Enzyme-induced staining of biomembranes with voltage-sensitive fluorescent dyes.
We consider the physicochemical basis for enzyme-induced staining of cell membranes by fluorescent voltage-sensitive dyes, a method that may lead to selective labeling of genetically encoded nerve cells in brain for studies of neuronal signal processing. The approach relies on the induction of membrane binding by enzymatic conversion of a water-soluble precursor dye. We synthesized an amphiphilic hemicyanine dye with and without an additional phosphate appendix at its polar headgroup. The fluorescence of these dyes is negligible in water but high when bound to lipid membranes. By fluorescence titration with lipid vesicles it was shown that the phosphate group lowers the partition coefficient from water to membrane by more than an order of magnitude. By isothermal titration calorimetry, we showed that the dye phosphate was a substrate for a water-soluble alkaline phosphatase following MichaelisMenten kinetics. In a suspension of lipid vesicles, the enzyme reaction led to a fluorescence increase due to enhanced membrane binding of the product dye in accord with the MichaelisMenten kinetics of the reaction and the partition coefficients of substrate and product. We successfully tested the staining method by fluorescence microscopy with individual giant lipid vesicles and with individual red blood cells. In both systems, the membrane fluorescence due to bound hemicyanine was enhanced by an order of magnitude, proving the feasibility of enzyme-induced staining with voltage-sensitive dyes.
Hinner
Marlon J
MJ
Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry, Martinsried/Mnchen, Germany 82152.
Hbener
Gerd
G
Fromherz
Peter
P