Atomic force microscopy of the erythrocyte membrane skeleton

Abstract

SummaryThe atomic force microscope was used to examine the cytoplasmic surface of untreated as well as fixed human erythrocyte membranes that had been continuously maintained under aqueous solutions. To assess the effects of drying, some membranes were examined in air. Erythrocytes attached to mica or glass were sheared open with a stream of isotonic buffer, which allowed access to the cytoplasmic membrane face without exposing cells to non-physiological ionic strength solutions. Under these conditions of examination, the unfixed cytoplasmic membrane face revealed an irregular meshwork that appeared to be a mixture largely of triangular and rectilinear openings with mesh sizes that varied from 35 to 100 nm, although few were at the upper limit. Fixed ghosts were similar, but slightly more contracted. These features represent the membrane skeleton, as when the ghosts were treated to extract spectrin and actin, these meshworks were largely removed. Direct measurements of the thickness of the membrane skeleton and of the lateral dimensions of features in the images suggested that, especially when air dried, spectrin can cluster into large, quite regularly distributed aggregates. Aggregation of cytoskeletal components was also favoured when the cells were attached to a polylysine-treated substrate. In contrast, the membrane skeletons of cells attached to substrates rendered positively charged by chemical derivatization with a 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 observed structures became shorter and thinner. Ghosts treated with Triton X-100 solutions to remove the bilayer revealed a meshwork having aggregated components resembling those seen in air. These findings support the proposition that the end-to-end distance of spectrin tetramers in the cell in the equilibrium state is much shorter than the contour length of the molecule and that substantial rearrangements of the spectrin-actin network occur when it is expanded by low ionic strength extraction from the cell. This study demonstrates the applicability of AFM for imaging the erythrocyte membrane skeleton at a resolution that appears adequate to identify major components of the membrane skeleton under near-physiological conditions.

Stabilities of folding of clustered, two-repeat fragments of spectrin reveal a potential hinge in the human erythroid spectrin tetramer

“…The tortuous trajectory of minimally perturbed erythroid spectrin in situ imaged by electron microscopy (45), and later confirmed by atomic force microscopy of dried (46) and hydrated (47,48) red cells, implies that spectrin can bend in a nonuniform fashion along its length. Likewise, rotary-shadowed human (49) and avian (50) spectrin dimers and tetramers exhibit different degrees of curvature, although not consistently in the same regions of the molecules.…”

PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

“…It has been reported that drying of the erythrocyte membrane for tapping-mode AFM in air causes a slight deformation of the attached cytoskeleton (30). This is a possibility for PHA inclusions, but because the network is apparently linked to the crystalline layer (otherwise it would be totally lost during sonication), it us unlikely that it deforms much beyond what occurs as the polymer crystallizes.…”

Imaging CFTR in its native environment

“…The membranes of RBC were isolated according to a modified method of Swihart et al [100]. In short, RBC attached to poly-L-lysine-coated glass were sheared open with a jet stream of isotonic phosphatebuffered saline (Fig.…”