Investigation of Intercellular Trafficking of Global Glycosylphosphatidylinositol‐Anchored Proteins Using Cell Metabolic Engineering and Transwell Coculture

Abstract

Abstract Glycosylphosphatidylinositols (GPIs) are essential for GPI‐anchored proteins (GPI‐APs), as the loose association of GPIs with the cell membrane is thought to facilitate GPI‐AP shedding and intercellular trafficking, which dictates cell communication. To explore this property, a novel method was developed based on metabolic engineering of GPI‐APs in live cells and coculture of labelled donor cells along with recipient cells in a Transwell system for straight observation of intercellular GPI‐AP transfer. Studies using this method have provided direct proof of intercellular GPI‐AP trafficking and its dependence on cell types. Notably, there is a preference for GPI‐AP transfer from cancer to normal cells. Lipid raft‐mediated endocytosis and macropinocytosis play a critical role in GPI‐AP trafficking and incorporation, with extracellular vesicles (EVs) involved as GPI‐AP transporters. It is further revealed that depleting cholesterol in the donor cell can enhance GPI‐AP shedding and trafficking. The method also allowed for in‐depth studies to gain insights into the regulatory mechanisms of GPI‐AP trafficking, such as its association with phospholipase A2 (PLA2), EV biogenesis, and membrane‐adjacent Ras and Rho signaling pathways. The results are important not only for understanding GPI biology but also for its biomedical applications, for example, GPI‐based cancer therapy.