Hagelberg, Carla; (1995) Vesiculation events and bilayer-cytoskeleton interactions in the human erythrocyte. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Changes in human erythrocyte morphology leading to echinocytosis and microvesicle release were studied. The results were generally consistent with the Bilayer Couple Hypothesis and it was calculated that expansion of the outer leaflet of the lipid bilayer by as little as 3% relative to the inner leaflet was sufficient to cause microvesicle release. The protein and phospholipid composition of microvesicles released as a result of ATP-depletion, during storage, or after treatment with merocyanine 540, dimyristoylphosphatidylcholine or Ca2+/A23187 was compared with the composition of the original cell membrane. These microvesicles were depleted in band 3, glycophorin and phosphatidylinositol 4,5-bisphosphate relative to phospholipid by 40% or more. This data was interpreted to mean that less than half of these membrane components are free to diffuse laterally in the lipid bilayer. Acetylcholinesterase was found to be enriched 2-3 fold in microvesicles, possibly because the removal of non-diffusing proteins from the vesiculating region of the lipid bilayer allows more space for freely-diffusing proteins like acetylcholinesterase to enter the microvesicle membrane. The phosphorylation of human erythrocyte membrane proteins was investigated in order to determine if phosphorylation affected cytoskeletal interactions which could alter shape or vesicle release. Protein kinase C was activated by phorbol myristate acetate and protein kinase A by the incubation of erythrocytes with cAMP. The extent of the polyphosphoinositide breakdown after ATP-depletion in control and cAMP-treated cells was compared. In the treated cells the phosphatidylinositol 4,5-phosphate breakdown was greatly increased. The phosphorylation of membrane proteins could cause a reduced interaction between this lipid and the cytoskeleton, leading to a greater proportion of the lipid being broken down. The microvesicles produced from cAMP-treated erythrocytes were analysed to ascertain if any phosphoproteins partitioned into the microvesicles. Band 7 was the only phosphoprotein, besides Band 3, found to be present in the microvesicles.

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