The myotubularin phosphatase MTMR4 negatively regulates PtdIns(3)P signalling in endosomal and phagosomal trafficking

Macrophages are white blood cells which as professional phagocytes are part of the innate immune control of infections, with additional roles in cytokine signalling and antigen presentation in adaptive immunity as well as tissue remodelling and inflammation. Phosphoinositide signalling is important in the regulation of cellular endosomal trafficking. PtdIns(3)P is present in the membrane of early and late endosomes and recruits effector proteins to the cytosolic face of endosomal membranes. In macrophages, PtdIns(3)P is also present on the membranes of newly formed phagosomes, where it recruits effector proteins containing FYVE domains and regulates phagosomal maturation. PtdIns(3)P is in part formed by the action of class III PI3-kinases on both endosomes and phagosomes. The myotubularins are a large family of proteins which possess in vitro 3-phosphatase activity to degrade PtdIns(3)P and PtdIns(3,5)P2, and there are differing reports on the localisation and function of different myotubularins within cells. Recombinant MTMR4, which possesses a FYVE domain, has been identified on endosomal structures where it alters EGFR trafficking. The aim of this thesis is to ascertain if MTMR4, as a putative regulator of phosphoinositide signalling, plays a role in endosomal and phagosomal biology. In this thesis, the localisation and function of MTMR4 in HeLa cells and RAW 264.7 macrophage cells has been characterised. A combination of fixed and live cell microscopy studies were used to demonstrate that MTMR4 localised to endosomes and also localised dynamically to phagosomes. MTMR4 regulated PtdIns(3)P on endosomes and on phagosomes. siRNA technology was used to assess the consequences of MTMR4 depletion. Knockdown of MTMR4 in HeLa cells decreased the amount of cellular transferrin loading, and produced a dispersed morphology to the endocytic recycling compartment, however, the recycling of transferrin was unaltered. MTMR4 negatively regulated the phagocytosis of IgG-opsonised latex beads as well as of serum opsonised zymosan. Knockdown of MTMR4 increased the maturation of Mycobacterium marinum phagosomes, however, this did not result in altered mycobacterial killing by RAW 264.7 cells. MTMR4 knockdown macrophages exhibited elevated F-actin and G-actin at phagocytic cups, and additionally had defects in lamellipodial spreading. Therefore this work has identified a novel role for MTMR4 in the regulation of endosomal and phagosomal PtdIns(3)P signalling and endosomal and phagosomal trafficking