Marlon J. Hinner, Gerd Hübener and Peter Fromherz
Optical recording of the electrical activity of individual neurons in culture or in a tissue requires cell-selective staining with a fluorescent voltage-sensitive dye. In a proof-of-principle experiment, we implement a novel approach of genetically targeted staining. The method relies on a water-soluble precursor dye and an overexpressed cell-surface enzyme, which transforms the precursor to a hydrophobic dye which binds to the targeted cell. We fused an alkaline phosphatase to a specifically designed general purpose membrane anchor. The fusion protein was expressed on the surface of HEK293 cells as proven by immuno- and histochemical staining. We synthesized an amphiphilic hemicyanine dye containing two enzymatically cleavable phosphate groups at its hydrocarbon tails. When the phosphate groups were removed, the binding to membranes was enhanced by a factor of thousand as shown by titration with lipid vesicles. We observed selective staining of enzymatically active cells by fluorescence microscopy in a mixed population of phosphatase transfected and untransfected HEK293 cells. The critical parameters of enzyme-induced cell-selective staining are elucidated by a simple kinetic model to guide further developments of the method.
Cell-selective staining by genetic targeting. Row (A). Identification of targeted cells. Stable clones of HEK293 cells expressing ETOLLIS-AP were cultured with cells that were stably transfected with EGFP. The first picture is a b rightfield image of all cells. The fluorescence image (excitation around 450 nm, emission at >510 nm) of the second picture shows the cells without phosphatase. The third picture obtained by virtual subtraction shows the cells expressing ETOLLIS-AP. Row (B). Enzyme induced staining with 400 nM of precursor dye Di-12P-ASPBS (4). Fluorescence images (excitation around 535 nm, emission around 610 nm) acquired before (-1 sec) and after addition of the precursor. The cells that express ETOLLIS-AP are stained with the hemicyanine dye Di-12A-ASPBS (3).
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