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

Nature Protocols volume 5, pages 1127–1137 (2010)Cite this article

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Abstract

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.

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Figure 1: Experimental design of the in vitro endosome docking/fusion and sorting/budding assay.
Figure 2: Imaging of multilabeled fluorescent beads.
Figure 3: Data obtained from docking/fusion and sorting/budding assays.

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Acknowledgements

We thank D. Brandhorst, D. Zwilling, U. Geumann and I. Bethani for the contribution to the development of the method presented in this study. We thank M. Lengauer and F. Kötting for the help with the ball homogenizer. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 523 and SFB 803) to R.J. S.V.B. has been the recipient of a fellowship of the International Max Planck Research School 'Molecular Biology'.

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Authors and Affiliations

  1. Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Sina V Barysch & Reinhard Jahn

  2. STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, Germany

    Sina V Barysch & Silvio O Rizzoli

Authors
  1. Sina V Barysch
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  2. Reinhard Jahn
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  3. Silvio O Rizzoli
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Contributions

S.V.B., R.J. and S.O.R. conceived the experimental strategy; S.V.B. and S.O.R. designed the experiments; S.V.B. and S.O.R. conducted the experiments; S.V.B., R.J. and S.O.R. wrote the article.

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Correspondence to Silvio O Rizzoli.

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The authors declare no competing financial interests.

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Barysch, S., Jahn, R. & Rizzoli, S. A fluorescence-based in vitro assay for investigating early endosome dynamics. Nat Protoc 5, 1127–1137 (2010). https://doi.org/10.1038/nprot.2010.84

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