( +info)Д®ndocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling.

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. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). 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. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen.

(1/933)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. Receptor-mediated targeting of fluorescent probes in living cells.

Qspace nrg 2011