Tagging transferrin receptor with a disulfide FRET probe to gauge the redox state in endosomal compartments

• Main Authors: Bi, Xiaobao, Yin, Juan, Zhang, Dingpeng, Zhang, Xiaohong, Balamkundu, Seetharamsing, Lescar, Julien, Dedon, Peter C., Tam, James P., Liu, Chuan-Fa
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2021
• Subjects: Science; Cell Membranes; Transferrin Receptors
• Online Access: https://hdl.handle.net/10356/148031
• Institution: Nanyang Technological University

Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.