Atomic force microscopy of the erythrocyte membrane skeleton

The atomic force microscope was used to examine the cytoplasmic surface of untreated as well as fixed human erythrocyte membranes that had been contin uously maintained under aqueous solutions. To assess the effects of drying, some membranes were examined in air. Erythrocytes attached to mica or glas s were sheared open with a stream of isotonic buffer, which allowed access to the cytoplasmic membrane face without exposing cells to non-physiologica l ionic strength solutions. Under these conditions of examination. the unfi xed cytoplasmic membrane face revealed an irregular meshwork that appeared to be a mixture largely of triangular and rectilinear openings with mesh si zes that varied from 15 to 100 nm, although few were at the upper limit. Fi xed ghosts were similar, but slightly more contracted, These features repre sent the membrane skeleton, as when the ghosts were treated to extract spec trin and actin, these meshworks were largely removed. Direct measurements o f the thickness of the membrane skeleton and of the lateral dimensions of f eatures in the images suggested that, especially when air dried, spectrin c an cluster into large, quite regularly distributed aggregates, Aggregation of cytoskeletal components was also favoured when the cells were attached t o a polylysine-treated substrate. In contrast, the membrane skeletons of ce lls attached to substrates rendered positively charged by chemical derivati zation with it cationic silane were much more resistant to aggregation. As steps were taken to reduce the possibility of change of the skeleton after opening the cells. the aggregates and voids were eliminated, and the observ ed structures became shorter and thinner. Ghosts treated with Triton X-100 solutions to remove the bilayer revealed a meshwork having aggregated compo nents resembling those seen in air. These findings support the proposition that the end-to-end distance of spectrin tetramers in the cell in the equil ibrium state is much shorter than the contour length of the molecule and th at substantial rearrangements of the spectrin-actin network occur when it i s expanded by low ionic strength extraction from the cell. This study demon strates the applicability of AFM for imaging the erythrocyte membrane skele ton at a resolution that appears adequate to identify major components of t he membrane skeleton under near-physiological conditions.