Membrane proteins at the interface of erythrocytes fused by treatment with polyethylene glycol.
Fusion of human red cells through the action of polyethylene glycol gives rise to pairs or higher clusters with a common membrane envelope, in which a barrier at the position of the original interface can be seen in phase contrast. At early times this septum contains lipids, as judged by labelling with a fluorescent lipophile, and transmembrane protein; this was shown by the presence of the preponderant component, band 3, detected by a fluorescent label, covalently attached before fusion at an extracellular site, or by immunofluorescence with anti-band 3 antibody. Ankyrin, which is bound to band 3, is also observed in the septum. The lipid thereafter disappears from the interface, carrying most of the band 3 with it. A continuous membrane skeletal network, defined by the presence of spectrin (detected by immunofluorescent staining in epifluorescence and confocal microscopy) appears to persist for long periods, but in many cells interruptions develop in the septum. In other fused pairs, particularly at longer times, the interface is seen to have vanished completely. Protease inhibitors have no discernible effect on any of these observations. The results suggest a model for the events that follow fusion. Covalent cross-linking of membrane proteins beyond a critical level causes inhibition of fusion, suggesting that proteins, probably the membrane skeletal network, regulate the fusion process. The efficiency of fusion is strikingly dependent on the composition of the isotonic medium, being relatively high at an orthophosphate concentration of 5 mM and minimal at 20 mM.
Litwa
M
M
Medical Research Council Muscle and Cell Motility Unit, King's College, London, U.K.
Maggs
A M
AM
Jin
C Z
CZ
Wang
W D
WD
Pedley
K
K
Beaven
G H
GH
Gratzer
W B
WB
D001457:Anion Exchange Protein 1, Erythrocyte / Q000187:drug effects; D017487:Ankyrins / Q000187:drug effects; D002459:Cell Fusion / Q000187:drug effects*; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects* / Q000648:ultrastructure; D006801:Humans; D008563:Membrane Lipids / Q000097:blood; D008565:Membrane Proteins / Q000187:drug effects*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D011092:Polyethylene Glycols / Q000494:pharmacology*; D013049:Spectrin / Q000187:drug effects
D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D008563:Membrane Lipids; D008565:Membrane Proteins; D013049:Spectrin; D011092:Polyethylene Glycols