使用全内反射荧光（TIRF）显微镜活细胞成像和单分子追踪技术研究内吞作用过程中质膜上受体的化学计量比。

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

机构信息

Group intracellular trafficking and tissue homeostasis. Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.

U1202, INSERM, Paris, France.

出版信息

Methods Mol Biol. 2021;2233:3-17. doi: 10.1007/978-1-0716-1044-2_1.

DOI:10.1007/978-1-0716-1044-2_1

PMID:33222124

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.

摘要

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。