Genetic Targeting of Individual Cells with a Voltage‐Sensitive Dye through Enzymatic Activation of Membrane Binding

Abstract

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.

Optical Recording of Fast Neuronal Membrane Potential Transients in Acute Mammalian Brain Slices by Second-Harmonic Generation Microscopy

“…Because the SHG emission is always at one-half the illumination wavelength, it can also be combined easily with functional fluorescent indicators such as Ca 2ϩ -and Na ϩ -sensitive dyes. With further improvements in the S/N, labeling methods (Hinner et al 2004), and a combination with faster and more flexible imaging modalities (Bullen and Saggau 1999;Iyer et al 2004;Kobayashi et al 2002;Otsu et al 2005;Roorda et al 2004;Sacconi et al 2003;Tsien and Bacskai 1995), SHG should move the imaging field closer to large scale high-resolution V m recordings of population activity deep in thick tissue preparations.…”

A Chemogenetic Approach for the Optical Monitoring of Voltage in Neurons

“…To circumvent these problems, hybrid voltage indicators have been proposed that combine the superior optical properties of small‐molecule fluorophores with genetically encoded voltage sensors . Examples include a precursor VSD that is converted to an active membrane‐bound dye by a genetically encoded enzyme, and click chemistry‐ and enzyme‐mediated ligation of organic fluorophores to rhodopsin to function as FRET donors …”

Genetically Encoded Voltage Indicators in Circulation Research

“…In addition to the fluorescent protein based voltage sensors (see below), it is worthwhile to mention the combination of genetic targeting and conventional organic chromophores [ 9 ]. In a proof of principle report, it was described that a membrane targeted phosphatase was able to cleave the hydrophilic phosphate group of a precursor dye, leading to a membrane bound voltage sensitive dye [ 10 ]. A further hybrid approach utilized the expression of a membrane bound GFP as Förster Resonance Energy Transfer (FRET) donor in combination with dipicrylamine (DPA), a synthetic voltage sensing molecule, as FRET acceptor [ 11 ].…”