The aim of this proposal is to understand how the mechanical properties of blood cells change when there are abnormalities or alterations in their structural components. In this particular proposal, the research focuses on the stability of the red blood cell membrane and the associations among the structural proteins of the membrane that give it its stability. Bilayer membranes by themselves are inherently unstable mechanically because they lack an elastic resistance to elongational deformation. Therefore, the presence of the membrane skeleton and its proper association with the bilayer are essential for cell survival. Abnormalities in membrane structural proteins or their interactions with one another are known to result in hemolytic anemia. In addition to developing more precise descriptions of the mechanical properties of the membrane skeleton in situ, the proposed research focuses primarily on the interaction between the membrane skeleton and the bilayer. Newly developed physical methods will be used to measure the strength of the bilayer skeletal interaction in normal cells and in cells with structural abnormalities or alterations. In addition, ultrastructural studies using antibodies or fluorescent labels that recognize specific membrane proteins will be used to determine the molecular events that are associated with the mechanical separation of bilayer and skeleton and to determine how these events are affected by structural changes in the intact membrane. The physical methods involve the formation of thin, cylindrical bilayer strands (tethers) from the cell surface in single cell micromechanical experiments. The forces applied to the cell and the corresponding changes in the cellular geometry are measured and interpreted quantitatively in terms of the energy of association between the bilayer and the skeleton. The structural alterations to be examined include: a) naturally occurring abnormalities in the membranes of patients with different forms of hemolytic anemia including hereditary spherocytosis, hereditary elliptocytosis, sickle cell disease and thalassemia; b) alterations in the structure of normal membranes as a result of chemical manipulations including low ionic strength, high pH and oxidation; and c) structural alterations induced by. incorporation of skeletal proteins or protein fragments to compete with specific native associations within the membrane skeleton and between the skeleton and the bilayer. It is our expectation that a more detailed and precise understanding of the effects that structural alterations have on the mechanical function (stability) of the membrane will result in a better understanding of the pathophysiology of hemolytic anemia and could ultimately lead to improved strategies for treating patients with hemolytic disease.
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