Abstract
A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. Synthesized conjugates of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with high affinity (approximately 5 nM) and specific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cells. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi provided direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties.
Highlights
Small-molecule fluorescent probes have been widely used to study protein localization, cytoplasmic ionic content [1], and solute diffusion [2]
Cells could be loaded at 4 °C, indicating that the conjugate entered the cells primarily by transmembrane diffusion and not by endocytosis. These results demonstrate the selective targeting of fluorescent probes to expressed sFv in living cells
The fluorophore targeting method reported here provides a new strategy for cellular labeling that complements existing methods
Summary
Small-molecule fluorescent probes have been widely used to study protein localization, cytoplasmic ionic content [1], and solute diffusion [2]. A cell labeling method is reported here that combines the site specificity conferred by genetically encoded targeting sequences with the excellent spectral and indicator properties of small chemical fluorophores. Fluorophore-hapten conjugates are added to the extracellular solution at low concentrations, diffuse to sites of sFv expression, and bind to the sFv. Conjugates of different indicator and spectral properties were synthesized (Fig. 1b), including phOx-Bodipy FL (green fluorescent), phOx-fluorescein (green fluorescent, pH-sensitive), and phOx-tetramethylrhodamine (red fluorescent).
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