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.

Phagocytosis and Motility in Human Neutrophils is Competent but Compromised by Pharmacological Inhibition of Ezrin Phosphorylation.

BACKGROUND AND OBJECTIVE: Ezrin links the cortical cytoskeleton to the plasma membrane and plays a role in regulating changes in cell shape. Recently, NSC668394 has been shown to inhibit a key step for its activity, i.e. phosphorylation at threonine 567. In neutrophils, another key regulatory step is the Ca2+-mediated cleavage of ezrin by calpain. METHODS: In this paper, we use NSC668394 as a pharmacological inhibitor to investigate the interplay between these two steps in regulating changes in neutrophil shape. RESULTS: NSC668394 reduced the amount of peripherally located ezrin in neutrophils, and increased Ca2+-dependent ezrin cleavage. Neutrophils with NSC668394-inhibited ezrin phosphorylation remained both phagocytic and chemotactically competent. However, phagocytosis was slightly impaired and chemotaxis could not be maintained over longer periods. The characteristic chemotactic morphology which neutrophils adopt was also aberrant. Although phosphorylation of ezrin plays a minor role in limiting the rapid changes in cell shape in neutrophils, inhibition of ezrin phosphorylation by NSC668394 prevented multiple and prolonged shape changes during extended chemotaxis. CONCLUSION: The susceptibility of prolonged chemotaxis to inhibition by NSC668394 may point to a useful target for anti-inflammatory therapy. Inhibition of neutrophil chemotaxis towards chronically inflamed sites without compromising their ability to undergo phagocytosis is a much sought after the effect of anti-neutrophil therapy.