Stoichiometry of Receptors at the Plasma Membrane During Their Endocytosis Using Total Internal Reflection Fluorescent (TIRF) Microscopy Live Imaging and Single-Molecule Tracking.

Stoichiometry of Receptors at the Plasma Membrane During Their Endocytosis Using Total Internal Reflection Fluorescent (TIRF) Microscopy Live Imaging and Single-Molecule Tracking. Methods Mol Biol. 2021;2233:3-17 Authors: Salavessa L, Sauvonnet N Abstract 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. PMID: 33222124 [PubMed - in process]
Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research