Hereditary spherocytosis (HS) is a common, inherited hemolytic anemia in which defects of spectrin or the proteins that attach spectrin to the lipid bilayer (ankyrin, band 3 and protein 4.2) lead to spheroidal, osmotically fragile cells that are selectively trapped in the spleen. The prevailing theory is that HS is caused by local disconnection of the skeleton and bilayer, followed by vesiculation of the unsupported surface components. The mechanism of this effect is not well understood. One possibility is that the lipid bilayer is stabilized directly by interactions with the membrane skeleton. Alternatively, the lipids may interact with the multiple transmembrane helices of band 3, which would indirectly anchor them to the skeleton. We will test these two hypotheses using the techniques of targeted gene deletion and gene replacement in mouse embryonal stem (ES) cells. We will first determine the consequences of deleting band 3 or protein 4.2 in mice. Preliminary data show that band 3-I- mice have a severe spherocytic hemolytic anemia, but a surprisingly intact membrane skeleton, which fits best with the second hypothesis. We will evaluate this more directly by replacing band 3 with: (1) a band 3 derivative that lacks anion transport function, (2) a chimera of the glucose transporter attached to the cytoplasmic domain of band 3, (3) the isolated cytoplasmic domain of band 3 anchored to the membrane by a single transmembrane helix or by isoprenylation (4) the isolated membrane domain of band 3, and (5) a combination of the membrane and cytoplasmic domains, dually expressed but unattached to each other. The effects of each mutation on survival, organ function and pathology, red cell lifespan and morphology, red cell membrane and membrane skeletal composition and organization, membrane stability, and the function and mobility of band 3 will be investigated. Other aspects of membrane function will be measured in selected mutants, including ankyrin and/or spectrin rebinding to membrane vesicles, anion transport function, acid- base balance, and analysis of shed membrane vesicles.