Group intracellular trafficking and tissue homeostasis. Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.
U1202, INSERM, Paris, France.
Methods Mol Biol. 2021;2233:3-17. doi: 10.1007/978-1-0716-1044-2_1.
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.
确定活细胞中的蛋白质化学计量对于理解基本的生物过程至关重要。这对于受体介导的胞吞作用尤其重要，受体介导的胞吞作用是一种高度调节的机制，需要许多因素的顺序组装。在这里，我们描述了一种定量分析质膜上受体聚集动力学的方法。我们的工作流程将 CRISPR-Cas9 编辑的细胞系在生理相关环境中表达 GFP 标记受体的 TIRF 活细胞成像、GFP 单分子分析的校准技术以及使用开源软件进行检测和跟踪相结合。该方法可以实时确定质膜上受体分子的数量。
