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A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface
Journal
Proceedings of the National Academy of Sciences of the United States of America
Volume
115
Number
27
Pages / Article-Number
E6227-E6236
Mesh terms
Adaptor Protein Complex 1, metabolism; Animals; Biological Transport, Active, physiology; Camelus; Cell Membrane, metabolism; DNA-(Apurinic or Apyrimidinic Site) Lyase, metabolism; Endosomes, metabolism, ultrastructure; Green Fluorescent Proteins, metabolism; HeLa Cells; Humans; Microscopy, Electron; Microscopy, Fluorescence; Receptor, IGF Type 2, metabolism; Single-Domain Antibodies, metabolism; trans-Golgi Network, metabolism, ultrastructure
Abstract
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.
