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. 2014 Aug;37(8):592-7.
doi: 10.14348/molcells.2014.0115. Epub 2014 Aug 18.

Atomic Force Microscopy of Asymmetric Membranes From Turtle Erythrocytes

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Free PMC article

Atomic Force Microscopy of Asymmetric Membranes From Turtle Erythrocytes

Yongmei Tian et al. Mol Cells. .
Free PMC article

Abstract

The cell membrane provides critical cellular functions that rely on its elaborate structure and organization. The structure of turtle membranes is an important part of an ongoing study of erythrocyte membranes. Using a combination of atomic force microscopy and single-molecule force spectroscopy, we characterized the turtle erythrocyte membrane structure with molecular resolution in a quasi-native state. High-resolution images both leaflets of turtle erythrocyte membranes revealed a smooth outer membrane leaflet and a protein covered inner membrane leaflet. This asymmetry was verified by single-molecule force spectroscopy, which detects numerous exposed amino groups of membrane proteins in the inner membrane leaflet but much fewer in the outer leaflet. The asymmetric membrane structure of turtle erythrocytes is consistent with the semi-mosaic model of human, chicken and fish erythrocyte membrane structure, making the semi-mosaic model more widely applicable. From the perspective of biological evolution, this result may support the universality of the semi-mosaic model.

Keywords: asymmetry; atomic force microscopy; membrane structure; turtle erythrocyte.

Figures

Fig. 1.
Fig. 1.
AFM topographic images of the smooth outer surface of the turtle erythrocytes. (A) The intact turtle erythrocyte is 27.5 ± 4.5 μm long, 15.2 ± 1.5 μm wide, and 1.4 ± 0.3 μm high. A black dashed circle encloses the nucleus (8.0 ± 3.1 μm long; 4.8 ± 1.2 μm wide). The inset is the correponding amplitude image. The bottom is the cross-sectional analysis along the black line. (B) The smooth edge of the intact turtle erythrocyte (47.2 ± 4.1 nm, thickness). The bottom is the cross-sectional analysis along the black line. (C) High-resolution image of the smooth edge. (D) High-resolution image of the prepared outer membrane leaflet. The average roughness of the outer membrane leaflet is 0.52 ± 0.11 nm. Scale bars: 5 μm in (A), 500 nm in (B), 200 nm in (C), 100 nm in (D).
Fig. 2.
Fig. 2.
Characterization of the protein-covered inner leaflet of the turtle erythrocyte membranes. (A) AFM topographic image of the inner membrane leaflet. (B) Magnification of the square area in (A). Arrows point to proteins on the substrate. (C) High-resolution image of the inner membrane leaflet. The average height and roughness of the membranes are 18.5 ± 2.4 nm and 4.0 ± 0.8 nm, respectively. (D) The diameter distribution of proteins in the inner membrane leaflet varies from 15 to 200 nm with 70% falling between 40 and 80 nm. (E) The height of proteins above the membrane is from 1.0 to 27.0 nm with the peak at 10–16 nm. Scale bars; 5 μm in (A); 2 μm in (B); 500 nm in (C).
Fig. 3.
Fig. 3.
Digestion of the inner leaflet of the turtle erythrocyte membranes by proteinase K. (A) AFM topographic image of the digested membrane. (B) (Top) High-resolution image of the edge of the digested membrane. Arrows point to the exposed lipid bilayer after digestion (2.5 ± 0.5 nm, thickness). The bottom is the cross-sectional analysis along the black line with single arrow pointing to the lipid bilayer and double arrows pointing to the remaining proteins or peptides. (C) The height distribution of proteins in the membrane after digestion is from 1.5 to 12.5 nm with the peak at 4–6 nm. Scale bars; 2 μm in (A); 200 nm in (B).
Fig. 4.
Fig. 4.
Detection of exposed amino groups on both leaflets of the turtle erythrocyte membranes. (A) The typical force curves acquired with a glutaraldehyde-functionalized AFM tip on the outer membrane leaflet, in which no force peak was observed (binding probability, 2.3%). (B) Representative force curves acquired on the inner membrane leaflet, showing multiple force peaks (binding probability, 86.2%). The inset shows the typical force curves obtained on the digested inner membrane leaflet (binding probability, 8.3%).

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