Receptor-mediated targeting of fluorescent probes in living cells

A strategy was developed to label specified sites in living cells with a wi de selection of fluorescent or other probes and applied to study pH regulat ion in Gels. cDNA transfection was used to target a single-chain antibody t o a specified site such as an organelle lumen. The targeted antibody functi oned 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, tetramethylrhoda mine, fluorescein) were bound with high affinity (similar to 5 nM) and spec ific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cell s. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging micr oscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06), Measureme nts of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi p rovided direct evidence that resting Golgi pH is determined by balanced lea k-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent prote in, the receptor-mediated fluorophore targeting approach permits specific i ntracellular fluorescence labeling. A significant advantage of the new appr oach is the ability to target chemical probes with custom-designed spectral and indicator properties.