A label-free tool to unravel the dynamics of lipid bilayers containing single membrane proteins: iGOR microscopy

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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.