Salzman N H, Maxfield F R
Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
J Cell Biol. 1988 Apr;106(4):1083-91. doi: 10.1083/jcb.106.4.1083.
A polyclonal anti-fluorescein antibody (AFA) which quenches fluorescein fluorescence has been used to distinguish between two models of intracellular vesicle traffic. These models address the question of whether sequentially endocytosed probes will mix intracellularly or whether they are carried through the cell in a sequential, isolated manner. Using transferrin (Tf) as a recycling receptor marker, we incubated Chinese hamster ovary (CHO) cells with fluorescein-Tf (F-Tf) which is rapidly endocytosed. After the F-Tf was completely cleared from the surface, AFA was added to the incubation medium and entered endocytic compartments by fluid phase endocytosis. Fusion of a vesicle containing AFA with the compartment containing F-Tf results in binding of AFA to fluorescein and the quenching of fluorescein fluorescence. When AFA was added to the culture medium 2 min after clearance of F-Tf from the surface, time dependent fluorescence quenching occurred. After 20 min, 67% saturation of F-Tf with AFA was observed. When the interval between F-Tf clearance and AFA addition was increased to 5 min only 41% saturation of F-Tf was found. These data indicate that there are some compartments which are accessible for mixing with subsequently endocytosed molecules, but the efficiency of mixing falls off rapidly as the interval between pulses is increased. In CHO cells Tf swiftly segregates to a collection of vesicles or tubules in the para-Golgi region, and at steady state most of the F-Tf is in this compartment. Using digital image analysis to quantify quenching in this region, we have found that F-Tf/AFA mixing is occurring either within this compartment or before transferrin enters it.
一种能淬灭荧光素荧光的多克隆抗荧光素抗体（AFA）已被用于区分两种细胞内囊泡运输模型。这些模型解决了一个问题，即依次内吞的探针在细胞内是否会混合，还是它们以一种依次、隔离的方式在细胞中传递。以转铁蛋白（Tf）作为循环受体标记物，我们用能被快速内吞的荧光素 - 转铁蛋白（F - Tf）孵育中国仓鼠卵巢（CHO）细胞。当F - Tf从细胞表面完全清除后，将AFA添加到孵育培养基中，它通过液相内吞作用进入内吞区室。含有AFA的囊泡与含有F - Tf的区室融合会导致AFA与荧光素结合，从而淬灭荧光素荧光。当在F - Tf从表面清除2分钟后将AFA添加到培养基中时，出现了时间依赖性的荧光淬灭。20分钟后，观察到F - Tf被AFA饱和了67%。当F - Tf清除与添加AFA之间的间隔增加到5分钟时，仅发现F - Tf被饱和了41%。这些数据表明，存在一些可与随后内吞的分子混合的区室，但随着脉冲间隔增加，混合效率迅速下降。在CHO细胞中，Tf迅速分离到高尔基旁区域的一组囊泡或小管中，并且在稳态下，大多数F - Tf都在这个区室中。使用数字图像分析来量化该区域的淬灭情况，我们发现F - Tf/AFA混合要么发生在这个区室内，要么发生在转铁蛋白进入该区室之前。
