Genetic targeting of individual cells with a voltage-sensitive dye through enzymatic activation of membrane binding.
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 to genetically targeted staining. The method relies on a water-soluble precursor dye and an overexpressed cell-surface enzyme that transforms the precursor into a hydrophobic dye that binds to the targeted cell. We fused an alkaline phosphatase to a specifically designed general-purpose membrane anchor, and the fusion protein was expressed on the surface of HEK293 cells, as was corroborated by immuno- and histochemical staining. We next synthesised 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 a 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 were elucidated by a simple kinetic model to guide further developments of the method.
Hinner
Marlon J
MJ
Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry, 82152 Martinsried/München, Germany.
Hübener
Gerd
G
Fromherz
Peter
P
D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D004396:Coloring Agents / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D018390:Gene Targeting; D006801:Humans; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism
D004396:Coloring Agents; D017931:DNA Primers; D010744:Phosphoric Monoester Hydrolases