Abstract
Endocytic trafficking and recycling are fundamental cellular processes that control essential functions such as signaling protein complexes transport and membrane identity. The small GTPase Rabs are indispensable component of the endosomal recycling machinery. The Rabs bind to effectors to mediate their functions, such as protein sorting and degradation, membrane tethering or lipid modification, and organelle motility. Due to the complex and dynamic nature of endosomal compartments and tracking route, detailed multiparametric analyses of three-dimensional data by quantitative methods are challenging. Here, we describe a detailed time-lapse imaging protocol designed for the quantitative tracking of single endosomal vesicles, using GFP-Rab4-positive recycling endosomes. This method permits automated tracking of single endocytic vesicles in three-dimensional live cell imaging, allowing the study of multiple parameters such as abundance, speed, directionality, and subcellular localization, as well as protein colocalization. This protocol can be broadly used in any kind of cellular models, under various contexts, including growth factors stimulation, gene knockdowns, drug treatments, and is suitable for high throughput screens.