Sample Preparation and Imaging of Erythrocyte Cytoskeleton with the Atomic Force Microscopy

Abstract

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

“…In view of the experimental data presented here, if the observed overcorrelation corresponds to the collective action of single kicking events of duration t z 0.1 s, assuming the active model of Fig. 5 B, on average, every elemental membrane patch undergoes a transverse displacement of length d z Dh h À Dh f z 25 nm, a distance compatible with the dimensions of the elemental cell of the cytoskeletal network (46).…”

“…The RBC membrane is composed of a flexible lipid bilayer, mainly composed of phospholipids and cholesterol, which is linked to a metabolically active cytoskeletal network. The native structure of unstressed membrane RBC skeleton has been determined by transmission electron microscopy (TEM) (45,46). TEM images of intact membranes reveal a foam-like meshwork of spectrin filaments with a gradual decrease in density from the center of the cell to the equatorial edge.…”

“…5 A) in which spectrin filaments are the main structural component (2,3). In vivo, the characteristic length of the network is a ¼ 46 5 15 nm (46), indicating that on average, the size of spectrin filaments in the native membrane skeleton is a fraction of its contour length (L z 190 nm) observed in membrane patches stretched on TEM grids (39). Spectrin filaments are pinned to the membrane Biophysical Journal 108(12) 2794-2806 via transmembrane-and membrane-associated proteins (47,48).…”

Combined Deletion of Mouse Dematin-Headpiece and β-Adducin Exerts a Novel Effect on the Spectrin-Actin Junctions Leading to Erythrocyte Fragility and Hemolytic Anemia

“…Erythrocytes were washed with PBS and examined within 5 h. Cell attachment on glass coverslips was performed as described previously (27). Briefly, a suspension of washed erythrocytes was brought into contact with chemically modified glass coverslips coated with lectin, erythroagglutinating phytohemagglutinin, for 30 min to allow the cells to attach.…”

“…AFM has been used previously to investigate erythrocyte cytoskeleton architecture and its stability without removing the plasma membrane by detergent treatment and without extending or staining the cytoskeleton (27,28). This method, which provides information on native skeleton structure, reveals differences in the skeletal structures between WT, single KO, and DAKO erythrocytes.…”