Spatiotemporal Analysis of GPCR-Dependent Rac Signalling in Neutrophils

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.

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.

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.