Spatiotemporal Analysis of GPCR-Dependent Rac Signalling in Neutrophils

Lead Research Organisation: Babraham Institute
Department Name: Signalling

Abstract

Rac is a protein that enables cells to do a myriad of things, such as moving around, secreting various factors, or eating and killing germs. In order to fulfil its many roles, Rac must become activated, which happens in response to signals such as hormones or growth factors that are received by the cell from the outside. Activation of Rac is done by a large group of proteins called GEFs which read these signals. We assume that so many different Rac-GEFs exist in order for each to induce a characteristic pattern of Rac activity within the cell, and that these patterns of Rac activity dictate how the cell reacts to a given signal. We have developed a tool that enables us to test this hypothesis, namely a mouse strain which carries a fluorescent dye that shows up colour patterns of Rac activity in living cells, the so-called Rac-FRET mouse strain. For this first project with the Rac-FRET mouse strain, we have chosen to study Rac activity in neutrophils, a type of white blood cell that defends us against bacterial and fungal infections. Rac activity is essential for neutrophils to function appropriately. When neutrophils don't work well, we get severe and repeated infections, or, when they work too hard, they can cause or worsen inflammatory disorders such as septic shock and rheumatoid arthritis. Therefore, although our project mainly aims to advance basic science, it also carries inherent importance for our health. We will measure Rac activity in neutrophils from the Rac-FRET mouse strain (we already know that this will work). Mostly, Rac activity will be assessed by microscopy, so we can measure both how much and when and where Rac is active. We will compare the patterns of Rac activity in normal Rac-FRET neutrophils to those which lack certain GEFs. The GEFs we chose for this purpose, namely P-Rex1/Vav1 and DOCK2, are known to be important in relaying a particularly type of signal, called GPCR signal, which enables many different neutrophil functions. If we can show that P-Rex1/Vav1 and DOCK2 cause distinct patterns of Rac activity in neutrophils in response to GPCR signals, we will have proof that they don't simply fulfil interchangeable roles. By correlating the patterns of Rac activity induced by each GEF with the responses that they induce in the cell, such as cell migration, we aim to proof that different pools of Rac activity within the cell can give rise to different neutrophil functions. In addition to our microscopy-based experiments, we will also use the Rac-FRET neutrophils in the development of an improved test-tube based method for testing reagents that stimulate or suppress Rac activity. Together, the results from this study will fundamentally advance our insight into Rac and neutrophil biology. It may also help us decide which GEFs to target in future for inhibiting inappropriate levels of Rac activity in inflammatory disorders while preserving Rac activity for immune-defence.

Technical Summary

The small G protein Rac is an essential controller of the actomyosin cytoskeleton, gene expression and oxygen radical formation. Rac is activated by a large number of GEFs. A central question is how Rac-GEFs translate extracellular signals into specific spatiotemporal patterns of Rac activity. We have developed a tool for quantifying Rac activity in living primary cells, a mouse strain that expresses a Rac activity reporter, Rac-FRET. In this first project using the Rac-FRET mouse, we will study the spatiotemporal regulation of Rac in neutrophils. Rac is pivotal for signal integration in neutrophils. Rac-deficiency causes severe neutrophil-dependent immunodeficiency, and inappropriate activation of Rac drives inflammatory disease. The Rac-FRET mouse enables us to quantify Rac activity in living primary neutrophils with excellent spatiotemporal resolution. Our hypothesis is that different GEFs activate spatiotemporally distinct pools of Rac. Use of the Rac-FRET mouse, alone or in combination with genetic deficiencies in selected neutrophil GEFs, will test this hypothesis. The selected Rac-GEFs, P-Rex1/Vav1 (P1V1) and DOCK2, are critical for neutrophil responses to GPCR activation. We will cross P1V1- and DOCK2-deficient mice to the Rac-FRET mouse and characterise Rac-dependent neutrophil GPCR responses and signalling pathways in P1V1, DOCK2 and Rac-FRET mice. We will develop a Rac2-FRET mouse strain for comparison of Rac1 and Rac2 activity. We will image the patterns of Rac activity in Rac-FRET, Rac2-FRET, P1V1/FRET and DOCK2/FRET neutrophils. This will show if P1V1 and DOCK2 generate spatiotemporally distinct pools of Rac activity in response to GPCR stimulation. Finally, we will develop a multi-sample assay using neutrophils from the Rac-FRET and Rac2-FRET mice to allow screening of a range of stimuli of Rac activity. The project will fundamentally advance our knowledge of small G protein and neutrophil regulation.

Planned Impact

Who will benefit from this research? The academic beneficiaries of this work are G protein and neutrophil research labs. An immediate beneficiary is the post-doc who will be doing the research. Other beneficiaries are the Babraham Institute and the BBSRC. The commercial sector is a possible future beneficiary. In the longer term, the healthcare sector, patients with acute or chronic inflammatory disorders and the UK economy are hoped to benefit. How will they benefit from this research? Our colleagues in G protein and neutrophil biology labs worldwide will benefit from this project as detailed in 'Academic Beneficiaries'. The main benefits for the post doc employed to work on this project will be the good publications which it promises to yield and the experience gained for a future career in scientific research. He/she will become an expert in G protein and neutrophil signalling, FRET imaging and mouse work. The post doc will be able to present his/her results at our weekly meetings with the other groups of the Inositide Lab, at our annual BI lab talks, and at international scientific conferences. Further opportunities to present his/her work will be at meetings which are regularly organised by Babraham's commercial affairs office (BBT) between companies and BI researchers to discuss potential areas of common interest. The Babraham Institute and the BBSRC will profit from the publications arising from this project and from our good standing in the scientific community, which this project is expected to consolidate further. At Babraham, the other groups in the Inositide Laboratory will benefit in particular, as several others of these also work on regulators of small G proteins or use neutrophils as their cell system of interest. Our project maps squarely into the BBSRC's strategic research priority 3 'basic bioscience underpinning health' by driving advances in fundamental bioscience aimed at leading to better health and improved quality of life, thus reducing the need for medical and social intervention Two of the BBSRC's key priorities for 2010-2015 are the 'development of model organisms and systems that provide insight into physiological processes that are key for maintaining health in humans' and the 'development of new tools in areas such as bio-imaging'. With the Rac-FRET mouse that forms the basis of this application, and with the generation of the Rac2-FRET mouse as part of this application, our project maps into both these BBSRC priorities. Possible impact on the commercial sector arises from our development of the multi-sample Rac activity assay as part of this project. Rac-GEFs are implicated in a variety of human diseases, mainly immune disorders, cancer and developmental disorders. Largely due to structural complexity, the interaction between GEFs and Rac has, until recently, not received much attention as a target for novel therapeutics. However, several groups have now succeeded in identifying small molecule inhibitors that target this interaction (Gao, 2004, PNAS 18:7618; Shutes, 2007, JBC 282:35666), although these early compounds still have weak potency. We have recently embarked ourselves on developing Rac-GEF inhibitors, with encouraging preliminary results. The multi-sample FRET-based Rac activity assay will be useful for testing our candidate compounds in vivo, and may move this project towards commercial exploitation. In the longer term, the healthcare sector, patients with acute or chronic inflammatory disorders and the UK economy are hoped to benefit from our work, because Rac activity is crucial for neutrophil function. It controls a fine balance between neutrophil-dependent immune-deficiency and inflammatory disorder. In the long term, we envisage a general strategy of targeting specifically the subset of regulators of Rac activity that promote inflammation while preserving the Rac activity required for immune function.

Publications

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Lindsay CR (2015) A Rac1-independent role for P-Rex1 in melanoblasts. in The Journal of investigative dermatology

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Pitchford S (2017) Platelets in neutrophil recruitment to sites of inflammation. in Current opinion in hematology

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Pantarelli C (2018) Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment. in European journal of clinical investigation

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Welch HC (2015) Regulation and function of P-Rex family Rac-GEFs. in Small GTPases

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Lissanu Deribe Y (2016) Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma. in Proceedings of the National Academy of Sciences of the United States of America

 
Description This project has elucidated the spatiotemporal distribution Rac activity in neutrophils and in other primary cell types, tissues and organs. We found both expected and unexpected patterns of Rac activity in neutrophils, the origins of which we are currently investigating in a follow-up study. We also monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators. This study evealed unexpected insights into Rac signaling during disease development. In a recent collaboration with the Garvan Institute, we have contributed to the generation and characterization of a similar mouse model.

Over the past two years, we have built on the data from this grant through our continued collaboration with Paul Timpson at the Garvan Institute in Sydney, which has given rise to 2 further joint publications.

The award has also formed the basis of a new BBSRC funded PhD project to study which Rac-GEF activates which subcellular pool of Rac during neutrophils adhesion and migration, making use of unpublished GEF-FRET mouse strains that we have generated using the original reporter mouse strain from this award.
Exploitation Route With the Rac-FRET mouse strain, we have generated a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments. We have made the mouse strain available to a number of labs worldwide already.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.babraham.ac.uk/our-research/signalling/heidi-welch
 
Description Our own currently unpublished follow-un work has shown that different types of Rac-GEFs activate different subcellular pools of Rac during different neutrophil responses. This work is ongoing and will give rise to at least two more primary publications in the future. In collaboration with us, others have used our mouse to monitor Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development.
First Year Of Impact 2014
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Title Collection of P-Rex-deficient and control tissues. 
Description This is available to interested research groups, mainly for testing of panels of new P-Rex antibodies. 
Type Of Material Biological samples 
Year Produced 2006 
Provided To Others? Yes  
Impact Better characterised research tools for the P-Rex research field. 
 
Title FRET-GEF mosue strains 
Description Rac-FRET reporter expressing GEF-deficient mouse strains 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact joint publications with several research groups in progress , pending 
 
Title P-Rex deficient mouse strains 
Description P-Rex deficient mouse strains 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Two publications, several more submitted or currently in preparation, many more expected in the future. Discovery unsuspected roles of P-Rex in cancer metastasis and in motor control. 
 
Title Rac-FRET mouse 
Description Rac activity reporter mouse strains 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact joint publications 
 
Title Role of P-Rex in cancer metastasis 
Description Our P-Rex deficeint mice crossed with mouse models of cancer metastasis 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2006 
Provided To Others? Yes  
Impact Provides the field with the research tools to investigate a role for P-Rex in metastasis formation 
 
Description Alexander Zarbock (Uni Muenster, Germany) 
Organisation University of Münster
Country Germany 
Sector Academic/University 
PI Contribution Role of P-Rex1 in neutrophil rolling and adhesion
Collaborator Contribution exchange of reagents joint paper
Impact joint paper
Start Year 2008
 
Description Beatson Institute, Glasgow: Characterisation of our Rac-activity Fret reporter mice 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution joint experiments exchange of reagents lab visits joint publications
Collaborator Contribution joint experiments exchange of reagents lab visits joint publications
Impact 2 joint papers
Start Year 2011
 
Description Jonathan Clark (BBT, Cambridge): 
Organisation Babraham Bioscience Technologies
Country United Kingdom 
Sector Private 
PI Contribution Development of a first-generation small-molecule P-Rex inhibitor
Collaborator Contribution Development of a first-generation small-molecule P-Rex inhibitor
Impact production of small molecule compounds to inhibit P-Rex
Start Year 2009
 
Description Marcus Thelen (IRB, Bellinzona, Switzerland) 
Organisation Institute for Research in Biomedicine (IRB)
Country Spain 
Sector Academic/University 
PI Contribution Marcus is interested in P-Rex activation through phosphorylation within cytokine signalling pathways. We've collaborated for many years, on a succession of project.
Collaborator Contribution exchange of reagents, lab visits, joint experiment, joint publication
Impact joint publications production of mAB 6F12
 
Description Role of P-Rex in metastasis 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide mice, reagents, and assay know-how. We meet and are in contact regularly.
Collaborator Contribution Our collaborators are using our P-Rex-deficient mice, constructs and antibodies to test the role of P-Rex in cancer cell migration.
Impact A joint publication is currently submitted.
 
Description Simon Pitchford and Clive Page (KCL, London) 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Role of platelets in leukocyte recruitment
Collaborator Contribution joint experiments lab visits joint publications
Impact joint papers in Blood and in Curr Opin In Hematol
Start Year 2009
 
Description Yonathan Lissanu-Deribe and Lynda Chin (Anderson Cancer Center, Texas University, USA): 
Organisation University of Texas
Department M. D. Anderson Cancer Center
Country United States 
Sector Academic/University 
PI Contribution Role of P-Rex2 in melanoma growth and metastasis
Collaborator Contribution joint experiments
Impact none yet
Start Year 2012
 
Description Editorial board member Cell Rep 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Editorial board member of
Cell Reports
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016,2017,2018,2019
 
Description European Phagocyte Workshop - Member of steering committee 
Form Of Engagement Activity A formal working group, expert panel or dialogue
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Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Steering committee of Annual European Phagocyte Workshop
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description Phagocytes.Net 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I am Editor of this website for fellow phagocyte researchers, postdocs, students and the general public that provides information on phagocyte-related events, research groups, and jobs. www.phagcytes.net

Better communication among research groups in the phagocyte field. Markedly increased numbers of participants at the annual meetings of the European Phagocyte Workshop.
Year(s) Of Engagement Activity 2006,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://www.phagcytes.net
 
Description Schools Days at Babraham 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact We host 10 GCSE or A level students for one day every year to conduct experiments.

Rasied image of "science" as a profession and of "scientists" among young people in the area.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description Signalling Escape Room 
Form Of Engagement Activity Participation in an activity, workshop or similar
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Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A PhD student from our lab was instrumental in devising and running the Signalling Escape Room, a fun activity where members of the public get "locked in" for 45 min and need to solve cell signalling pathway themed puzzles in order to escape. We took this event to the Cambridge Science Festival, the Latitude Music Festival , as well as running it locally for visitors, including the Head of the BBSRC. Everyone who participated loved it. The event was booked up in advance wherever we took it.
Year(s) Of Engagement Activity 2019