373324 Electroporation-Delivered Protein Biosensors for Study of Molecular Activity
Fluorescence resonance energy transfer (FRET) based biosensors have been widely used for visualization of molecular activities in living cells in real time with high spatiotemporal resolution. FRET occurs when two fluorophores in close proximity undergo non-radiative transfer of energy from an excited donor fluorophore (more blue shifted) to an acceptor fluorophore (more red shifted). FRET-based biosensors exploit this physical phenomenon and translate a specific biochemical event (e.g. protein phosphorylation, etc.) into a conformational change in the biosensor (i.e. alteration in the distance and/or orientation between the two fluorophores) and subsequently a change in the optical signal. However, the use of protein biosensors has been largely limited to genetically modified cell lines created by delivering and expressing a plasmid form of the biosensor. Such genetic encoding requires a sizable cell population for successful transfection and high cell viability and functionality after the procedure for strong gene expression. Such limitation renders the application of protein biosensor technology to rare primary cells impractical. Primary cells from animals and humans are in general harder to transfect than cell lines because they divide slowly or do not divide. This hurdle needs to be removed in order to extend the use of protein biosensors to clinical diagnosis and prognosis.
In this study, we deliver biosensor in its protein form into cells via electroporation. Electroporation allows the delivered biosensor to be directly exposed to and interact with intracellular molecules, thus to reflect cellular events by checking the corresponding FRET effect. This method does not require the transfection and expression of protein in target cells, so that is applicable to almost all cell types, including rare primary cells. In our work, Src reporter in its protein form was first produced, extracted and purified from E.Coli, and then it was loaded into mammalian cells by electroporation. Src is a protein tyrosine kinase which plays critical roles in a variety of cellular activities, including cell adhesion, migration and cancer invasion and metastasis. We used a sensitive Src FRET biosensor that contained a Src SH2 domain, a flexible linker, and a Src substrate peptide, concatenated between enhanced cyan fluorescent protein (ECFP) and a variant of yellow fluorescent protein (YPet). The design of the Src reporter allows the juxtaposition of ECFP and YPet to yield a high FRET emitting yellow fluorescence. Upon pervanadate (PVD) stimulation, which is a tyrosine phosphatase inhibitor, the Src reporter is phosphorylated by Src in vitro, which separates YPet from ECFP and induces the decrease of FRET, hence cyan fluorescence increased at the expense of yellow fluorescence emission. In this way, the activity of Src within cells is able to be analyzed by monitoring the fluorescence. We demonstrated that electroporation-delivered Src biosensor reports Src activity in the cytoplasm with very similar sensitivity and spatial resolution to that obtained with genetic encoding. We envision that our biosensor approach will be useful for examining primary cell samples with clinical relevance.