Fracture-flip/Triton X-100 reveals the cytoplasmic surface of human erythrocyte membranes.

Abstract

We propose the use of fracture-flip combined with Triton X-100 extraction to visualize the cytoplasmic surface of plasma membranes.Unfixed human erythrocytes were freeze-fractured, carbon-cast, and thawed. The carbon casts, along with attached freeze-fractured erythrocytes, were treated with 2% Triton X-100 to solubilize unfractured plasma membranes and to release haemoglobin. After repeated washing, the carbon-casts, along with attached protoplasmic and exoplasmic membrane halves, were picked on grids, flipped, and Pt-shadowed.Our method leads to extended views of the cytoplasmic surface revealing the fibrilar network that laminates the inner surface of the erythrocyte membrane. Spectrin immunogold labelling of fractured, carbon cast erythrocytes shows that the colloidal gold particles are associated with the fibrilar network at the cytoplasmic surface.Removal of membrane skeletal elements including spectrin by treatment with a low ionic strength buffer containing EDTA leads to loss of the network and reveals globular particles on the cytoplasmic surface of the membrane. These globular particles contained band 3, as shown by immunogold labelling.Our method can be extended to both the ultrastructural observation and the cytochemistry of the cytoplasmic surfaces of other biomembranes.Recently, we have developed 'Fracture-Flip', a method that provides extended, macromolecular resolution views of cell surfaces (1,2,5,14). Fracture-flip proceeds as follows: (1) Freezing: Fixed or unfixed cells are frozen.(2) Freeze-fracture: Freeze-fracture splits the membranes into protoplasmic and exoplasmic halves. (3) Carbon-fixation: The exposed EF faces are evaporated with carbon. This produces a carbon cast with stabilized membrane halves. (4) Washing, flipping (inversion), and Pt-shadowing of the carbon cast: The carbon casts are thawed. Contrary to conventional freeze-fracture, the carbon casts are not digested with acids or bases. Instead, they are repeatedly rinsed in distilled water to wash away unfractured cells. The exoplasmic halves of membranes remain attached to the carbon casts, which are transferred to electron microscope grids. The carbon casts on grids are flipped (inverted). Pt-shadowing creates the informative three-dimensional images of membrane surfaces.As developed, fracture-flip cannot reveal the inner, cytoplasmic surface of the membrane because the cell body remains attached to the inner, protoplasmic membrane half. We report here a method to overcome this obstacle. Briefly, we use unfixed cells that, after freeze-fracture and carbon-stabilization of apolar domains, are treated with Triton X-100. We reasoned that while the detergent would dissolve unfixed, unfractured membranes, it would not reach, micellize, and extract split membranes, as their apolar domains were now positioned against, and stabilized by, their carbon casts. After washing, carbon casts with their stabilized membrane halves are imaged by Pt/C evaporation. Our results provide views of the membrane skeleton of erythrocytes in situ. Immun...

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application

“…Figure 11 shows atomic force micrographs of Pt/C-coated, SDS-digested (unlabeled) erythrocyte membrane halves showing closely packed globular particles (10-30 nm in diameter) on the inner (cytoplasmic) surfaces (B) and the much smoother outer surfaces (A). Our findings are consistent with the macromolecular resolution views of the erythrocyte membrane surface seen in previous studies (Fujimoto and Pinto da Silva 1992). The combination of transmission electron microscopy and atomic force microscopy will make it possible to examine the surface and interior of the same membrane for more precise identification and distribution of membrane components.…”

“…Split membrane halves are physically fixed by Pt/C shadowing SDS-FRL is based on the hypothesis that the split membrane halves are physically stabilized (fixed) by C-shadowing onto the apolar fracture face, thus the C-fixed membrane halves would not be extracted with organic solvents or detergents (Andersson-Forsman and Pinto da Silva 1988a; Fujimoto and Pinto da Silva 1988, 1989, 1992Fujimoto and Ogawa 1991). To test this hypothesis, we first applied SDS-FRL to the immunogold labeling of intercellular junction proteins, e.g., gap junction protein (connexins) and tight junction protein (occludin); the findings suggested the reliability and the potential significance of SDS-FRL in the immunocytochemical labeling of integral membrane proteins (Fujimoto 1995).…”

Fracture-flip and fracture-flip cytochemistry to study macromolecular architecture of membrane surfaces: Practical procedures, interpretation and application.

“…In order to overcome this limitation, the use of fracture-flip combined with Triton X-100 extraction has been proposed (6,7). In principle, fractureflip/Triton X-100 makes it possible to visualize the exoplasmic luminal surfaces of cell organelles as well as the cytoplasmic surfaces of plasma membranes.…”

“…Its final images can easily be shown in stereo, and thus provide interpretable threedimensional information. The most important advantage is that the combination of fracture-flip with cytochemical methods, fracture-flip cytochemistry, will provide high-resolution views of the distribution of antigens and receptors on the cell surfaces (2,3,(7)(8)(9).…”

Connexin Methods and Protocols

“…The aim of such technological development was to study in parallel the ultrastructural features and the biochemical nature of the membrane constituents (11)(12)(13)(14). The freeze-fracture simultaneously splits the two opposite junctional membranes in a stepwise fashion.…”