No Need to FRET

Abstract

Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study, due to their requirement of a lipid membrane for function. Our mathematical model shows that polarization fluorescence microscopy using fluorescent proteins can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. We have now experimentally demonstrated that polarization microscopy can be used for imaging of G-protein activation, changes in intracellular calcium concentration, and other cellular processes, in living cells, with sensitivity comparable to, or even exceeding that of current FRET probes. Crucially, in contrast to FRET, polarization fluorescence microscopy only requires presence of a single fluorescent protein. Therefore, as both our theoretical and experimental work shows, many existing constructs can be used as optical probes of molecular processes involving membrane proteins. Apart from utilizing existing constructs, polarization microscopy offers a clear path towards development of new genetically encoded optical probes of membrane protein function, including a usable genetically encoded optical sensor of cell membrane voltage. Our results indicate that in many biological applications, FRET is likely to be complemented or even replaced by polarization microscopy.