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

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  • FUJIMOTO KAZUSHI
    Section of Membrane Biology, Laboratory of Mathematical Biology, National Cancer Institute, National Institutes of Health
  • SILVA PEDRO PINTO DA
    Section of Membrane Biology, Laboratory of Mathematical Biology, National Cancer Institute, National Institutes of Health

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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.

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