B cell deficiency and anaemia caused by mutations in the murine atp11c gene

Organization of the plasma membrane into specialised substructures in lymphocytes facilitates important biological functions including the initiation of crucial intracellular signalling cascades at the plasma membrane. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. Membrane-bound lipid transporters are believed to generate and dynamically maintain the lipid asymmetry between the two leaflets of the cell membranes, but not much is known about the role of these transporters in a variety of biological systems in mammals. This thesis examines the effect of two ENU-induced mutations of the murine Atp11c gene, which encodes a member of the P4-type ATPase family thought to serve as 'flippases' that mediate the translocation of specific aminophospholipids to the cytoplasmic leaflet of cell membranes. Loss of ATP11C in mice led to a severe B cell deficiency due to a developmental arrest at the pro-B cell stage during early B cell development in the bone marrow. The number of splenic follicular B cells and peritoneal B1 cells was also severely reduced in mutant mice. However, marginal zone B cells as well as other haematopoietic lineages including T, NK and myeloid cells appeared to accumulate normally in mutant mice. Moreover, the requirement for ATP11C in B cells was cell autonomous, and could not be corrected by the expression of pre-rearranged immunoglobulin transgenes or enforced expression of the pro-survival protein BCL-2 or by transgenic expression of IL-7. Further analysis of mutant mice revealed that while the IL-7R-mediated signalling pathway appears mostly intact, the ATP11Camb mutation leads to a defect in the expression and/or signalling through the pre-BCR, which provides essential signals for the development of pre-B cells. In contrast, B cells from B cell receptor (BCR) transgenic ATP11C-deficient mice were able to form germinal centres upon immunisation when adoptively transferred into wild-type host, indicating an intact in vivo signalling through the mature BCR in mutant animals. Functional analysis using fluorescently labelled phospholipid derivatives revealed that cells of the immune system from ATP11C-deficient mice exhibit impaired aminophospholipid flippase activity compared to those from control animals, indicating for the first time that ATP11C functions as a phospholipid flippase in biological membranes. Although the first phospholipid translocase activity was described in erythrocytes about 30 years ago, the characterisation of enzyme activity and its effect on the membrane asymmetry, development and survival of erythrocytes remains to be unveiled. Intriguingly, ATP11C-deficient mice developed anaemia due to a shortened erythrocyte lifespan, exhibited a large proportion of abnormal-shaped erythrocytes, and increased phosphatidylserine exposure on their surface due to impaired flippase activity. Thus, these findings identified ATP11C as a candidate for aminophospholipid translocation activity in erythrocytes. In conclusion, the findings of this thesis provide novel insights into the role of the putative phospholipid transporter ATP11C in B cell development and erythrocyte survival, and suggest a new candidate for inherited B cell deficiency and anaemia in humans