Sina V Barysch1,2, Reinhard Jahn1 & Silvio O Rizzoli2
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 (~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.
Early endosomes represent the first sorting station of newly internalized material in all eukaryotic cells. On endocytosis, material is taken up into small carrier vesicles that are targeted to early endosomes, to which they dock and fuse. Endocytosed material is then sorted within the endosome. Depending on the final destination, such cargoes are either packed into budding vesicles, or they remain within early endosomes until they mature into late endosomes and then fuse with lysosomes for degradation of their content. Carrier vesicles budding from early endosomes are delivered to different intracellular destinations such as the plasma membrane, the recycling endosomes or the trans-Golgi network. Thus, early endosomes undergo continuous remodeling due to a steady influx of material by fusion with incoming endocytotic vesicles, which is balanced by an efflux of outgoing vesicles. Moreover, early endosomes frequently fuse with each other (termed homotypic fusion).
Of all these processes, fusion is the only one to be extensively studied. The reason is that fusion can be followed in vitro by biochemical assays relying on contents mixing. In these assays, two sets of endosomes are labeled separately by endocytosis with two markers that possess an intrinsic affinity toward each other (such as antigen and antibody, or avidin and biotin). After cell lysis, endosomes are allowed to fuse in vitro in the presence of an externally added quencher that prevents complex formation between markers escaping from the organelles. The amount of fusion is determined by measuring the degree of binding between markers (reviewed in ref. 1). Such assays have defined our current knowledge of fusion dynamics, and most of the proteins involved in homotypic fusion were identified in such in vitro fusion experiments. However, these assays cannot address the vital step that precedes fusion—docking. Furthermore, measuring sorting and budding in vitro has been much more difficult, with most assays relying on the separation of small carrier vesicles from larger donor organelles. For example, permeabilized cells were used to measure endosomal budding of recycling vesicles. In contrast to the endosomal donor organelle, these vesicles can escape from cells though pores in the plasma membrane2, 3. Moreover, separation through density gradients has been used to study the budding of several types of vesicles from endosome precursors4, 5, 6, 7, 8. However, these assays only function when there is a substantial difference in buoyancy between the precursor and budded organelles—which explains the paucity of budding assays in the literature.
We have recently developed a new technique for measuring docking and fusion, which is based on the separate labeling of two sets of endosomes with two fluorescent cargo molecules. In vitro fusion of organelles results in mixing of the dyes, producing double-labeled organelles that can be conveniently quantified by fluorescence microscopy9. Furthermore, we found that measuring the distance between the centers of intensity of the differentially labeled organelles (see Experimental design) allows for monitoring fusion and docking in parallel10, 11. Sorting and budding can be investigated by a variation of the technique: organelles are simultaneously labeled with multiple, differently colored markers, and the segregation of markers is followed by monitoring the production of single-color carrier vesicles12 (see Experimental design).
Our assay has several fundamental advantages over the assays discussed above. First, to the best of our knowledge, our in vitro assay is the only one to date that is able to measure the docking of small organelles quantitatively. Second, it can analyze (and differentiate) docking and fusion in parallel. Third, our budding and fusion assays have no inherent buoyancy requirements, and therefore can be used for investigating virtually any type of organelles (we have already used them for endosomes from various cell types and even synaptic vesicles13). Finally, compared with the biochemical in vitro fusion and budding assays, our technique has the advantage of analyzing cargo-containing endosomes on a single-organelle basis. This has allowed, for example, the investigation of protein composition on single sorting organelles, by immunostaining12. From a technical point of view, the assay can be conveniently upscaled, with up to 20 reactions being run in parallel, in a much easier manner than upscaling, for example, density-gradient assays.
The reliance of the assay on identification of different fluorescently labeled cargoes is an advantage, as indicated above; however, it also introduces several constraints. The fusion of two differently colored organelles is accurately reported, but the fusion of two identically colored organelles is not. In addition, only one double-labeled spot appears when several organelles of different colors fuse together, with the assay thus ignoring multiple fusion events. For the sorting reaction, formation of small vesicles containing both of the fluorescently labeled cargoes is not detected, as they are still double labeled.
The workflow of the in vitro early endosome docking/fusion or sorting/budding assay is depicted in Figure 1. For these assays, cytosol (here we describe the preparation of cytosol from adult rat brains, Steps 1–8) and fluorescently labeled endosomes are needed (we label early endosomes by incubating cultured cells with externally added fluorescent cargo molecules by endocytotic uptake, Steps 9–24). After labeling, cells are cracked with a ball homogenizer and a postnuclear supernatant (PNS), which contains the labeled endosomes, is prepared (Steps 25–30). For docking/fusion assays, two separate sets of differently labeled endosomes are needed. For sorting/budding assays, endosomes are simultaneously labeled with at least two endocytic markers that are sorted into different vesicular pathways. As our laboratory focuses on neuronal membrane trafficking, we use for our assays the neuroendocrine cell line PC12; however, the assay can be adapted to other cell lines.
For docking/fusion (left), two sets of cells are allowed to take up different fluorescent cargoes (dextran-Alexa 488 and dextran-Alexa 594) by endocytosis for 5 min, leading to the fluorescent labeling of early endosomes with two colors. Cells are cracked and postnuclear supernatants (PNSs) are prepared, which contain fluorescently labeled endosomes. In vitro incubation of the two PNS fractions in the presence of cytosol and an ATP-regenerating system leads to docking and fusion of organelles, which can be visualized by immobilizing them on glass coverslips and subsequent imaging (in which double-labeled organelles appear). Data analysis shows the precise position of each endosome and measures distances between them, thus allowing the quantification of docked and fused endosomes. For sorting/budding (right), cells are simultaneously labeled with two fluorescent cargoes and PNS is prepared. In vitro incubation of the PNS fraction in the presence of cytosol and an ATP-regenerating system leads to cargo sorting and separation because of budding, resulting in a decrease in double-labeled organelles. As for the docking/fusion assay, this can be visualized by fluorescence microscopy and quantified by image analysis.