Live-cell Imaging of Endocytic Transport using Functionalized Nanobodies in Cultured Cells

Endocytosis of receptors and other transmembrane proteins from the cell surface to endosomes and beyond is critical for homeostasis, physiology, and function. To investigate endocytic uptake and retrograde protein trafficking, we have established a versatile toolkit comprising functionalized nanobodies to monitor transport from the cell surface to the trans-Golgi network (TGN) by means of fixed and live-cell imaging, electron microscopy, and gel electrophoresis combined with autoradiography. We developed derivatized nanobodies targeting green fluorescent protein (GFP) or mCherry - monomeric, non-crosslinking, high-affinity protein binders - that can be added to cell lines expressing membrane proteins of interest bearing the corresponding extracellular fluorescence tags. Upon binding to GFP- or mCherry-tagged transmembrane reporters, the nanobodies are specifically internalized and trafficked in parallel with the reporters' endogenous sorting routes. These nanobodies were functionalized with selected fluorophores to track retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to resolve ultrastructural localization by electron microscopy, and with tyrosine sulfation motifs to quantitatively assess TGN arrival kinetics. In this methodological study, we detail the general protocol for bacterial expression and purification of functionalized nanobodies, as well as the generation of stable GFP-reporter cell lines. We exemplify the utility of this approach for live-cell imaging by employing an mCherry-modified anti-GFP nanobody (VHH-mCherry) to analyze the endocytic uptake of the transferrin receptor (TfR) and the cation-dependent mannose-6-phosphate receptor (CDMPR).