A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface

Buser, Dominik P. and Schleicher, Kai D. and Prescianotto-Baschong, Cristina and Spiess, Martin. (2018) A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface. Proceedings of the National Academy of Sciences of the United States of America, 115 (27). E6227-E6236.

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Official URL: https://edoc.unibas.ch/67431/

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Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of; trans; -Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters' homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the; trans; -Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.
Faculties and Departments:05 Faculty of Science
05 Faculty of Science > Departement Biozentrum
05 Faculty of Science > Departement Biozentrum > Services Biozentrum > Imaging Core Facility (Biehlmaier)
01 Faculty of Theology > Ehemalige Organisationseinheiten Theologie > Altes Testament und semitische Sprachwissenschaft (Mathys)
05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biochemistry (Spiess)
UniBasel Contributors:Schleicher, Kai and Spiess, Martin
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:National Academy of Sciences
Note:Publication type according to Uni Basel Research Database: Journal article
Identification Number:
Last Modified:27 Apr 2020 13:39
Deposited On:27 Apr 2020 10:38

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