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Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells
Published on: February 21, 2019
Quantitative Methods to Study Endocytosis and Retrograde Transport of Cargo Proteins
Massiullah Shafaq-Zadah1, Estelle Dransart1, Ludger Johannes2
1Institut Curie, PSL Research University, Cellular and Chemical Biology unit, Endocytic Trafficking and Intracellular Delivery team, U1143 INSERM, UMR3666 CNRS, 26 rue d'Ulm, Paris Cedex 05, France.
Abstract:
Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517-533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5β1 integrin, we here describe generic methods to differentiate (1) internalized from cell surface-accessible cargo proteins and (2) endocytic cargo proteins that have reached the Golgi apparatus via the retrograde route from those that have not. The choice of antibodies or natural ligands allows to adjust these methods to virtually any chosen biological system.
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