The network of red cell skeletal proteins facilitates oxygen and electrolyte exchange by enabling the red cell to squeeze through capillaries a third the resting diameter of the cell without disintegrating. Genetic deficiencies in the amounts and kinds of these cytoskeletal components lead to defective control of cell shape and elasticity and, therefore, a reduced number of cells or anemia. The most abundant protein in the cytoskeleton, spectrin, is also most important functionally, since it displays the unique elasticity fundamental to red cell deformability. Isoforms of spectrin also exist in nerve cells, lymphocytes and many other types of cells, the different shapes and functions of which indicate a role for spectrin throughout the whole organism.
The aim of this proposal is to understand the structure of spectrin and how its mutual interactions with other cytoskeletal and membrane components effect changes in cell shape while ensuring the stability of the cell membrane. Two models exist for the elasticity of erythroid spectrin: the """"""""entropy spring"""""""" model, in which the structure of unstretched spectrin is more random and, therefore, an enthalpically favored, folded conformation. These models will be tested using fluorescence spectroscopy to measure dynamics of internal motion of spectrin and to detect possible conformational changes occurring upon extension of the molecule. Electron microscopy will be used to investigate the static structure of spectrin under physiological conditions. Elongation flow field birefringence will be used to directly measure the force required to stretch spectrin under conditions that can differentiate between the different elasticity models. Mutual constraints on the mobility of spectrin and its membrane binding sites will be determined by fluorescence photobleaching recovery microscopy. The longterm goal of this project is the reconstitution of isolated cytoskeletal proteins and their membrane anchor proteins on an artificial lipid vesicle which will undergo deformation like an intact cell. This model system will be used to analyze unambiguously not only the roles of the cytoskeletal components, spectrin in particular, and the lipids of the bilayer, but also how exposure to environmental factors affect cell shape and cell surface stability.
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