Evidence from pathologic red blood cells suggests that the coupling of skeletal proteins with the overlaying membrane is essential for normal cell integrity and dynamic behavior. Although extensive biochemical and electron microscopic studies have provided an initial concept of the skeletal structure, the geometry of the skeleton's relationship to the overlying bilayer is not well under-stood. Our goal is to use biophysical approaches to obtain a detailed perspective of the skeleton:bilayer interface in the intact cell that can eventually be studied under dynamic conditions. Such studies should provide insight into the instability of the membrane in certain hemolytic disorders. To define the molecular geometry of this interface, we plan to measure various skeleton:bilayer distances using fluorescence energy transfer and condensed phase radioluminescence techniques. For this purpose, the following steps will be carried out: (1)Labelling and incorporation of membrane elements: Site-directed fluorescent labelling of spectrin will be achieved using a novel cleavable, fluorescent, photoaffinity crosslinking reagent. In situ specific labelling of actin, band 3, and 4.1 will also be conducted. Fluorescent and tritiated lipophilic compounds will be obtained commercially. The labelled protein and lipid species will then be incorporated in to inside- out vesicles and ghosts. Proper biochemical incorporation of proteins will be assessed by:determination of their binding affinity and reversibility of binding to the membrane, competitive displacement with antibodies or appropriate functional protein fragments, and electron microscopic localization with anti-fluorescein immuno-gold conjugates. Proper functional incorporation of proteins will be assessed by monitoring membrane deformability and fragility by ektacytometry. (2)Measurement of skeleton:bilayer distance in normal cells: Short distances (within 20-80 A) between the fluorescently-labelled skeletal proteins and the plane of the inner leaflet membrane lipids will be measured by fluorescence energy transfer. Longer distances (between 80 to 500 A) will be measured by condensed phase radioluminescence using fluorescently-labelled skeletal proteins and tritiated membrane lipids. (3)Assessment of the effects of physiologic perturbations on the skeleton:bilayer distances: The changing interactions between the skeleton and the overlying bilayer will be examined during various membrane stresses (e.g., shear stress, hypotonic expansion, echinocytogenesis of different types, and mild oxidation with Heinz bodily induction). The localized topography of skeleton:bilayer distances (e.g., in the spikes of echinocytes or adjacent to Heinz bodies) will be measured in individual cells using a laser scanning confocal fluorescence dual channel microscope coupled to an image processing system.
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