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

Abstract

The intracellular events underlying phagocytosis, a crucial event for innate immunity, are still unresolved. In order to test whether the reservoir of membrane 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 (phagocytically-competent cell line RAW264.7), by 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 fluo-Tags would result. Experimentally-induced Ca2+influx triggered cleavage of peripherally located ezrin; which was temporally associated with cell expansion. Ezrin cleavage was also observed during phagocytosis, localised to the phagocytic pseudopodia. 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 becomes available for cell spreading and phagocytosis.