Determination of protein stoichiometry in living cells is key to understanding basic biological processes. This is particularly important for receptor-mediated endocytosis, a highly regulated mechanism that requires the sequential assembly of numerous factors. Here, we describe a quantitative approach to analyze receptor clustering dynamics at the plasma membrane. Our workflow combines TIRF live imaging of a CRISPR-Cas9-edited cell line expressing a GFP-tagged receptor in a physiological relevant environment, a calibration technique for single-molecule analysis of GFP, and detection and tracking with an open-source software. This method allows to determine the number of receptor molecules at the plasma membrane in real time.
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First, we would like to thank Dr. Alexandre Grassart for training us with the technologies of CRISPR/Cas9 gene edition and single-molecule analysis and for all helpful discussion. We thank the PBI (Imagopole) platform of Institut Pasteur for microscope maintenance and technical help. LS is part of the Pasteur Paris University (PPU) International PhD Program and has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 665807.
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