Burgert H G, Thilo L
Exp Cell Res. 1983 Mar;144(1):127-42. doi: 10.1016/0014-4827(83)90447-0.
An analysis was made of the pinocytosis-derived internalization and recycling of membrane in the macrophage cell line, P388D1. Plasma membrane glycoconjugates, reversibly labelled with [3H]galactose, were used as a membrane marker. Label internalized with the plasma membrane was no longer accessible to release by externally added beta-galactosidase and could therefore be distinguished quantitatively from label remaining on the cell surface. Direct experimental evidence for membrane recycling was obtained by demonstrating that previously internalized label reappeared at the cell surface. The composition of labelled membrane glycoconjugates, as analysed by SDS-polyacrylamide gel electrophoresis, remained unaltered before and after internalization. The label remained membrane-bound in an unmodified way during the entire period of 8 h investigated, corresponding to about twenty-four cycles of membrane flow. Membrane flow led to a steady-state distribution of label between the plasma membrane and intracellular membranes. The redistribution of label occurred with biphasic kinetics, which could be described as the sum of two exponential functions. This behavior is explained by presenting a model of membrane flow between the plasma membrane and two consecutive intracellular membrane compartments, which we assume to consist of pinosomal membranes and of pinosome-derived membrane of secondary lysosomes. The relative membrane surface areas turn out to be in the ratio of 100:12.5:7.3, respectively. At the observed rate of pinocytosis, the equivalent of the plasma membrane is internalized once every 21 min, in the form of primary pinosomes of the size 0.24 micrometer. The residence time of membranes in the pinosome compartment is about 3 min. The rate at which membranes enter the lysosomal compartment is 31 times lower than the rate of membrane internalization. We conclude that only 3% of the amount of membrane internalized at any one time subsequently enters the secondary lysosome compartment. After a residence time of 49 min this membrane fraction is finally recycled to the cell surface. The results are discussed in terms of mixing and sorting-out of pinosomal and lysosomal membranes.
对巨噬细胞系P388D1中膜的胞饮作用衍生的内化和再循环进行了分析。用[3H]半乳糖可逆标记的质膜糖缀合物用作膜标记物。质膜内化的标记物不再能被外部添加的β-半乳糖苷酶释放，因此可以与细胞表面残留的标记物进行定量区分。通过证明先前内化的标记物重新出现在细胞表面，获得了膜再循环的直接实验证据。通过SDS-聚丙烯酰胺凝胶电泳分析，标记的膜糖缀合物的组成在内化前后保持不变。在所研究的8小时整个期间，标记物以未修饰的方式保持与膜结合，这相当于约二十四个膜流动循环。膜流动导致标记物在质膜和细胞内膜之间达到稳态分布。标记物的重新分布具有双相动力学，可以描述为两个指数函数的总和。通过提出质膜与两个连续的细胞内膜隔室之间的膜流动模型来解释这种行为，我们假设这两个隔室分别由吞噬体膜和次级溶酶体的吞噬体衍生膜组成。相对膜表面积分别为100:12.5:7.3。以观察到的胞饮作用速率，相当于质膜的部分每21分钟以内化一次，以内径为0.24微米的初级吞噬体形式存在。膜在吞噬体隔室中的停留时间约为3分钟。膜进入溶酶体隔室的速率比膜内化速率低31倍。我们得出结论，任何时候内化的膜量中只有3%随后进入次级溶酶体隔室。在停留49分钟后，这部分膜最终再循环到细胞表面。根据吞噬体膜和溶酶体膜的混合和分选对结果进行了讨论。
