Voltage Imaging
Optical imaging of membrane voltage offers several advantages over traditional patch clamp recording techniques. We created a palette of fluorescent voltage-indicating proteins that span much of the visible spectrum. These sensors are based on a novel voltage-sensing mechanism called electrochromic FRET (eFRET). In eFRET, a fluorescent protein is the FRET donor and the retinal chromophore in a rhodopsin protein is the FRET acceptor. Membrane voltage shifts the absorption spectrum of the retinal chromophore, thereby modulating the FRET efficiency. By measuring the changes in fluorescent protein emission, we could detect membrane action potential spikes in neurons via fluorescence microscopy.
Proteomic Mapping
In our previous work, we have invented a spatially-restricted protein labeling method for mapping subcellular proteome. Traditional proteomic approach requires serial physical isolations of cellular components, which are often tedious and sometimes impossible to achieve. Our new method utilizes an engineered peroxidase, APEX, to covalently tag the relevant proteome in living cells with a small molecule handle. Thereafter, we identify the labeled proteome via mass spec analysis.
APEX catalyzes the one-electron oxidation of phenol substrates into highly reactive and short-lived phenoxyl free radicals. These free radicals readily react with biomolecules such as proteins to form covalent adduct labels. Due to their short life-time in the aqueous solution, phenoxyl radicals have a diffusion radius on the order of tens of nanometers, which restricts the labeling reaction to the vicinity of the APEX enzyme. We applied this highly spatial-specific enzyme-mediated protein labeling strategy to map the proteome of mitochondrial sub-compartments in living cells. We are currently extending this approach to label ribosomes at the neuronal synapse. Combined with the high sensitivity of RNA sequencing, this would enable us to investigate local protein synthesis via ribosome profiling.
