The μ-calpain-ezrin axis : a potential target for therapy in inflammatory disease

• Main Author: Roberts, Rhiannon Emma
• Published: Cardiff University 2017
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.732288

Neutrophils are the most abundant class of white blood cell in humans. They are among the first inflammatory cells to respond during infection or tissue damage, and their excessive recruitment to synovial joints in rheumatoid arthritis has been implicated in the progression of the disease. The process of neutrophil extravasation from blood vessels involves a rapid expansion of the available cell surface area. This ~200% increase is facilitated by the release of cell membrane microridges. As ezrin forms crucial cross-links between the plasma membrane and cortical F-actin, in neutrophils and other myeloid cells, it is thought to maintain the structure of these microridges, which act as a reservoir of plasma membrane for spreading. It has been suggested that elevating the cytosolic Ca2+ concentration, thereby activating the Ca2+-activated cysteine protease μ-calpain, would break the ezrin link and permit cell spreading. This thesis has investigated the relationship between, μ-calpain, ezrin and Ca2+ concentration. Through immunocytochemistry, confocal microscopy of live phagocytosis and chemotaxis experiments, observations in neutrophils have been complemented by investigations in a RAW 264.7 model cell line, amenable to transfection. By generating the novel genetically encoded Ca2+ indicator EPIC (Ezrin Peripheral Indicator of Ca2+), it has been possible to experimentally measure the cytosolic Ca2+ concentration in ezrin-rich microdomains beneath plasma membrane microridges, in transfected myeloid cells, during Ca2+ influx. It has thus been found that sub-membranous cytosolic Ca2+ in these microridges reaches highmicromolar concentrations, well within the range required to activate μ-calpain and result in ezrin cleavage. The work in this thesis contributes to the understanding of the molecular mechanisms which govern neutrophil morphological changes during events such as phagocytosis and extravasation. It is hoped that these findings will help contribute towards the body of research aimed at influencing the design of novel therapeutics to treat autoimmune conditions.