Biozentrum, University of Basel, Basel, Switzerland.
Elife. 2021 Nov 30;10:e70982. doi: 10.7554/eLife.70982.
Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.
细胞间通讯是生命过程中的一个基本过程，内体作为调节信号分子摄取和分泌的关键细胞器发挥作用。被内吞的物质被分拣内体接受，在那里它要么被分拣进行回收，要么随着内体成熟在溶酶体中降解。由于大多数细胞内系统中内体的体积小且运动迅速，对内体成熟过程的研究受到了阻碍。在这里，我们报告了一种简单通用的活细胞成像测定法来监测内体成熟动力学，该方法可应用于多种哺乳动物细胞类型。急性离子载体处理导致早期内体隔室扩大，成熟为晚期内体，并与溶酶体融合形成内溶酶体。该测定法重现了 Rab5 到 Rab7 的转换以及 PI(3)P 的形成和周转，并且可以使用标准宽场显微镜观察到。我们使用这种方法表明，Snx1 和 Rab11 阳性的再循环内体的募集发生在整个内体成熟过程中，与 Rab 转换无关。相比之下，有效的内体酸化依赖于 Rab 转换。该测定法为进一步揭示内体成熟的各个方面提供了一个有力的工具。
