Abstract

Super-resolution microscopy techniques have the potential to transform the imaging of cellular protein complexes and protein localisation, potentially offering widely applicable techniques for determining markers of receptor engagement and activation of downstream signalling pathways. In addition to target engagement, these methods may be applicable to target validation via characterisation of the multi-protein complexes that comprise the target within its native cellular environment. Techniques based on the localisation of single fluorescent molecules, such as STORM (stochastic optical reconstruction microscopy), offer the highest resolutions which approach the scale of individual proteins and protein complexes. At MDC we have built super-resolution imaging capabilities in STORM and the lower resolution but simpler technique of Airyscan imaging. Using the well-characterised epidermal growth factor receptor as a test system, we have imaged and quantified properties of ligand-evoked clustering and also investigated the effects on receptor clustering of a known inhibitor of receptor internalisation PD158780. Alexa-647 conjugated epidermal growth factor stimulated rapid clustering of EGFR across a range of scales from 20-200nm. Interestingly, the internalisation inhibitor PD158780 also affected clustering dynamics, reducing the density of EGFR within clusters. These initial studies pave the way to perform target engagement and target validation studies in more complex ways and in cell systems such as human iPSC or primary tissues.