Receptor-mediated targeting of fluorescent probes in living cells
Receptor-mediated targeting of fluorescent probes in living cells
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DOI：
10.1074/jbc.274.12.7603
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发表时间：
1999-03-19
影响因子：
4.8
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Farinas, J;Verkman, AS
开发了一种策略，用广泛选择的荧光或其他探针标记活细胞中的特定位置，并应用于研究高尔基体的pH调节。将单链抗体靶向特定的部位，如细胞器管腔。靶向抗体作为高亲和力受体捕获细胞透过性的半抗原-荧光团结合物。合成的半抗原(4-乙氧亚甲基-2-苯基-2-恶唑啉-5-酮，Phox)与荧光探针(BODIPY F1、四甲基罗丹明、荧光素)高亲和力(约5 nM)结合并特异性定位于中国仓鼠卵巢细胞内质网、高尔基体和质膜中表达的单链抗体。利用pH敏感的荧光素-荧光素结合物和比率成像显微镜，测量了高尔基体管腔内的pH(pH 6.25+/-0.06)。对高尔基体液泡H+/ATPase泵活性和H+泄漏的测量提供了直接证据，证明静止的高尔基体pH是由平衡的泄漏泵动力学决定的，而不是H+/ATPase无法逆电化学梯度泵出。与绿色荧光蛋白的表达一样，受体介导的荧光团靶向方法允许特定的细胞内荧光标记。新方法的一个显著优势是能够针对具有定制设计的光谱和指示剂属性的化学探针。
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 Golgis. 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 (similar to 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.