Monitoring Endosomal Cargo Retrieval to the Trans-Golgi Network by Microscopic and Biochemical Approaches

The endosomal recycling pathway plays a crucial role in diverse physiologically important biological processes such as cell-to-cell signaling, nutrient uptake, immune response, and autophagy. A selective subset of these recycling cargoes, mostly transmembrane proteins, is retrieved from endosomes to the trans-Golgi network (TGN) by a retrograde transport process. Endosome-to-TGN retrograde trafficking is crucial for maintaining cellular homeostasis and signaling by preventing proteins and lipids from degradation in the lysosome. Many of the membrane sorting machinery, such as the retromer complex and sorting nexins (SNXs) are involved in endosomal retrieval and recycling of various transmembrane proteins. Recent technological advances in the resolution of light microscopy and unbiased analytical approaches in quantitative image analysis enable us to explore and understand the regulation of membrane trafficking pathways in greater detail. In this chapter, we describe quantitative imaging-based methods for analyzing the roles of proteins involved in the retrograde trafficking in retromer dependent or independent fashion, using cation-independent mannose-6-phosphate receptor (CIM6PR) as an example.