Ca²⁺-activated cleavage of ezrin visualised dynamically in living myeloid cells during cell surface area expansion

The intracellular events underlying phagocytosis, a crucial event for innate immunity, are still unresolved. In order to test whether the reservoir of membrane required for the formation of the phagocytic pseudopodia is maintained by cortical ezrin, and that its cleavage is a key step in releasing this membrane, the cleavage of cortical ezrin was monitored within living phagocytes (the phagocytically competent cell line RAW264.7) through expressing two ezrin constructs with fluorescent protein tags located either inside the FERM or at the actin-binding domains. When ezrin is cleaved in the linker region by the Ca²⁺-activated protease calpain, separation of the two fluorophores would result. Experimentally induced Ca²⁺ influx triggered cleavage of peripherally located ezrin, which was temporally associated with cell expansion. Ezrin cleavage was also observed in the phagocytic pseudopodia during phagocytosis. Thus, our data demonstrates that peripheral ezrin is cleaved during Ca²⁺-influx-induced membrane expansion and locally within the extending pseudopodia during phagocytosis. This is consistent with a role for intact ezrin in maintaining folded membrane on the cell surface, which then becomes available for cell spreading and phagocytosis.