Dynamics of membrane skeleton in fused red cell membranes

The dynamic nature of the red cell membrane cytoskeleton was examined by the method of fluorescence redistribution after fusion (FRAF). Ghosts, labelled at the interior membrane surface, and intact cells, labelled at the outer surface with a different fluorophore, were fused and the passage of the fluorescent label into the unlabelled part of the membrane was followed in the fluorescence microscope. To achieve specific labelling of a cytoskeletal component only, a fluorescent analogue of phalloidin was used to label the actin. It was shown that there was no dissociation of the phalloidin during the time of the experiment, and no exchange of phalloidin between labelled and unlabelled actin in unsealed mixtures of ghosts. In the fused membrane pairs at 37 degrees C the phalloidin-linked fluorescence diffused in the plane of the membrane and became distributed through the membrane envelope in 1-2 h. It was shown by gel electrophoretic analysis that no detectable loss of or damage to membrane proteins resulted from the fusion process. It is concluded that, at least in membrane pairs treated with fusogen, exchange of actin or actin-containing elements occurs within the cytoskeletal network.