Single-molecule microscopy is a powerful tool for investigating functional events at the plasma membrane. With the state-of-the-art microscopy method, changes in protein mobility can be correlated to cellular events or structures. Such experiments offer quantitative data that is impossible to yield from ensemble methods as they render the individual functional event in a protein’s life tractable to investigation and analysis. We can visualize changes in molecular behaviour with respect to triggered or observed cellular events and thus understand the role of the observed molecule in these processes. However, setting up the conditions required for investigating stoichiometry is challenging. Single-molecule tracking requires a sparse population of the protein of interest for single-molecule visualization. This requirement is met by partially labelling the protein of interest, which occludes information like the stoichiometry of the protein and how oligomeric protein influence function. We propose control of the number of proteins at the plasma membrane by controlling its transport to achieve a low population of fully labelled proteins of interest at the site of its action. We developed an optogenetic tool for the light-controlled delivery of functional soluble and transmembrane proteins to the plasma membrane. We show that small amounts of proteins can be released optogenetically and efficiently transported to the plasma membrane using an optically cleavable fluorescent protein. Our method allows for the controlled delivery of proteins, including functional ion channels, to the plasma membrane in amounts compatible with single-molecule imaging. Single-molecule microscopy and the developed optogenetic tool were applied to study the unconventional secretion of Fibroblast Growth Factor (FGF2). FGF2 is an essential growth factor involved in cell growth, differentiation, and development. FGF2 skips the conventional ER-Golgi route for secretion and instead adopts an unconventional secretion pathway. Through biochemical reconstitution experiments, it is known to directly interact with the plasma membrane, where it undergoes phosphorylation and oligomerization. It is hypothesized that FGF2 oligomers form a pore in the plasma membrane where FGF2’s interaction with the extracellular heparan sulphate proteoglycans facilitates its extraction from the plasma membrane. To elucidate steps in FGF2 secretion in the context of live cells and to test the current model, I aimed to visualize the FGF2 secretion using live cell single-molecule microscopy. I employed the developed optogenetic tool to synchronize FGF2 release in the cytosol to study the initial phase of FGF2 secretion. Furthermore, by observing the mobility and intensity of FGF2 molecules via single molecule tracking, I dissected the sequence of events in the FGF2 secretion process.
