Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome.
Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome.
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DOI：
10.1083/jcb.109.6.3303
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发表时间：
1989-12
期刊：
影响因子：
--
通讯作者：
Maxfield FR
中科院分区：
文献类型：
--
作者：
Dunn KW;McGraw TE;Maxfield FR
为了研究内吞区室的融合和分离，我们使用数字图像分析来量化在持续内吞1-20分钟期间内体中荧光配体的积累。荧光标记的转铁蛋白（Tf）和低密度脂蛋白（LDL）分别作为循环受体和溶酶体定向配体的标记。通过测量单个核内体的强度，我们发现每个核内体的LDL含量在1到10分钟之间增加30-40倍，然后达到稳定。相反，每个核内体的Tf量在2分钟内达到稳定状态，其水平仅为1分钟的3至4倍。我们使用脉冲追逐双标记方法来证明Tf在LDL积聚的隔室中循环。当Tf和LDL同时加入细胞2分钟时，几乎所有的内体都含有这两种标记。在没有外部配体的情况下，再孵育2-4分钟，含ldl的室室就会耗尽Tf，因为Tf被定向到旁高尔基再循环内体。然而，如果在LDL脉冲后2-4分钟将Tf添加到培养基中，大多数含LDL的内体都被Tf标记。数据表明，至少有30-40个含有Tf和LDL的内吞囊泡在5-10分钟内与内体腔室融合。LDL在这个隔室内积累，Tf同时被去除。简单的数学模型表明，这种类型的迭代分拣可以导致非常高效的排序。
To study the fusion and separation of endocytic compartments, we have used digital image analysis to quantify the accumulation of fluorescent ligands in endosomes during continuous endocytosis for periods of 1-20 min. Fluorescently labeled transferrin (Tf) and low density lipoproteins (LDL) were used as markers of recycling receptors and lysosomally directed ligands respectively. By measuring the intensity of individual endosomes, we found that the amount of LDL per endosome increases 30-40-fold between 1 and 10 min and then plateaus. In contrast, the amount of Tf per endosome reaches a steady state within 2 min at a level that is only three to four times that at 1 min. We used pulse-chase double label methods to demonstrate that Tf cycles through the compartment in which the LDL accumulates. When both Tf and LDL are added to cells simultaneously for 2 min, nearly all endosomes contain both labels. With 2-4 min further incubation in the absence of external ligands, LDL-containing compartments become depleted of Tf as Tf is directed to para-Golgi recycling endosomes. However, if Tf is added to the medium 2-4 min after a pulse with LDL, most of the LDL-containing endosomes become labeled with Tf. The data indicate that at least 30-40 endocytic vesicles containing both Tf and LDL fuse with an endosomal compartment over a period of 5-10 min. LDL accumulates within this compartment and Tf is simultaneously removed. Simple mathematical models suggest that this type of iterative fractionation can lead to very high efficiency sorting.