A fluorescence-based in vitro assay for investigating early endosome dynamics.

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.