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.
Organisations
- Babraham Institute (Lead Research Organisation)
- Babraham Bioscience Technologies (Collaboration)
- The University of Texas at San Antonio (Collaboration)
- University of Münster (Collaboration)
- Institute for Research in Biomedicine (IRB) (Collaboration)
- Beatson Institute for Cancer Research (Collaboration)
- KING'S COLLEGE LONDON (Collaboration)
People |
ORCID iD |
Heidi Welch (Principal Investigator) |
Publications
Pan D
(2016)
Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1.
in The Journal of biological chemistry
Barber MA
(2012)
The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1a.
in The Biochemical journal
Lawson CD
(2022)
Small-molecule inhibitors of P-Rex guanine-nucleotide exchange factors.
in Small GTPases
Pajic M
(2015)
The dynamics of Rho GTPase signaling and implications for targeting cancer and the tumor microenvironment
in Small GTPases
Welch HC
(2015)
Regulation and function of P-Rex family Rac-GEFs.
in Small GTPases
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
Srijakotre N
(2020)
PtdIns(3,4,5)P3-dependent Rac exchanger 1 (P-Rex1) promotes mammary tumor initiation and metastasis.
in Proceedings of the National Academy of Sciences of the United States of America
Campbell A
(2013)
P-Rex1 Cooperates with PDGFRß to Drive Cellular Migration in 3D Microenvironments
in PLoS ONE
Crainiciuc G
(2022)
Behavioural immune landscapes of inflammation.
in Nature
Lindsay C
(2015)
A Rac1-Independent Role for P-Rex1 in Melanoblasts
in Journal of Investigative Dermatology
Hornigold K
(2022)
Age-related decline in the resistance of mice to bacterial infection and in LPS/TLR4 pathway-dependent neutrophil responses.
in Frontiers in immunology
Machin P
(2023)
Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis
in Frontiers in Immunology
Pantarelli C
(2018)
Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.
in European journal of clinical investigation
Warren SC
(2018)
Removing physiological motion from intravital and clinical functional imaging data.
in eLife
Baker MJ
(2016)
Small GTPases and their guanine-nucleotide exchange factors and GTPase-activating proteins in neutrophil recruitment.
in Current opinion in hematology
Pitchford S
(2017)
Platelets in neutrophil recruitment to sites of inflammation.
in Current opinion in hematology
Floerchinger A
(2021)
Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging.
in Cell reports
Nobis M
(2017)
A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.
in Cell reports
Johnsson AE
(2014)
The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues.
in Cell reports
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 | Devising and Running of Signalling Escape Room - Elizabeth Hampson |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
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 |
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 |
Part Of Official Scheme? | No |
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 |