Receptor-mediated Targeting of Fluorescent Probes in Living Cells

Javier Farinas; A. S. Verkman

doi:10.1074/jbc.274.12.7603

Receptor-mediated Targeting of Fluorescent Probes in Living Cells*

Javier Farinas‡ and
A. S. Verkman

From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521

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 (∼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.

Footnotes

↵* This work was supported by National Institutes of Health Grants DK43840 and DK35124 and National Cystic Fibrosis Foundation Grant R613. We also acknowledge the University of California, San Francisco mass spectrometry facility, which is supported by National Institutes of Health Grant RR01614.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Cardiovascular Research Inst., 1246 Health Sciences East Tower, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: javier{at}itsa.ucsf.edu;http://www.ucsf.edu/verklab.