Turner, Eleanor Jane Holland; (2002) Vesiculation and rafts in stomatocytic red cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes work on ATP-dependent endocytic vesiculation and sphingomyelin and cholesterol-rich lipid rafts in the membrane of normal red cells and those with 'hereditary stomatocytosis' (HSt) syndromes, a set of rare congenital human haemolytic anaemias which show a 'leak' to the univalent cations Na+ and K+ and in some cases the deficiency of a 32 kDa raft protein, 'stomatin'. The work explores the idea that the pathophysiology of these diseases may be secondary to an abnormality in cellular processes of vesiculation based on membrane rafts. It was found that an in vitro process of ATP-dependent vesiculation, which occurs in normal red cell membranes, was completely absent in two pedigrees with overhydrated HSt (OHSt), the most severe version of these diseases. Both this defect and the cation leak were corrected by the cross-linking reagent, dimethyl adipimidate. In eleven other HSt pedigrees with different variants of the stomatocytosis syndromes ATP-dependent endocytic vesiculation was present. It was determined that lipid rafts existed in the normal erythrocyte membrane and that stomatin was partially associated with the rafts. Rafts were found to exist in the OHSt membrane in spite of the lack of stomatin. The degree of raft association of various membrane proteins, was investigated and the proportion of raft associated actin was found to be significantly reduced in OHSt cells. It was found that in normal red cell membranes, the process of ATP- dependent vesiculation could be inhibited by incubating erythrocyte ghosts with an antibody to stomatin. The raft disrupters nystatin, filipin, digitonin, saponin and methyl-[beta]-cyclodextrin were also found to inhibit this process. Further work exploring the mechanism of ATP-dependent endocytosis was performed. This work is consistent with the suggestion that the basic defect in the leaky OHSt cell may lie in a raft-based process of endocytosis, which may involve the stomatin protein.

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