Emans N, Biwersi J, Verkman A S
Department of Medicine, University of California, San Francisco 94143-0521, USA.
Biophys J. 1995 Aug;69(2):716-28. doi: 10.1016/S0006-3495(95)79947-7.
A fluorescence assay of in vivo endosome fusion was developed and applied to define the kinetics of endosome fusion in baby hamster kidney (BHK) fibroblasts. The assay is based on an approximately 10-fold enhancement of the green fluorescence of BODIPY-avidin upon biotin binding. The BODIPY-avidin fluorescence enhancement occurred in < 25 ms, was pH-independent, and involved a BODIPY-tryptophan interaction. For endocytosis in vivo, BHK fibroblasts were pulse-labeled with BODIPY-avidin together with a red (rhodamine) fluorescent fusion-independent chromophore (TMR). After specified chase times in a nonfluorescent medium, a second cohort of endosomes was pulse-labeled with biotin-conjugated albumin, dextran, or transferrin. Fusion of biotin-containing endosomes with avidin-containing endosomes was quantified by ratio imaging of BODIPY-to-TMR fluorescence in individual endosomes, using imaging methods developed for endosome pH studies. Analysis of BODIPY-to-TMR ratio distributions in avidin-labeled endosomes exposed to zero and maximum biotin indicated > 90% sensitivity for detection of endosome fusion. In avidin pulse (10 min) -chase-biotin albumin pulse (10 min) studies, both fused and unfused endosomes were identified; the fractions of avidin-labeled endosomes that fused with biotin-labeled endosomes were 0.48, 0.21, 0.16, and 0.07 for 0-, 5-, 10-, and 20-min chase times. Fitting of fusion data to a mathematical model of in vivo endosome fusion required the existence of an intermediate fusion compartment. Pulse-chase studies performed with biotin-transferrin to label the early/recycling endosomes indicated that after a 10-min chase, avidin-labeled endosomes reached a compartment that was inaccessible to biotin-transferrin. The assay was also applied to determine whether endosome fusion was influenced by temperature, pH (bafilomycin A1), second messengers (cAMP agonists, phorbol 12-myristate 13-acetate, staurosporine), and growth-related factors (platelet-derived growth factor, genistein). The results establish a sensitive fluorescence assay to quantify the fusion of vesicular compartments in living cells.
我们开发了一种用于体内内体融合的荧光测定法，并将其应用于确定幼仓鼠肾（BHK）成纤维细胞中内体融合的动力学。该测定法基于生物素结合后BODIPY-抗生物素蛋白绿色荧光增强约10倍。BODIPY-抗生物素蛋白荧光增强在<25毫秒内发生，与pH无关，并且涉及BODIPY-色氨酸相互作用。对于体内的内吞作用，用BODIPY-抗生物素蛋白以及红色（罗丹明）荧光融合无关发色团（TMR）对BHK成纤维细胞进行脉冲标记。在非荧光培养基中经过特定的追踪时间后，用生物素偶联的白蛋白、葡聚糖或转铁蛋白对第二批内体进行脉冲标记。使用为内体pH研究开发的成像方法通过对单个内体中BODIPY与TMR荧光的比率成像来定量含生物素的内体与含抗生物素蛋白的内体的融合。对暴露于零生物素和最大生物素的抗生物素蛋白标记的内体中BODIPY与TMR比率分布的分析表明内体融合检测灵敏度>90%。在抗生物素蛋白脉冲（10分钟）-追踪-生物素白蛋白脉冲（10分钟）研究中，鉴定出融合和未融合的内体；对于0、5、10和20分钟的追踪时间，与生物素标记内体融合的抗生物素蛋白标记内体分数分别为0.48、0.21、0.16和0.07。将融合数据拟合到体内内体融合的数学模型需要存在中间融合区室。用生物素-转铁蛋白进行脉冲-追踪研究以标记早期/再循环内体表明，在10分钟的追踪后，抗生物素蛋白标记的内体到达了生物素-转铁蛋白无法进入的区室。该测定法还用于确定内体融合是否受温度、pH（巴弗洛霉素A1）、第二信使（cAMP激动剂、佛波酯12-肉豆蔻酸13-乙酸酯、星形孢菌素）和生长相关因子（血小板衍生生长因子、染料木黄酮）的影响。结果建立了一种灵敏的荧光测定法来定量活细胞中囊泡区室的融合。
