Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Nat Protoc. 2010 Jun;5(6):1127-37. doi: 10.1038/nprot.2010.84. Epub 2010 May 27.
Early endosomes receive material from the plasma membrane by fusion with endocytotic vesicles. This material is sorted within endosomes and directed to subdomains at which carrier vesicles bud. These vesicles are then transported toward different cellular destinations. In this article, we describe a protocol for the cell-free reconstitution of endosome docking/fusion and sorting/budding, which is based on labeling of endosomes by endocytotic uptake with fluorescent cargoes. The protocol includes (i) the preparation of fluorescently labeled endosomes, (ii) assays for docking/fusion and for sorting/budding in vitro and (iii) imaging of the reaction mix by fluorescence microscopy to quantify docking, fusion, cargo sorting and budding using counting of single organelles. Production of endosome stocks requires approximately 1 d. The in vitro reactions can then be performed separately (approximately 1 d) and are conveniently carried out with multiple samples in parallel. The assay can be adapted for studying the dynamics of organelles other than endosomes.
早期内涵体通过与内吞小泡融合接收来自质膜的物质。这些物质在内体中进行分拣，并被定向到载体小泡出芽的亚区。然后这些囊泡被运输到不同的细胞目的地。在本文中，我们描述了一种无细胞体系中内涵体停泊/融合和分拣/出芽的重建方法，该方法基于通过荧光货物的内吞摄取对内涵体进行标记。该方案包括：（i）荧光标记内涵体的制备，（ii）体外停泊/融合和分拣/出芽测定，以及（iii）通过荧光显微镜对反应混合物进行成像，以使用单个细胞器的计数来定量停泊、融合、货物分拣和出芽。内涵体储存库的制备大约需要 1 天时间。然后可以分别进行体外反应（大约 1 天），并且可以方便地在平行的多个样品中进行。该测定可以适应于研究除内涵体以外的细胞器的动力学。
