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

Lead Research Organisation: University of Exeter
Department Name: Biosciences


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.

Technical Summary

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.

Planned Impact

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.
Description We have completed several objectives from our work plan and have published one manuscript (Histochem Cell Biol. 2020 Nov;154(5):507-519) and a second manuscript has been accepted in Nature Communications.

Abstract: Cell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.

Abstract: Wnt signalling regulates cell proliferation and cell differentiation as well as migration and polarity during development. However, it is still unclear how the Wnt ligand distribution is precisely controlled to fulfil these functions. Here, we show that the planar cell polarity protein Vangl2 regulates the distribution of Wnt by cytonemes. In zebrafish epiblast cells, mouse intestinal telocytes and human gastric cancer cells, Vangl2 activation generates extremely long cytonemes, which branch and deliver Wnt protein to multiple cells. The Vangl2-activated cytonemes increase Wnt/ß-catenin signalling in the surrounding cells. Concordantly, Vangl2 inhibition causes fewer and shorter cytonemes to be formed and reduces paracrine Wnt/ß-catenin signalling. A mathematical model simulating these Vangl2 functions on cytonemes in zebrafish gastrulation predicts a shift of the signalling gradient, altered tissue patterning, and a loss of tissue domain sharpness. We confirmed these predictions during anteroposterior patterning in the zebrafish neural plate. In summary, we demonstrate that Vangl2 is fundamental to paracrine Wnt/ß-catenin signalling by controlling cytoneme behaviour.
Exploitation Route Exploitation and application
We organized three workshops for the users of the GW4 universities including Bristol, Cardiff and Bath Universities.

Communications and Engagement
We ensured that all findings are disseminated to the academic and user community, via publication of data in acclaimed high impact journals (see details above) and depositing in appropriate repositories and databases (e.g. Dryard), crafting of succinct reviews (see details above) and via presentations at local, national and international conferences - due to COVID19 virtual only.

Impact Milestones
We organized/took part in the following events to present data obtained by the SMD:
Outreach events for the general public (2019, 2020)
Workshop Sep 2020
EMBO Workshop 2021
Sectors Pharmaceuticals and Medical Biotechnology

Description Function of cytonemes in the mouse intestinal crypt 
Organisation Duke-NUS Graduate Medical School
Country Singapore 
Sector Academic/University 
PI Contribution We have now investigated how cytonemes form using a combination of state-of-the-art genetic and high-resolution imaging techniques. In initial experiments involving zebrafish cells that were grown in the laboratory, we found that the Wnt proteins kick start their own transport; before they travel to their destination, they act on the cells that made them. Wnt proteins activate the receptor Ror2 and Vangl2 on the surface of the signal-producing cell. Ror2/Vangl2 then triggers signals inside the cell that begin the assembly of the cytonemes. The more Vangl2/Ror2 is activated, the more cytonemes the cell makes, and the more Wnt signals it can send out. eLife : https://elifesciences.org/articles/36953 Nature Communications: accepted
Collaborator Contribution Together with the group of Prof DM Virshup, we have shown that cytonemes are regulated by Ror2 and Vangl2.This mechanism operates in various tissues: Ror2/Vangl2 also controls the cytoneme transport process in living zebrafish embryos, and in the mouse intestine. This knowledge will help us to develop new ways to control Wnt signalling, which could help to produce new treatments for diseases ranging from cancers (for example in the stomach and bowel) to degenerative diseases such as Alzheimer's disease.
Impact Publication in eLife, and Nature Communications. Collaboration is multi-disciplinary : cell biology, developmental biology, biochemistry.
Start Year 2017