury. HSC administration attenuated this inflammatory response.

Bulluck H et al, (2016), Reducing myocardial infarct size: challenges and future opportunities. Heart, 102:341-348.

Kopp H G et al, (2005), The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda), 20:349-356.

P14 Dissecting the spatio-temporal dynamics of GPCR during memory formation in Drosophila neurons. Isabella Maiellaro1, Robert Kittel2, Martin Lohse1,3

1 Institute of Pharmacology and Toxicology, University of Würzburg 2 Institute of Physiology Dpt. of Neurophysiology, University of Würzburg 3 Max Delbrück Center for Molecular Medicine, Berlin Synaptic plasticity is believed to underlie the formation of learning and memory in organisms from insects to man. G protein-coupled receptors (GPCRs) are known in neurons to modulate synaptic plasticity. It has been shown that GPCRs exert this role via the second messenger Cyclic adenosine monophosphate (cAMP) and its effector Protein Kinase A (PKA). Little is known about the spatio-temporal dynamics of the GPCR-cAMP-PKA pathway activation within living neurons, despite the strong possibility that GPCR-linked pathways subserve the fundamental basis of learning and memory.

In Drosophila it has been shown that the activation of presynaptic octopamine receptors (GPCR orthologous of β2-Adrenergic Receptor) leads to structural synaptic changes via activation of the cAMP response element-binding protein transcription factor (CREB) in the soma (Koon et al, 2011). Intriguingly our previous work demonstrated that cAMP is incapable of diffusing from the presynaptic site to the soma (Maiellaro et al, 2016) -therefore it is presently unknown how cAMP signals originating in the periphery can reach the soma of neurons to activate CREB.

Here, we study whether PKA is responsible for the dissemination of cAMP signals from the synaptic bouton to the soma of the neurons. Using transgenic Drosophila larvae that express the PKA sensor, we used Förster Resonance Energy Transfer (FRET) to follow PKA activation, in real time, at high spatio-temporal resolution. Additionally, the retrograde diffusion of the endogenously tagged catalytic subunits of the PKA (PKA-C) was monitored with the intent to quantify the kinetics of PKA-C diffusion. Dissecting these cAMP-PKA dynamics during synaptic plasticity will help to clarify fundamental and still poorly understood mechanisms at the basis of learning and memory. Furthermore, this new knowledge may yield attractive pharmacological targets to modulate these important processes in vivo.

Koon AC et al, (2011), Autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signalling. Nat Neurosci, 14:190-9.

Maiellaro et al, (2016), cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons. Cell Reports, 17:1238-1246.

P15

Regulation of the platelet collagen receptor GPVI by ADAM10 and tetraspanins.

Alexandra Matthews1, Connie Koo1, Peter Noy1, Esme Gardiner2, Natalie Poulter1, Michael Tomlinson1

1 University of Birmingham, UK 2 Australian National University, Australia

The platelet collagen receptor GPVI is a critical mediator of arterial thrombosis, but has a minor role in haemostasis. The ubiquitously-expressed transmembrane metalloprotease ADAM10 is the major sheddase for GPVI following platelet activation. Shedding removes the GPVI extracellular region and renders the platelet non-responsive to collagen. We have shown that ADAM10 substrate specificity is regulated by the TspanC8 subgroup of tetraspanins, comprising Tspan5, 10, 14, 15, 17 and 33. Human platelets express Tspan14, 15 and 33. The aim of this study is to determine whether any TspanC8 specifically regulates the ADAM10-mediated shedding of GPVI. We have found that overexpression of Tspan14, but not other TspanC8s, in HEK293T cells results in reduced GPVI shedding (Nov et al, 2016). Tspan14 interaction with ADAM10 is not necessary for its capacity to reduce GPVI shedding, and Tspan14 co-immunoprecipitates GPVI. To elucidate the mechanism by which Tspan14 suppresses GPVI shedding, we established the megakaryoblastic cell line HEL as a model system. HELs express Tspan14 in addition to four other TspanC8s: Tspan5, 15, 17 and 33 (Matthews et al, 2017). Consistent with the idea that ADAM10 is the major sheddase for GPVI, no shedding is observed in ADAM10 knockout HELs. GPVI shedding is unaffected by loss of Tspan14, despite substantially reduced ADAM10 surface expression levels. In the absence of Tspan14, GPVI shedding is likely to be mediated by the remaining Tspan15/ADAM10 and Tspan33/ADAM10 complexes. We hypothesise that Tspan14/ADAM10 complexes cannot shed GPVI, but Tspan15/ADAM10 and Tspan33/ADAM10 complexes can, and this may be related to the capacity for Tspan14 to interact with GPVI. Further understanding of GPVI shedding by TspanC8/ADAM10 complexes may enable us to therapeutically target a specific TspanC8, to induce shedding and protect a patient from heart attack or stroke.

This work is supported the BBSRC Midlands Integrative Biosciences Training Partnership and COMPARE University of Birmingham and University of Nottingham Midlands.

Noy PJ et al, (2016), TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions: Evidence For Distinct Binding Mechanisms For Different TspanC8 Proteins. J Biol Chem, 291:3145-57.

Matthews AL et al, (2017), Regulation of A disintegrin and metalloproteinase (ADAM) family sheddases ADAM10 and ADAM17: The emerging role of tetraspanins and rhomboids. Platelets, 28:333-341.

P16

Development of a fluorescent antagonist binding assay for the Gastrin Releasing Peptide BB2 Receptor. Liciane Medeiros1, Marco Filho Silva1,2, Danielly Olguins1,2, Rafael Roesler2, SarahMistry1, Nicholas Holliday1

1 University of Nottingham, UK 2 Federal University of Rio Grande do Sul, Brazil Gastrin releasing peptide (GRP) and its G protein coupled receptor BB2 are implicated in pain perception, gastrointestinal regulation, chemoattraction, and tumour proliferation. We report a novel BB2 fluorescent ligand (SM1292; BODPIY630/650-[DPhe6, DAla11]Bombesin6-13-NH2), based on the amide of BIM26226, and its use in a whole cell BB2 competition binding assay for BB2 based on high content imaging.

HEK293 cells stably expressed SNAP-tagged human BB2 receptor (HEK BB2) or BB2 S304A mutation; Fluo4 calcium mobilisation and fluorescent ligand assays were as described (Liu et al, 2016). Whole cell binding in 96 well imaging plates used SM1292 (1-1000nM) plus or minus competing peptides in HBS / 0.1% BSA / H33342 (2µg ml-1). After 30min at 37°C, H33342 nuclear, SNAP-labelled BB2 and BODIPY630/650 ligand images were acquired using an MDC IX Ultra plate-reader and quantified by granularity analysis. pEC50/pKD/pKi values (Graphpad Prism 7) are mean±s.e.m. from 4-5 experiments.

In calcium mobilization assays, the unlabelled ligand (DPhe6, DAla11]Bombesin6-13-NH2) was a competitive antagonist of GRP signaling (control pEC50 9.45±0.18), with a pKD of 7.2 estimated by Schild analysis for BB2 receptors; S304A mutation, in extracellular loop 3,reduced the effect of the peptide antagonist but not GRP. SM1292 saturation analysis in the absence or presence of 1µM GRP, estimated its pKD as 7.32±0.18 for BB2 and 6.87±0.21 for BB2 S304A. Competition binding for BB2 using 10nM SM1292 generated pKi estimates for GRP (8.55±0.06), neuromedin B (6.24±0.52), DPhe6, DAla11]Bombesin6-13-NH2 (7.04±0.21) and a second peptide antagonist RC3095 (7.55±0.66).

Characterization of SM1292 demonstrates that fluorophore modification of DPhe6, DAla11] Bombesin6-13-NH2 can be achieved whilst retaining nanomolar affinity for the BB2 receptor, and SM1292 competition binding experiments obtain ligand affinities expected for BB2. Ser304 plays a key role in fluorescent and unlabelled peptide antagonist binding, as previously reported (Tokita et al, 2001).

Liu M et al, (2016), Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15. Bioconjug Chem, 27:2166-75.

Tokita et al, (2001), Molecular basis for selectivity of high affinity peptide antagonists for the gastrin-releasing peptide receptor. J Biol Chem, 276:36652-63.

P17

Novel fluorescent β-adrenoceptor ligands. Sarah Mistry, Joëlle Goulding, Stephen Hill, Barrie Kellam

University of Nottingham, UK Fluorescent ligands have been increasingly used as pharmacological probes, alongside techniques such as Fluorescence Correlation Spectroscopy (FCS) and confocal microscopy, due to their ability to provide an in-depth insight into receptor pharmacology.

We have designed and synthesised nine fluorescent analogues of the β2-adrenoceptor (β2AR) antagonist ICI-118,551 consisting of a pharmacophore, linker and a fluorophore. Seven of these analogues contain dipeptide linkers, as previous work in our group has demonstrated that dipeptide linkers can confer receptor subtype-selectivity, increases in affinity and also enhanced imaging properties (Vernall et al, 2013). As fluorescent ligands are novel chemical entities they therefore need to be fully pharmacologically characterised. Previous studies have shown that their properties can differ considerably from those of the parent ligand (Baker et al, 2010).

To determine the affinity of the novel fluorescent ligands for the β2ARs, a Bioluminescence Resonance Energy Transfer (BRET) saturation binding assay was employed. Preliminary data obtained indicate that linker/fluorophore combinations have a significant effect on β2AR affinity and also their imaging properties.

It is anticipated that these novel ligands will be utilised in combination with FCS to investigate the pharmacology of β2ARs expressed at endogenous levels.

Vernall AJ et al, (2013), Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers. Org. Biol. Chem, 11:5673-82.

Baker JG et al, (2010), Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands. Br. J. Pharmacol, 159:772–786.

P18

Expansion microscopy as a tool for 3D super-resolution imaging.

Robert Neely, Emma Faulkner, Ruth Densham, Jo Morris

University of Birmingham, UK

Expansion microscopy (Chen et al, 2015) is a form of super-resolution fluorescence microscopy with an upper bound on the limit of resolution that approaches 10 nm (Chang et al, 2017). However, unlike every other super-resolution approach, expansion microscopy can be implemented on a standard optical system and requires no significant post-processing to generate high-resolution, 3-dimensional images of a sample. As the name suggests expansion microscopy works by swelling the ‘sample’ by a factor of four to twenty times prior to imaging. In order to do this the cell is fixed and stained as usual but the fluorophores are subsequently cross-linked into a hydrogel that is built around the sample. Finally, the biological material is digested and the gel swollen to expand the fluorophores, which trace the position of the features of interest in the gel. The gel is optically clear and as a result, samples can be readily imaged in 3D using both light sheet and confocal microscopes.

Here, we present initial work on the development of expansion microscopy as a tool for imaging the genome and those proteins involved in the DNA damage response. We will discuss limitations of the chemistry of expansion microscopy and the ways in which we have begun to manipulate this in order to develop novel approaches for imaging in gels.

Chen F et al, (2015), Optical imaging. Expansion microscopy. Science, 347:543–548.

Chang JB et al, (2017), Iterative expansion microscop. Nat. Methods, 14:593–599.

P19

The configuration of GPVI drives the binding with fibrin and collagen and its role in thrombus formation. Marie-Blanche Onselaer 1, Alexander Hardy1, Clare Wilson2, Ximena Sanchez3, Jeanette Miller4, Callum Watson1, Stephanie Watson1, Helen Philippou2, Andrew Herr4, Diego Mezzano3, Robert Ariëns2 and Steve Watson1

1 University of Birmingham, UK 2 University of Leeds, UK 3 Pontificia Universidad Catolica de Chile, Chile 4 Center for Systems Immunology, & Infectious Diseases, Cincinnati, USA Fibrin, the final product of coagulation cascade, has been shown to bind the platelet receptor GPVI which is more widely known as the major signalling receptor for collagen. This interaction enhances platelet recruitment and activation. In the present study, we have mapped the interaction between fibrin and GPVI to generate reagents that can be used to evaluate the physiological significance of this interaction in vitro and in vivo.

Patients and mice deficient for GPVI were used for platelet aggregation and spreading studies. Recombinant GPVI proteins and fibrin(ogen) fragments were used in solid-phase binding assays and surface plasmon resonance studies.

We show that fibrin-induced human platelet spreading and aggregation are abolished in patients deficient in GPVI confirming that it is the major receptor underlying platelet activation by fibrin. We demonstrate by surface plasmon resonance and solid-phase binding assays that fibrin binds selectively to monomeric GPVI with a KD of 302 nM; in contrast, collagen binds selectively to dimeric GPVI. Using a series of proteolytic fibrin fragments, we show that D-dimer (the D-D interface part from fibrin) but not the E-fragment (the central part from fibrin) binds to soluble GPVI. Moreover, immobilised D-dimer induces platelet spreading which is inhibited by Src and Syk inhibitors. In suspension, D-dimer inhibits platelet aggregation induced by fibrin or by low concentrations of collagen but not by a collagen-related peptide or by thrombin.

These results confirm GPVI as a major signalling receptor for fibrin in platelets. Furthermore, we provide evidences that fibrin and collagen bind to a distinct configuration of GPVI raising the possibility of development of agents that selectively inhibit binding of fibrin or collagen.

This work was supported by the BHF (RCAZ17520).

Onselaer M-B et al, (2017), Fibrin and D-dimer bind to monomeric GPVI. Blood Advances, 1:1495-1504.

P20

Receptor Clustering Prevents GPVI Shedding And Leads To Sustained Signalling.

Chiara Pallini1, Elizabeth Gardiner2, Robert Andrew3, Steve Watson1, Natalie Poulter1

1University of Birmingham, UK 2Australian National University, Australia 3Monash University, Australia

GPVI is the main signalling receptor for collagen on platelets. GPVI binds to collagen as a dimer and signals through the FcRγ ITAM culminating in platelet activation and thrombus formation. GPVI has been shown to form dimers and higher oligomers upon activation thereby increasing the avidity for collagen and enhancing signalling (Poulter et al, 2017). GPVI is cleaved in response to collagen stimulation in cell suspensions thereby limiting platelet activation. In the present study, we show that collagen induces sustained (hours) activation of GPVI in platelets in association with minimal shedding. We propose that the absence of shedding is due to clustering of GPVI and exclusion of the sheddase ADAM10 from the signalling complex.

Using dSTORM combined with Ripley’s K-function based cluster analysis (Owen et al, 2010), we demonstrate that GPVI is highly clustered along collagen fibres. SR-Tesseler segmentation was used to confirm clustering of GPVI. Immunoblotting showed that approximately 15% of GPVI is shed in platelets that have adhered to collagen over 3h. Furthermore, phosphorylation of LAT and Syk, hallmarks of GPVI signalling, was maintained over this time frame. Dual-colour confocal microscopy revealed that GPVI colocalises with pLAT and pSyk for up to 3h. Inhibition of Src kinases by PP2 in platelets spread on collagen did not affect their spreading, whereas treatment with the Syk inhibitor PRT-060318 decreased the spreading extent. This indicates that Syk, but not Src kinases, is required for the sustained signalling.

Our work supports the hypothesis that GPVI signalling is further regulated by higher-order clustering, acting to protect GPVI from shedding and sustaining the signalling via Syk tyrosine kinase.

This work is supported by the British Heart Foundation.

Owen DM et al, (2010), PALM imaging and cluster analysis of protein heterogeneity at the cell surface. J Biophotonics, 3:446-54.

Poulter NS et al, (2017), Clustering of GPVI dimers upon adhesion to collagen as a mechanism to regulate GPVI signalling in platelets. J Thromb Haemost, 15:549-564.

P21

Measuring binding of fluorescent variants of VEGF165a, VEGF165b and VEGF121a to VEGFR2 using NanoBRET.

Chloe Peach1, Laura Kilpatrick1, Rachel Friedman-Ohana2, Matthew Robers2, Jeanette Woolard1, Stephen Hill1

1University of Nottingham, UK 2Promega Corporation, Wisconsin, USA

Vascular endothelial growth factor receptor 2 (VEGFR2) is a key mediator of angiogenesis and vascular permeability (Koch et al, 2011) Alternative splicing of the Vegfa gene produces VEGF ligand isoforms with different signalling and physiological outcomes (Woolard et al, 2009), such as the recently identified VEGF-Ax (Eswarappa et al, 2014).

Quantitative information on the binding affinities of these isoforms to full-length VEGFR2 in living cells is currently lacking. Here we used bioluminescence resonance energy transfer (BRET) to quantify binding of fluorescent variants of VEGF165a, VEGF165b and VEGF121a to N terminal NanoLuc-tagged VEGFR2 in HEK293 cells. Stably expressing NanoLuc-VEGFR2 HEK293 cells were seeded 24 hours prior to experimentation in 96-well plates.

For saturation experiments, increasing concentrations of selective VEGF isoforms single-site labelled with the fluorophore tetramethylrhodamine (TMR; VEGF165a–TMR, VEGF165b-TMR and VEGF121a-TMR) were added in the presence and absence of a high concentration of unlabelled VEGF in Hanks buffered saline solution/0.1% bovine serum albumin.

In competition experiments, cells were co-stimulated with fixed concentrations of VEGF165a–TMR, VEGF165b-TMR or VEGF121a-TMR and increasing concentrations of unlabelled VEGF. Following 60min stimulation at 37°C, the NanoLuc substrate furimazine (10μM) was added and BRET ratios were recorded using a BMG Pherastar. Each ligand isoform had a clear saturable component of binding with little evidence of non-specific binding. Equilibrium binding constants (Kd) derived for each fluorescent ligand from saturation assays show VEGF165a-TMR, VEGF165b- TMR and VEGF121a-TMR bind to NanoLuc-VEGFR2 with nanomolar affinity.

All fluorescent ligands were displaced by a panel of unlabelled VEGF isoforms, for which derived affinity constants (Ki) were comparable ranging between 0.2nM and 1.4nM. Fluorescent variants of VEGF165a, VEGF165b and VEGF121a enabled ligand binding affinities to be quantified for the first time in living cells expressing full-length VEGFR2 without endogenous co-receptors present, suggesting distinct signalling between VEGF isoforms does not arise from binding alone.

Koch S et al, (2011), Signal transduction by vascular endothelial growth factor receptors.

Biochem. J, 437:169-183.

Woolard J et al, (2009), Molecular diversity of VEGF-A as a regulator of its biological activity. Microcirculation, 7:572-92 .

Eswarappa SM et al, (2014), Programmed translational readthrough generates antiangiogenic VEGF-A., Cell, 157:1605-18.

P22

Persistent homology as a tool to classify topology in single molecule localization microscopy. Jeremy Pike, Abdullah Khan, Steve Thomas, Iain Styles

University of Birmingham, UK

Single-molecule localization microscopy techniques such as dSTORM and PALM can achieve spatial resolution of the order of tens of nanometers. The resulting data from these techniques is a list of molecule positions. There are several existing clustering algorithms which have been applied to localization microscopy datasets to segment and visualize biological structures. However these algorithms do not quantify the topology of the structures. For example, the classification of holes or gaps within induvial clusters. Here, we report an unbiased approach for the classification of topology in single molecule localization data. Specifically, we compute the persistent homology of simplicial complexes (Grist et al, 2008). The use of this novel approach is demonstrated on both simulated and real datasets.

Ghrist R et al, (2008), Barcodes: The persistent topology of data. Bull. Amer. Math. Soc., 45:61–75.

P23

The podoplanin-CLEC-2 axis inhibits inflammation in sepsis.

Julie Rayes, Siân Lax, Surasak Wichaiyo, Stephanie K. Watson, Ying Di, Beata Grygielska, Steve P. Watson

University of Birmingham, UK

Platelets are recognized to play a critical role in vascular inflammation through the podoplanin and collagen/fibrin receptors, C-type-lectin-like-2 (CLEC-2) and glycoprotein VI (GPVI), respectively. Both receptors regulate endothelial permeability and prevent peri-vascular bleeding in inflammation (Boulaftali et al, 2013), (Gros et al, 2015). Here, we investigate the role of both receptors in two models of sepsis in mice, intra-peritoneal lipopolysaccharide (I.P. LPS) and cecal ligation and puncture (CLP).

Platelet-deletion of CLEC-2 but not GPVI increases the clinical severity of sepsis in both models leading to enhanced systemic inflammation and accelerated organ injury. CLEC-2 deficiency is associated with a reduction in podoplanin-expressing macrophages despite an increase in cytokines and chemokines in the infected peritoneum. An activating podoplanin antibody reduces inflammation in both models providing evidence that activation of podoplanin underlies the anti-inflammatory action by platelet CLEC-2.

These observations identify podoplanin as a novel anti-inflammatory target regulating immune cell recruitment and activation in sepsis.

Boulaftali Y et al, (2013), Platelet ITAM signaling is critical for vascular integrity in inflammation. The Journal of clinical investigation, 123:908-916.

Gros A et al, (2015), Single platelets seal neutrophil-induced vascular breaches via GPVI during immune-complex-mediated inflammation in mice. Blood, 126:1017-1026.

P24

The use of Fluorescence Correlation Spectroscopy to investigate ligand-receptor interaction at the Neuropeptide Y Y1 receptor. Rachel Richardson1, Marleen Groenen1, Simon Mountford2, Mengjie Liu2, Philip Thompson2, Stephen Briddon1, Nicholas Holliday1

1 University of Nottingham, UK 2 Monash University, Australia

Fluorescent ligands allow insight into ligand-receptor interactions when used in combination with Fluorescence Correlation Spectroscopy (FCS) to measure free and receptor-bound diffusion properties. Here we describe this approach to study novel Neuropeptide Y Y1 receptor (NPY; Y1R) fluorescent monomeric antagonists BIDA84 (([Lys2-sulphatedCy5, Arg4]BVD15), BIDA81 (([Lys2-RhoB, Arg4]BVD15), and BIDB13, a rhodamine derivative of a BVD15 dimer (Liu et al, 2016). We characterised the behaviour of each fluorescent peptide by FCS and autocorrelation analysis, to obtain their diffusion co-efficients (D) in solution, and when bound to living cells expressing the Y1 receptor.

FCS solution measurements were taken using 1-100nM peptide in HBSS/0.1%BSA at 21°C. For receptor binding, HEK293 cells expressing the SNAP-tagged Y1R were pre-incubated with 10nM peptide in HBSS/BSA (30mins; 37°C; 0%CO2), and FCS measurements on the upper cell membrane were taken as previously described (Kilpatrick et al, 2012). In solution (10nM), D values were 129±8μm2/s (n=4) for BIDA84, 10.8±0.5μm2/s (n=4) for BIDA81 and 10.4±0.5μm2/s (n=4) for BIDB13. Cell membrane measurements revealed receptor-bound and free ligand by the detection of slower diffusing species. BIDA84 exhibited a faster bound D of 0.62±0.06μm2/s (n=26 cells: 4 experiments) compared to BIDA81, 0.18±0.02μm2/s (n=56, 4), BIDB13 0.37±0.06μm2/s (n=43, 4) or 0.2μM SNAPsurface AF488/647 labelled Y1R (0.22±0.01μm2/s n=59, 4; 0.59±0.05μm2/s, n=32, 4). Hence FCS detects Y1R fluorescent ligand binding, and for both Cy5- and Rho-labelled ligands, the diffusion co-efficient of receptor-bound species closely reflect those of SNAP fluorophore labelled receptors.

Supported by the Nottingham-Monash PhD program

Liu M et al, (2016), Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15. Bioconjugate Chem, 27, 2166.

Kilpatrick LE et al, (2012), Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors. Biochim Biophys Acta, 1823:1068-1081.

P25

EP4 receptor agonists inhibit cellular responses in airway remodelling. Elizabeth Rosethorne, Steven Charlton University of Nottingham, UK Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial lung disease with unknown cause that leads to progressive respiratory failure and death within 5 years post diagnosis. IPF is characterised by the formation of fibroblast and myofibroblast foci that excrete excessive amounts of extracellular matrix resulting in airway stiffening and loss of pulmonary function. Recent work has demonstrated a link between a decrease in prostaglandin E2 (PGE2) signalling and fibrotic diseases, and restoration of this pathway can inhibit, and in some cases reverse, remodelling processes associated with fibrosis (Garrison et al, 2013). Here we have investigated the potential for EP4 receptor agonists to inhibit processes associated with airway remodelling.

cAMP accumulation (AlphaScreen), inhibition of PDGF-mediated ERK phosphorylation (Immunoflourescence) and inhibition of PDGF- or serum-mediated cell proliferation (DELFIA BrdU incorporation) were monitored in primary human bronchial smooth muscle cells (hBSMc; Promocell) and primary human lung fibroblasts (HLF; Lonza) after treatment with a range of EP receptor agonists.

The pan-EP receptor agonists PGE2 and misoprostol, and the EP4-selective agonist AGN205204, but not the EP2-selective agonists butaprost or AH13205, were able to promote robust cAMP accumulation, with PGE2 being the most potent and efficacious in both cell types. Despite being a partial agonist with respect to PGE2, AGN205204 was the most efficacious of all the EP receptor agonists in inhibiting cell proliferation. In comparison, the full agonist PGE2 was only partially effective at inhibiting these processes.

Together these data demonstrate that the EP4 receptor has the potential to strongly inhibit processes associated with airway remodelling, making it an interesting new target for the treatment of IPF. From these results it appears that the long-term phenotypic effects of the EP4 receptor may not be solely dependent on global cAMP accumulation, but may be determined by the kinetics and localisation of intracellular signals.

Garrison G et al, (2013), Reversal of Myofibroblast Differentiation by Prostaglandin E2. American Journal of Respiratory Cell and Molecular Biology, 48:550-558.

P26

Unnatural amino acid mutagenesis and photoaffinity cross-linking to determine the binding site for CGRP at its receptor. John Simms, David Poyner

Aston University, Birmingham, UK

Calcitonin gene related peptide (CGRP) is a very abundant neuropeptide, especially in sensory nerve fibres and given the plethora of effects of CGRP, it is not surprising that the calcitonin like receptor has emerged as an important therapeutic target. However, despite the huge amount of mutagenesis derived data from our lab and others combined with structural data such as X-ray crystallography the structure of the bound complex is still elusive.

In order to tackle this problem, we have employed an unnatural amino acid approach combined with molecular modelling to understand how the agonist CGRP binds to its receptor. In this approach we have substituted each of the residues in extracellular loop 2 (ECL2) with the amber stop codon and genetically incorporated azido-phenylalanine (AzF) into the receptor structure. Subsequent UV exposure of the bound complex revealed whether a particular position is in contact with the ligand by forming a covalent complex with it. Using this approach we have identified 2 positions in ECL2 with contacts with the ligand and using this data have generated a molecular model of the docked agonist complex. Interestingly, the final position of the ligand is very consistent with the recent low resolution cryoEM structure of the Calcitonin receptor. This approach could be important in understanding how accessory protein such as receptor activity modifying proteins influence the conformation changes associated with conformational change with different agonists at this receptor.

P27

Structural insights into GPCR activation using essential dynamics and molecular dynamics simulations. John Simms, David Poyner

Aston University, Birmingham, UK

Insights into receptor activation have been helped over the last few years with the increased number of structures in distinct conformational states. The transition pathway between conformations remains poorly understood but is key to understanding the structural basis of efficacy and signalling bias. We have used a computational approach to predict the activation pathway of the β2a Adrenergic receptor (β2aR) using Isoproterenol as a model compound.

A series of Molecular dynamics simulations of the active and inactive states of the β2aR (PDB 3SN6 and 2RH1, respectively) were performed in an implicit bilayer from which a concatenated trajectory was built and used for principle component analysis. A set of eigenvectors with a non-zero eigenvalues was used to generate a trajectory from active to inactive conformations. Ligand docking of Isoproterenol to snapshots along the trajectory was performed using a modified version of Autodock with subsequent complexes being refined using an in house scoring function. Virtual ligand screening (VLS) was used to assess whether the ligand specific refined complexes enriched docking results for a particular structural class when tested against a range of decoy structures.

Interestingly, we have found that using this approach is able to highlight a plausible transitional pathway between the active and inactive states of GPCR conformational change and furthermore the resulting structures were gave insights into the structural changes that occur during agonist and antagonist binding.

P28

Investigating the regulatory mechanisms of actin nodule formation and turnover in platelets. Victoria Simms, Natalie Poulter, Stephen Thomas

University of Birmingham, UK

The dynamic functioning of the actin cytoskeleton plays a crucial role in enabling platelets to adhere, spread and form stable aggregates to prevent blood loss at sites of vascular damage. During the process of spreading, platelet actin undergoes distinct morphological changes including the formation of nodules, filopodia, lamellipodia and stress fibres (Calaminus et al, 2008). We have previously shown that platelets lacking actin nodules do not form proper aggregates under flow and we therefore hypothesise that actin nodules are important for this process (Poulter et al, 2015). Using live-cell total internal reflection fluorescence (TIRF) imaging combined with specific inhibitors we aim to understand the role GTPases play in regulating actin nodule formation and turnover.

Monitoring the formation, lifetime and organisation of the actin nodules in platelets spreading on fibrinogen using live-cell TIRF imaging, has revealed that inhibiting different GTPases has diverse effects on actin nodule dynamics. Inhibiting RhoA, ROCK1 and non-muscle myosin II (proteins in the RhoA signalling pathway involved in stress fibre formation), using Rhosin hydrochloride, Y27632 and blebbistatin respectively, causes an increase in actin nodule formation and turnover. These results suggest that the RhoA signalling pathway is important for maintaining stable actin nodules. Conversely, inhibition of Rac1, which is known to participate in lamellipodia formation, using EHT-1864, leads to decreased actin nodule formation but extended nodule lifetime. Moreover, these platelets form elongated filopodia. These results indicate that Rac1 signalling is critical for actin nodule formation as well as turnover in platelets.

Actin nodule formation and regulation is differentially regulated by the Rho GTPases RhoA and Rac1. Future work will aim to understand the interplay between these molecules and the consequence of altering actin nodule dynamics on platelet aggregate formation.

This work is supported by the British Heart Foundation.

Calaminus SD et al, (2008), Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading. Journal of Thrombosis and Haemostasis, 6:1944-1952.

Poulter N et al, (2015), Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3. Nature Communications, 6:1-15.

P29

Platelet membrane receptor glycoprotein Ib (GP)Ib-IX complex analysed using dSTORM super-resolution light microscopy. Alex Slater1,2, Natalie Poulter2, Jonas Emsley1

1 University of Nottingham, UK 2 University of Birmingham, UK The platelet glycoprotein (GP) Ib-IX complex is a crucial receptor for the recruitment of platelets to an injured vessel (Massberg et al, 2003). Initial tethering of platelets to a region of

exposed collagen on the endothelium (Kroll et al, 1991). Further interactions of GPIb-IX with thrombin, contact factors high molecular weight kininogen and factor XI, and leukocyte integrin Mac-1 link this receptor complex with platelet activation, the coagulation pathway and leukocyte-platelet recruitment (Andrews et al, 2004), highlighting the multiple roles that this receptor plays in haemostasis. The ability of collagen, fibrinogen and various high molecular weight kininogen derived peptides to activate platelets was initially quantified using a spreading assay. Spread platelets were then subject to the single molecule super resolution microscopy technique direct Stochastic Optical Reconstruction Microscopy (dSTORM) to analyze, for the first time, the distribution and clustering of the GPIb-IX receptor on the surface of platelets adhering and spreading on different ligands involved in platelet activation. GPIb appears to be highly clustered in response to all the ligands tested so far. Future work will employ two-colour dSTORM to analyze the co-localization of GPIb-IX with thrombin and FXI. We aim to use advanced imaging to help further our understanding of the multiple functions of the GPIb receptor complex and whether it can be used as a potential drug target for thrombotic cardiovascular disease.

Massberg S et al, (2003), A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med, 197:41-9.

Kroll MH et al, (1991), von Willebrand factor binding to platelet GpIb initiates signals for platelet activation. J Clin Invest, 88:1568-73.

Andrews RK et al, (2004), Platelet interactions in thrombosis. IUBMB Life, 56:8-13.

P30 DNA origami: a tool for single molecule localisation microscopy. Darren Smith, Robert Neely University of Birmingham, UK

Despite providing remarkable insight into a variety of biological systems, the full potential of single molecule localisation microscopy (SMLM) has not been realised. In part, this is because interpretation of the pointilistic data that is generated is very challenging. After successfully determining the correct position of each individual fluorophore (which in itself is difficult), the relationship between localised points must be accurately reconstructed to reveal the true underlying structure within the system. To this aim, there has recently been great effort in developing analysis methods that can correctly cluster SMLM data (Baddeley, 2015). A barrier to the optimisation of such methods is the difficulty of providing realistic data for training and testing. Synthetic data can easily be generated but suffers from the lack of systematic errors observed experimentally. Standardised structures are therefore required to push forward the developments of SMLM analysis strategies.

DNA origami provides a means to produce nanoscale objects with precisely controlled geometry. In addition, the use of DNA as a scaffold means that the many technologies that have been developed to modify DNA can easily be incorporated to create samples with specific physical properties. Here, we produce fluorescent origami structures by incorporating staple strands containing fluorophores or by utilising the DNA-PAINT approach (Jungmann et al, 2010), which allows SMLM by transient binding of short imager strands to specific docking sites on the structure. The resulting data can be used directly to test clustering algorithms, but can also inform the generation of more realistic synthetic data for more rigorous evaluation of these methods.

Baddeley D, (2015), Detecting nano-scale protein clustering. Nat. Methods, 12: 1019–1020.

Jungmann R et al, (2010), Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett, 10:4756–61.

P31

Using NanoBRET to quantify antibody binding to a thermostabilised turkey β1-adrenoceptor. Mark Soave, Jeanette Woolard, Stephen Hill University of Nottingham, UK

The nature of antibody-GPCR interactions can be complex, so require full pharmacological analysis. Previously, a monoclonal antibody (mAb3) was shown to bind to the second extracellular loop of the turkey β1-adrenoceptor (tβ6-m23) (Hutchings et al, 2014). Our group utilised a luciferase (NanoLuc) fused to the N-terminus of several GPCRs to measure fluorescent ligand binding using a BRET proximity assay (NanoBRET) (Stoddart et al, 2015). Here, we investigate the ability to measure mAb3 binding to tβ6-m23 using the NanoBRET technique.

Saturation and competition binding experiments were performed on HEK293 cells transiently transfected with 100ng/well NanoLuc-tβ6-m23 construct. Media was removed and replaced with HBSS with the relevant concentration of fluorescent ligand and, if used, mAb3 was added 30 minutes prior to this. Non-specific binding was determined with 10µM propranolol. Cells were incubated for 1 hour at 37°C before the addition of the NanoLuc substrate, furimazine. Filtered light from each well was simultaneously measured using 460 nm (80-nm bandpass) and >610 nm longpass filters using a PheraSTAR plate reader. The raw BRET ratio was calculated by dividing the>610 nm emission by the 460 nm emission. To directly measure mAb3 binding, cells were fixed in 3% paraformaldehyde before treated with a concentration range of mAb3 (1 to 250nM) at 37°C for 1 hour. Cells were then incubated with a rhodamine-tagged secondary antibody for 1 hour in the dark before measuring the BRET ratio after furimazine addition.

Direct binding of mAb3 could be measured with NanoBRET (KD 19.4 ± 5.2nM, n=4). MAb3 binding resulted in a decrease in the Bmax of CGP-12177-TMR (39.3 ± 9.3% total binding, n=4) with no change in affinity. Competition NanoBRET experiments showed good agreement with these data (mAb3 pKB 7.71 ± 0.19, n=4) and confirmed tβ6-m23 binding pharmacology (propranolol pKD 7.45 ± 0.19, n=4). These data demonstrated the capability to quantify specific antibody-GPCR binding using the NanoBRET approach.

Hutchings CJ et al, (2014), Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment. MAbs, 6:1–16.

Stoddart LA et al, (2015), Application of BRET to monitor ligand binding to GPCRs. Nature Methods, 3:1–5.

P32

Large-scale screening for membrane protein interactions involved in platelet-monocyte interactions Yi Sun, George Rainger, Steve Watson

University of Birmingham, UK

Beyond the classical roles in haemostasis and thrombosis, platelets are important in the initiation and development of various thromboinflammatory diseases. In atherosclerosis and deep vein thrombosis, for example, platelets bridge monocytes with endothelium and form heterotypic aggregates with monocytes in the circulation. This can alter monocyte phenotype by inducing their activation, stimulating adhesion and migration. These interactions involve cell surface receptor-ligand pairs on both cells. This list is likely incomplete as new interactions of importance to platelet biology are continuing to be discovered as illustrated by our discovery of PEAR-1 binding to FcεR1α.

We have developed a highly sensitive avidity-based assay to identify novel extracellular interactions among 126 recombinantly-expressed platelet cell surface and secreted proteins involved in platelet aggregation. In this study, we will use this method to identify novel platelet-monocyte interactions. We aim to identify ligands for orphan receptors and novel partners of well-known proteins. Identified interactions will be studied in preliminary functional assays to demonstrate relevance to the inflammatory processes supporting atherogenesis.

Platelet-monocyte interactions are essential for the development of thromboinflammatory disease. Up until relatively recently, technologies only allow us to limit our studies on each individual protein interaction at a single time. These studies propose for the first time to study the cell surface platelet-monocyte interactions in a systematic large-scale approach using a reliable screening method we have developed. If successful, this will likely to identify previously unknown ligands for important receptors that will be investigated in details and also provide a list of novel interactions for the field. This should stimulate studies on developing alternative therapeutic strategies to treat vascular inflammatory disorders such as atherosclerosis, DVT and sepsis and other clinically important inflammatory conditions.

Sun Y et al, (2015), A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit alpha (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand. Molecular & Cellular Proteomics, 14:1265-1274.

P33

Role of endothelial cell ADAM10 and TspanC8 tetraspains in angiogenesis. Justyna Szyroka, Michael Tomlinson

University of Birmingham, UK Angiogenesis is the naturally occurring process of the formation of new blood vessels from pre-existing vessels, and is important in health, such as wound healing and in diseases, such as cancer and atherosclerosis. ADAM10 (A Disintegrin and Metalloprotease 10) is a well-recognised ‘molecular scissor’ that cleaves major angiogenic surface proteins including Notch, VE-cadherin and VEGFR2. ADAM10 forms complexes with TspanC8 tetraspanins (Tpan5, 10, 14, 15, 17 and 33), which may direct the protease to the aforementioned substrates (Matthew et al, 2017), (Reyat et al, 2017). However, ADAM10 and TspanC8 functions in angiogenesis have not been well characterized. Thus, we hypothesised that specific endothelial TspanC8s regulate angiogenesis by controlling ADAM10 activity, surface localization and substrate specificity. Pharmacological and genetic inactivation of ADAM10 in primary human endothelial cells was used in conjunction with in vitro tube formation, proliferation and scratch wound assays to investigate its role in angiogenesis. To date, the results showed that ADAM10 promotes endothelial cell proliferation and migratory responses, but suppresses tube formation.

Future studies will aim to determine which endothelial TspanC8s promote ADAM10 regulation of angiogenesis, and which ADAM10 substrates contribute to it. Since angiogenesis is crucial for pathogenesis of many diseases, targeting ADAM10-TspanC8 complexes could emerge as a potential therapeutic strategy that will benefit human health.

Matthews AL et al, (2017), Scissor sisters: regulation of ADAM10 by the TspanC8 tetraspanins. Biochem Soc Trans, 45:719-730.

Reyat JS et al, (2017), ADAM10-Interacting Tetraspanins Tspan5 and Tspan17 Regulate VE-Cadherin Expression and Promote T Lymphocyte Transmigration. The Journal of Immunology, 15:666-676.

P34 Tetraspanin Tspan18 promotes haemostasis and thrombo-inflammation through regulation of Orai1-dependent store-operated Ca2+ entry. Michael G Tomlinson1, Peter Noy1, Rebecca Gavin1, Dario Colombo1, Jasmeet Reyat1, Alexander Brill1, Holly Payne1, Ban Dawood1,

Ina Thielmann2, Elizabeth Haining1, Jing Yang1,

Francesco Michelangeli1, Ed Rainger1, Bernhard Nieswandt3, Neil Morgan1, Stephen Watson1

1 University of Birmingham, UK 2 University of Würzburg, Germany

The induced release of von Willebrand factor (vWF) from endothelial cells is important for haemostasis, but also promotes thrombo-inflammation which can initiate deep vein thrombosis. The mechanism of induced vWF release is not fully understood, but is dependent on intracellular Ca2+ signalling. The tetraspanins are a superfamily of 33 four-transmembrane proteins which interact with specific ‘partner proteins’, and regulate their trafficking to the cell surface, clustering and lateral diffusion in the plasma membrane. The aim of this study was to perform the first characterisation of human and mouse tetraspanin Tspan18.

Tspan18 was expressed at relatively high levels by endothelial cells, but also by platelets and T-lymphocytes. Tspan18-deficient mice had defective haemostasis due to the loss of Tspan18 from non-haematopoietic cells. The mice were protected from deep vein thrombosis, and histamine-induced release of vWF from endothelium was impaired. In model cell lines, over-expression of Tspan18, but not other tetraspanins, promoted Ca2+ signalling, and Tspan18 specifically interacted with the store-operated Ca2+ entry channel Orai1. Tspan18 in human umbilical vein endothelial cells was essential for Orai1 trafficking to the plasma membrane and Ca2+ signalling, and subsequent vWF release and platelet adhesion. Finally, a rare human Tspan18 sequence alteration variant, M241V, found in two heterozygote family members with bleeding disorders, did not activate Ca2+ signalling or interact with Orai1.

In summary, Tspan18 is a novel regulator of store-operated Ca2+ entry on endothelial cells by interacting with Orai1 and promoting its trafficking to the cell surface. Tspan18 may be important in human bleeding disorders and thrombo-inflammation.

This work was supported by the British Heart Foundation and the Biotechnology and Biological Sciences Research Council.

P35

Production of antibodies against the SMALP-solubilised adenosine 2a receptor by phage display. Romez Uddin1, Richard Logan2, Jack Charlton2, Sarah Routledge1, Phillippe Diderich3, Abbey Lightfoot3, Anthony Scott-Tucker3, Charlotte Deale3, Michael Wright3, Tom Crabbe3, David Poyner1, Mark Wheatley2

1 Aston University, UK 2 University of Birmingham, UK 3 UCB Pharma Ltd, Slough, UK

Antibodies are invaluable tools for researching G protein coupled receptors (GPCRs) but are difficult to produce due to the limited exposure of epitopes. Furthermore, detergents used to solubilise GPCRs can strip lipids that are essential for function (Wheatley et al, 2016). Styrene-maleic acid co-polymer-lipid particles (SMALPs) were used instead of detergents to solubilise GPCRs with the annular lipid bilayer intact. We report the successful use of SMALP-solubilised adenosine 2a receptors (A2AR) for antibody discovery using phage display. The A2AR-SMALP was prepared and purified using nickel-charged affinity resin. The A2AR-SMALP was then immobilised on ELISA plates for phage display. A naïve llama VHH phage display library (unpublished) was first incubated with ‘empty’ (DMPC)-SMALPs to subtract SMALP binding phage. The VHH-expressing phage were then applied to A2aR-SMALPS for positive selection against the target. The ELISA plate containing the phage and the target protein was washed multiple times to remove non-binding phage. The A2AR binding phage was eluted from the target protein and was incubated with TG-1 E.coli to expand the A2AR phage library. The process was repeated two more times with increasing wash cycles to generate phage specific to A2AR. After the final biopanning round, the phage was expanded in E.coli, which was subsequently plated to obtain single colonies, each containing monoclonal phage. The individual clones were screened by monoclonal phage ELISA. From 31 monoclonal antibody-producing phage examined, six bound to the DMPC-SMALP and were rejected due to non-specific binding to lipids and SMA. No phage bound to the PBS-containing wells. Seven clones showed specific binding to the A2AR only. Thus the method appears to have discovered antigen specific VHHs against the A2AR, which retained its native conformation.

Wheatley M et al, (2016), GPCR-styrene maleic acid lipid particles (GPCR-SMALPs): their nature and potential. Biochem Soc Trans, 44:619-23.

P36

The inner workings of a GPCR: Signalling-specific conformations of vasopressin V2 receptor. Dmitry Veprintsev1,2, Franziska Heydenreich1,2, Tilman Flock1,2,3, Bianca Plouffe4, Jean Duchaine4, Xavier Deupi1, Michel Bouvier4

1 Paul Scherrer Institute, Switzerland 2 ETH Zürich, Switzerland 3 Fitzwilliam College, Cambridge, UK 4 University of Montréal, Canada The physiological effects of G protein-coupled receptor activation depend on their ability to activate effector proteins, primarily G proteins and β-arrestins. In many cases, beneficial and detrimental pharmacological effects are mediated by different effectors. Here we determined the function of each individual amino acid of vasopressin V2 receptor in signalling through several G protein subtypes and β-arrestins. We identified residues involved in common and effector-specific allosteric connections of ligand and effector binding sites. We show how changes in the ligand binding pocket bias the receptor towards specific signalling pathways. Our data suggest the existence of signalling-specific micro-conformations, which are unique for each activated effector. These microstates explain the molecular basis for functional selectivity and biased signalling by G protein-coupled receptors and outline the path to rational engineering of biased therapeutics.

P37 Utilising CRISPR/Cas9 and NanoBRET to monitor ligand binding at G protein-coupled receptors under endogenous promotion.

Carl White1,2, Elizabeth Johnstone2, Birgit Caspar1, Kevin Pfleger2, S Steve Hill1

1 University of Nottingham 2 University of Western Australia, Australia

Bioluminescence resonance energy transfer (BRET) is widely used to investigate protein or ligand interactions with membrane receptors and/or between cellular proteins. However a fundamental limitation of current BRET techniques is the requirement for ectopic expression of fusion proteins, which precludes the direct application of this method to study endogenous protein interactions in their native cellular environments. Recently, we have described the development of CRISPR/Cas9-mediated genomic engineering for BRET assays (White et al, 2017), as well as a highly sensitive NanoBRET ligand binding assay (Stoddart et al, 2015). By combining these technologies we aimed to develop a methodology to observe ligand binding at GPCRs under endogenous promotion in model cell lines.

CRISPR/Cas9-mediated homology-directed repair was used to insert Nluc into the ADORA2B or CXCR4 genomic locus in HEK293 and/or Hela cells, resulting in Nluc fused to the N-terminus of the receptors. In-frame insertion was confirmed via Sanger sequencing. Bioluminescence imaging was performed using a LV200 imaging system (Olympus). For saturation, competition and kinetic ligand binding studies, live cells containing Nluc fusion receptors were incubated with a fluorescent ligand in the absence or presence of unlabelled ligands. Luminescence was generated by the addition of furimazine and filtered light emissions were measured using a LUMIstar or PHERAstar plate reader (BMG Labtech).

Following CRISPR/Cas9 genome engineering, detectable levels of luminescence were observed in cells expressing Nluc/ADORA2B or Nluc/CXCR4. Relative expression levels of the receptors were in agreement with published data. Binding affinities (Kd) of XAC-X-BY630 at genome-edited Nluc/ADORA2B could be determined from saturation or kinetic binding analysis. IC50 values of unlabelled adenosine receptor ligands were obtained by configuring the NanoBRET assay in a competition ligand binding mode. Binding affinities of CXCL12-AF647 at gene-edited Nluc/CXCR4 in HEK293 and HeLa cells were determined via saturation binding experiments, as were IC50 values of unlabelled CXCR4 ligands from competition binding assays.

Using CRISPR/Cas9 mediated genome engineering we have shown that the NanoBRET proximity assay can be used to observe fluorescent ligand binding at GPCRs found endogenously in model cell lines.

White CW et al, (2017), Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time. Sci Rep, 7:3187

Stoddart LA et al, (2015), Application of BRET to monitor ligand binding to GPCRs. Nat Methods, 12:661–663.

P38 Light sheet microscopy: revealing platelet biology in 4 dimensions. Malou Zuidscherwoude, Natalie Poulter, Stephen Thomas, Stephen Watson University of Birmingham, UK Rapid developments in fluorescent light microscopy have advanced our ability to investigate platelet biology in living cells. In light sheet microscopy a thin sheet of light is swept through the cell while it is being imaged, building up 3D volumes of the cell over time. In contrast to other microscopy techniques, only the plane within the cell that is directly imaged is illuminated. This property makes light sheet microscopy ideal for live cell imaging, because the dose of light and thus phototoxicity is minimized whilst allowing for rapid 3D volume imaging.

We introduce two advanced light sheet based microscopes. Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM) (Wu et al, 2013) uses two perpendicular light sheets sequentially. The 3D volume is reconstructed using information from both angles. The poor axial resolution in each angle is compensated for by the better lateral resolution from the other angle, resulting in isotropic resolution. The Lattice Lightsheet Microscope (Chen et al, 2014) creates an exceptionally thin light sheet, which allows for superior optical sectioning and minimal background caused by out of focus light. With both of these methods, cells can be imaged in 3D for extended periods of time with very low doses of light.

Applying diSPIM imaging to the challenge of visualising 3D thrombus architecture has allowed us to pinpoint the exact location and boundaries of individual platelets within aggregates in 3D by overcoming the problem of poor axial resolution associated with conventional imaging techniques. Moreover, diSPIM enabled us to visualize platelet aggregation under flow and the structure of these aggregates in 3D. Lattice Lightsheet Microscopy has enabled us to track protein dynamics in individual cells in 4D without signs of phototoxicity or cell stress.

Light sheet microscopy will provide an unprecedented insight into platelet biology including mechanisms underlying platelet formation, platelet receptor activation and aggregate heterogeneity.

Wu Y et al, (2013), Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nature biotechnology, 31:1032-1038.

Chen BC et al, (2014), Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science, 346: 1257998.

P39

Expansion Microscopy (ExM) as a tool for investigating DNA repair Emma Faulkner, Rob Neely, Jo Morris, Ruth Densham

University of Birmingham, UK Protection of genomic integrity and stability is crucial for cellular and organismal function. However, DNA is subject to continuous assault by exogenous (e.g. ionising radiation, ultraviolet light) or endogenous (e.g. replication errors, intracellular reactive oxygen species) DNA damaging agents. These can cause double strand breaks (DSBs), single strand breaks (SSBs) and base damage. As a result, cells have evolved intricate interwoven mechanisms termed the DNA damage response (DDR). DDR initiates cellular responses upon sensing of DNA damage such as cell cycle checkpoint, senescence, apoptosis and DNA repair (Soutoglou E et al, 2009). Detailed molecular, biochemical and genetic analyses have allowed determination of the complex molecular framework responsible for DNA repair pathways. However, it is evident that there is crucial spatial and temporal organisation of the DNA repair machinery during the recognition of DNA lesions, and in the assembly of DNA repair complexes (Soutoglou E et al, 2009). Determining the role of spatial and temporal regulation of repair proteins during DNA repair events remains a significant challenge. Expansion Microscopy (ExM) is applied here to enable development of a visual model of molecular choreography occurring at sites of DNA damage. ExM allows visualisation of 3D biological specimens with nanoscale resolution on diffraction-limited microscopes. Samples are embedded in a polyelectrolyte gel, which is then physically expanded by the addition of water. This expansion allows visualisation of subdiffraction limit features by expanding them to be greater than the diffraction limit. Expansion is isotropic allowing resolution of cellular structures with an effective resolution of ∼70nm (Geertsema H et al, 2016). We have demonstrated ExM can be performed on nuclear DNA in an isotropic, reproducible manner. We have applied use of antibodies and fluorescent proteins to investigate key proteins in the repair of DSBs in the context of chromatin.

Soutoglou E et al, (2009), The Emerging role of nuclear architecture in DNA repair and genome maintenance. Nature Reviews. 10: 243-254.

Geertsema H et al, (2016), Expansion Microscopy passes its first test. Nature methods 13, 481-482.

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PARTICIPANTS

55

Sharmaine Afferion [email protected]

Juliana Akinaga [email protected]

Diana Alcobia [email protected]

Nuha Anajirih [email protected]

Elaine Anderson [email protected]

Maria Agusta Arruda [email protected]

Anastasia Arvaniti [email protected]

Fiona Ashford [email protected]

Julia Ast [email protected]

Kurt Ballmer-Hofer [email protected]

Phil Bardelang [email protected]

Arisbel Batista-Gondin [email protected]

Nick Beazley-Long [email protected]

Tom Bellamy [email protected]

Andrew Benest [email protected]

Tobias Benkel [email protected]

Joanne Berry [email protected]

Monica Bouzo-Lorenzo [email protected]

Steve Briddon [email protected]

Nigel Bunnett [email protected]

Davide Calebiro [email protected]

Meri Canals [email protected]

Kathleen Caron [email protected]

Jessica Carpenter [email protected]

Birgit Caspar [email protected]

Vicky Chapman [email protected]

Steven Charlton [email protected]

Stefania Cioato [email protected]

Joanne Clark [email protected]

Samantha Cooper [email protected]

Africa Couto [email protected]

Federica Cuozzo [email protected]

Sebastien Dekkers [email protected]

Alessandro di Maio [email protected]

Jonas Emsley [email protected]

Emma Faulkner [email protected]

Nicholas Fine [email protected]

Hester Franks [email protected]

Karolina Gherbi [email protected]

56

Amalia Goula [email protected]

Joëlle Goulding [email protected]

Rachael Grime [email protected]

Nick Groenewoud [email protected]

Rob Hall [email protected]

Steve Hill [email protected]

David Hodson [email protected]

Nick Holliday [email protected]

Aaron Horsey [email protected]

Wafa Hourani [email protected]

Neena Kalia [email protected]

Dee Kavanagh [email protected]

Barrie Kellam [email protected]

Abdullah Khan [email protected]

Laura Kilpatrick [email protected]

Connie Koo [email protected]

Evi Kostenis [email protected]

Becky Lewis [email protected]

Jack Lochray [email protected]

Adam Lokman [email protected]

Yi Long [email protected]

Isabella Maiellaro [email protected]

Saori Makaia [email protected]

Robert Markus [email protected]

Alexandra Matthews [email protected]

Christine McGrath [email protected]

Liciane Medeiros [email protected]

Raquel Mesquita-Ribeiro [email protected]

Sarah Mistry [email protected]

Neil Morgan [email protected]

Georgiana Neag [email protected]

Rob Neely [email protected]

Marie-Blanche Onselaer [email protected]

Tom O'Sullivan [email protected]

Chiara Pallini [email protected]

Chloe Peach [email protected]

Jeremy Pike [email protected]

Natalie Poulter [email protected]

David Poyner [email protected]

57

Chidinma Raymond [email protected]

Steve Rees [email protected]

Raphael Reher [email protected]

Rachel Richardson [email protected]

Anne Ridley [email protected]

Elizabeth Rosethorne [email protected]

Andrea Sabbatini [email protected]

Ian Sayers [email protected]

Tim Self [email protected]

Victoria Simms [email protected]

John Simms [email protected]

Alex Slater [email protected]

Darren Smith [email protected]

Leigh Stoddart [email protected]

Sarah Storr [email protected]

Iain Styles [email protected]

Yi Sun [email protected]

Jason Swedlow [email protected]

David Sykes [email protected]

Justyna Szyroka [email protected]

Steve Thomas [email protected]

Lorna Thorne [email protected]

Mike Tomlinson [email protected]

Romez Uddin [email protected]

Sally Utton [email protected]

Dmitry Veprintsev [email protected]

Steve Watson [email protected]

Andy West [email protected]

Mark Wheatley [email protected]

Carl White [email protected]

Jeanette Woolard [email protected]

Joanne Wright [email protected]

Zhi Yuan [email protected]

Iwona Ziomkiewicz [email protected]

Malou Zuidscherwoude [email protected]

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COMPARE Directors and PIs

DIRECTORS

Steve Hill, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

Steve Watson, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham

DEPUTY DIRECTORS

Iain Styles, School of Computer Science, College of Engineering and Physical Sciences, University of Birmingham

Jeanette Woolard, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

PRINCIPAL INVESTIGATORS

Jillian Baker, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

David Bates, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham

Roy Bicknell, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham

Steve Briddon, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

Alex Brill, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham

Davide Calebiro, Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham

Meritxell Canals, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

Steven Charlton, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

Chris Denning, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham

Jonas Emsley, Division of Biomolecular Sciences and Medicinal Chemistry, School of Pharmacy, University of Nottingham

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Peter Fischer, Division of Biomolecular Sciences and Medicinal Chemistry, School of Pharmacy, University of Nottingham

Ian Hall, Division of Respiratory Medicine, School of Medicine, University of Nottingham

David Hodson, Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham

Neena Kalia, Institute of Cardiovasular Sciences, College of Medical and Dental Sciences, University of Birmingham

Barrie Kellam, Division of Biomolecular Sciences and Medicinal Chemistry, School of Pharmacy, University of Nottingham

Rob Lane, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

Paula Mendes, Chemical Engineering, College of Engineering and Physical Sciences, University of Birmingham

Robert Neely, Department of Chemistry, College of Engineering and Physical Sciences, University of Birmingham

Natalie Poulter, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham

Yotis Senis, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham Dr Yi Sun (Receptor-Ligand Interactions, UoB)

Steve Thomas, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham

Mike Tomlinson, Department of Biosciences, College of Life and Environmental Sciences, University of Birmingham

Dmitry Veprintsev, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham

Mark Wheatley, Department of Biosciences, College of Life and Environmental Sciences, University of Birmingham

COMPARE FELLOW

Yi Sun, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham

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International Advisory Board

Membership:

Chair Patrick Vallance GlaxoSmithKline

Deputy Steve Rees AstraZeneca

Members Kurt Ballmer-Hofer University of Basel

Nigel Bunnett Columbia University

Kathleen Caron University of North Carolina,

Evi Kostenis University of Bonn

Anne Ridley Kings College London

Jason Swedlow University of Dundee

Victor Tybulewicz The Francis Crick Institute

Strategic Oversight Group

David Adams University of Birmingham

John Atherton University of Nottingham

Jessica Corner University of Nottingham

Tim Softley University of Birmingham

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Support Team

For all initial enquiries about COMPARE,please email: [email protected]

Web: www.birmingham-nottingham.ac.uk/compare/index.aspx

Sally Utton, Programme Operations Manager Tel: +44 (0)121 414 4511 or +44 (0)115 823 0105

Email: [email protected] Christine McGrath, Administrator, Nottingham Tel: +44 (0)115 823 0398

Email: [email protected] Sharmaine Afferion, Administrator, Birmingham Tel: +44 (0) 121 414 4511

Email: [email protected] University of Birmingham The University of Nottingham Edgbaston University Park Birmingham Nottingham B15 2TT NG7 2RD