Approximately 30% of the proteins in a given organism are membrane proteins. These represent more than 60% of all known drug targets and play a critical role in both infection and immunity. The organization of membrane proteins into complexes, their segregation in lipid domains, and their effect on membrane shape is known to influence processes such as intracellular transport, cell division, cell migration, and signal transduction. Despite this importance, understanding the underlying principles of protein organization and function directly in the membrane lipid environment is severely limited by the lack of suitable non-invasive techniques with sufficient spatio-temporal resolution and sensitivity.

Optical imaging has distinct advantages compared to contact-based techniques such as atomic force microscopy in terms of non-invasiveness, and can offer localisation precision at the nanoscale beyond the optical diffraction limit. To achieve enough sensitivity and specificity, optical methodologies mostly rely on fluorescence labelling with the drawbacks of limited observation periods due to photobleaching, phototoxicity, and most importantly the need for chemical or genetic sample manipulation/modification which raises the question if the observed behaviour is real or artifactual.

Here, we propose the development of a novel optical imaging method which we have called interferometric gated off-axis reflectance (iGOR) microscopy suitable for fast tracking of single unlabelled biomolecules with millisecond time resolution, via their intrinsic scattered light, in suspended bilayer membranes. Notably, the technique will enable to quantify the elasto-mechanical properties of the lipid membrane, through precise measurement of the layer topography and its fluctuation dynamics, simultaneously with tracking single protein diffusion in 3D on the millisecond time scale, thus revealing new insights into membrane-protein interactions. iGOR will be developed as an optical set-up hardware and associated quantitative data analysis toolkits, to extract time-dependent position coordinates of single proteins correlated with time-dependent membrane axial position and thickness maps, with unprecedented sensitivity and precision.

As a biologically-relevant test of iGOR's capabilities, we will investigate the diffusion of integral membrane proteins (P2X receptors) into a suspended lipid membrane. P2X receptors are cell-surface ion channels which are activated by extracellular ATP. Activation leads to downstream signalling events which have important consequences for nerve transmission, pain sensation, inflammation and control of smooth muscle tone. Therefore, P2X receptors are important drug targets for analgesic or anti-inflammatory actions. Notably, growing evidence, albeit based on indirect biochemistry assays, indicates that they partition into lipid-ordered compartments. iGOR is therefore an ideal technology to directly address the question of how P2X receptors diffuse and partition into lipid microdomains.

We expect that inserting the protein will lead to a local deformation of the membrane, which can be sensitively measured by our technique. It was recently suggested that this deformation results in effective repulsive and attractive interactions between proteins, mediated by the membrane, similar to a Coulomb interaction between charges. The unique possibilities offered by our iGOR method will thus open the exciting prospect of understanding these fundamental aspects of membrane biophysics, which are attracting a lot of interest.

In future work, iGOR could be upgraded to include an electrophysiology assay to measure membrane voltages and ion fluxes, which will pave the way toward addressing long-standing questions in the membrane-protein research field, e.g. whether ion channel function varies depending if these proteins are in lipid rafts or non-raft compartments.

Technical Abstract:
Our programme of work has three workpackages. Firstly, we will develop and build iGOR's experimental set-up. Key points will be i) the use of an external off-axis reference light field, and ii) the time-gated detection of the scattered field from the sample via its interference with the reference field using short optical pulses (femtosecond laser sources available in house). Notably, the off-axis interference enables the retrieval of the scattered field in amplitude and phase. This offers additional 'topography' information with sub-nanometer sensitivity, since changes in the phase of the scattered field are proportional to the relative axial position of the object in reflection geometry, while the amplitude of the scattered field is proportional to the object dry mass.
Secondly, we will develop the quantitative data analysis toolkit to extract time-dependent axial position and thickness maps of a suspended lipid membrane, and the subsequent analysis to derive the elasto-mechanical properties of the membrane. Algorithms will be implemented in C and/or MATLAB, and will adopt mathematical approaches based on analysing the data in Fourier domain. We will demonstrate this analysis and the performance of iGOR on lipid membranes suspended in aqueous environment, fabricated in-house in the form of "half-sphere" giant unilamellar vesicles (hsGUVs) partly attached to the glass support during electroformation, such that they are translationally not mobile but a substantial part of their surface is suspended.
Thirdly, as a biologically-relevant test of iGOR's capabilities for single molecule tracking, we will investigate the diffusion of integral membrane proteins (P2X receptors, for which we have substantial expertise) into a suspended hsGUV. We will further develop our data analysis toolkit in order to extract time-dependent position coordinates of single proteins correlated with time-dependent axial position and thickness maps of the surrounding membrane.

Potential Impact:
Identified impacts

Society: Membrane proteins represent the target of about two thirds of currently marketed pharmaceuticals and play a critical role in both infection and immunity. As such, the development of the next generation enabling technologies to directly address their organisation and function at the lipid membrane, and potentially resolve key open questions - e.g. the highly debated 'lipid-raft hypothesis' in many viral diseases - will contribute to the development of a more healthy nation.

Economy: Microscope manufacturers will be interested in this novel label-free non-invasive imaging technique based on a simple layout that can be easily combined with commercially available wide-field microscope instruments. In addition, our work may help with structural studies which can lead to structure-based drug designs important for the pharmaceutical sector.

Knowledge: This project will deliver an enabling technology with the potential to significantly advance our understanding of the role that lipid-lipid and lipid-protein interactions play in many important processes such as intracellular transport, cell division, cell migration, and signal transduction. The outcomes of this work will be very relevant to groups working on membrane proteins worldwide, trying to understand their behavior at the nanoscale and in real time. This work will also contribute towards the development of better membrane protein expression and purification protocols.

People: This project will appoint one postdoctoral researcher who will be trained in optical microscopy technology developments beyond state-of-the-art for applications in molecular cell biology. This researcher will be given the opportunity to co-supervise final year undergraduate and/or MSc students performing final year projects in our labs (our group typically has >6 lab project students each year). Hence this project will generate a legacy of highly trained, skilled people at the physics/life sciences interface.

A description of the pathways to impact is given in the corresponding attachment.