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Integral Protein Linkage and the Bilayer-Skeletal Separation Energy in Red Blood Cells

James Butler ^*, Narla Mohandas  and Richard E. Waugh ^*

^* Department of Biomedical Engineering and Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York 14642; and New York Blood Center Laboratory of Red Cell Physiology, New York, New York 10021

Correspondence: Address reprint requests to Richard E. Waugh, Dept. of Biomedical Engineering, University of Rochester, River Campus, Box 270168, Rochester, NY 14627. Tel.: 585-275-3768; Fax: 585-276-1999; E-mail: waugh{at}seas.rochester.edu.

Stabilization of the lipid bilayer membrane in red blood cells by its association with an underlying membrane-associated cytoskeleton has long been recognized as critical for proper red blood cell function. One of the principal connections between skeleton and bilayer is via linkages between band 3, the integral membrane protein that transports anions across the cell surface, and membrane skeletal elements including ankyrin, adducin, spectrin, and the junctional complex of the skeleton. Here, we use membrane tether formation coupled with fluorescent labeling of membrane components to examine the importance of band 3 in stabilizing the bilayer-skeletal association. In membranes from a patient deficient in band 3, the energy associated with the bilayer skeleton is approximately zero, whereas when band 3 is immobilized by ligation with the monoclonal antibody R10, the energy of association approximately doubles. Fluorescence images of tethers reveal that 40% of the band 3 on the normal cell surface can be pulled into the tether, confirming a lateral segregation of membrane components during tether formation. These results validate a critical role for band 3 in stabilizing the bilayer-skeletal association in red cells.

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