Open AccessA versatile nanobody-based toolkit to analyze retrograde transport from the cell surface
Author(s) -
Dominik P. Buser,
Kai D. Schleicher,
Cristina PrescianottoBaschong,
Martin Spiess
Publication year - 2018
Publication title -
proceedings of the national academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.1801865115
Subject(s) - endosome , golgi apparatus , microbiology and biotechnology , live cell imaging , green fluorescent protein , transport protein , homing (biology) , chemistry , protein subcellular localization prediction , compartment (ship) , biophysics , biology , biochemistry , cell , intracellular , oceanography , ecology , gene , geology
Significance Retrograde transport from the cell surface to intracellular compartments is essential for homeostasis and cell physiology. We developed derivatized nanobodies to follow proteins from the cell surface to endosomes and thetrans -Golgi network. Nanobodies are an emerging class of protein binders with many advantages over conventional antibodies: they are small and noncross-linking, and can be produced in bacteria. Using a nanobody against GFP, our tool is widely applicable to any GFP-tagged protein of interest. It allows the quantitative analysis of endocytic and retrograde transport biochemically, by fixed- and live-cell imaging and by electron microscopy. As proof-of-principle, we applied them to determine the contribution of adaptor protein-1/clathrin in retrograde transport of the mannose-6-phosphate receptors to thetrans -Golgi.
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