A Single Molecule Detection Platform (SMD) for a Leica SP8 TCS to analyse protein-protein interactions in living specimen.

The discovery of the fluorescent protein GFP (green fluorescent protein) from certain jellyfish, has revolutionized our ability to study protein localization, protein dynamics and interactions of proteins. These and related fluorescent proteins allow us to investigate protein function within the complex environment of the living cell. Parallel to the developments in fluorescent protein biology, there have been advances in fluorescence imaging methods and microscopical systems that make it possible to localize proteins linked with such fluorescent proteins, to quantitate their abundance and to probe their mobility and interactions with each other. Recently, two imaging methods such as Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have been modified so that they can be done on user-friendly, commercially available laser scanning microscopes, replacing the need for complex custom-built microscopes. The combined advances in GFP biology and imaging methods are providing a massive stimulus for investigating the dynamic properties of proteins in living cells.

The University of Exeter excels in many aspects of biomedical research, from fungal-related plant disease research to signalling biology in vertebrate embryonic development. In the past, biochemical approaches have been used to investigate protein-protein interactions. These observations can now be complemented and extended in real time in living cells. The latest generation of single molecule detectors is therefore a game changer for protein biology - in bacteria, fungi, plants and animals. It is now possible to generate dynamic maps of protein interactions in living cells using a fluorescence microscope. In this application, we seek support to purchase a modern single molecule detection platform (SMD), which will complement the existing facilities at the Exeter Bioimaging Centre.

The development of the many different genetically encoded fluorescent proteins has sparked a revolution in optical imaging in biomedical research. These fluorescent proteins have expanded the repertoire of imaging applications from multi-colour imaging of protein co-localization and behaviour inside living cells to the detection of changes in intracellular activities, such as pH or ion concentration. Two technologies have taken centre stage in analysing these biological processes, Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS).

On the one hand, FRET is the process by which energy absorbed by one donor fluorophore is transferred directly to another nearby acceptor molecule through a non-radiative pathway. Only when the donor-acceptor pairs get close enough during specific protein-protein interactions would FRET result. The quenching of the donor as well as the sensitized acceptor emission can be used to quantify energy transfer, most sensitively measured through changes of the fluorescence lifetime. On the other hand, FCS has the ability to measure concentrations and diffusion constants, as well as to detect several diffusing species. The dual-colour variation, termed Fluorescence Cross-Correlation Spectroscopy (FCCS), is utilized to probe two proteins labelled with different fluorophores. FCCS can extend investigations to examine reactions between two partners, such as kinetics, and fractions of binding proteins.

This application seeks funds to purchase a modern single molecule detection platform (SMD) to

(i) investigate the molecular proximity of the donor- and acceptor-labelled proteins using FRET by fluorescence lifetime imaging (FLIM)
(ii) measure concentrations and diffusion constants, as well as to detect several diffusing species by FCS.

It will be accessible to a wide range of internal and external users and, therefore, will significantly improve the research infrastructure at Exeter and within the UK.

The gene encoding green fluorescent protein (GFP) has become an important visual marker of protein localisation in unicellular and multicellular organisms. In 2008, the Nobel Prize in Chemistry has been awarded for the discovery and development of GFP and related fluorescent protein tools. Parallel to the developments in GFP biology, there have been advances in imaging technologies that make it possible to localize proteins fused with GFP by sensitive imaging technologies. And indeed, six years later the Nobel Prize in Chemistry was awarded for the development of super-resolved fluorescence microscopy. We base our application on these fundamental discoveries. The impact of our proposed work is therefore timely and significant on the world's stage with regard to protein-protein interactions cross-species. Our work will draw together the disciplines of cell and developmental biology, plant disease biology, ecosystem health, cell movement dynamics and effector biology

Our work will impact upon:
i) Knowledge - giving a greater understanding of the protein-protein interactions in living cells
ii) UK science - enhancing the profile of the Investigators on the national and international stage
iii) Interdisciplinary science - endowing the Investigators with new awareness and widened interdisciplinary skills
iv) Fostering broadened industrial links with Leica Microsystems.
v) Fuelling greater research effort across the GW4 universities.
vi) Enhancing skills of experimental officer and training new staff and students
vii) Promoting public awareness of science and raising awareness of importance of fluorescence imaging technologies in life science.