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An improved unroofing method consisting of tearing off the cell membrane using an adhesive electron microscopy (EM) grid instead of vitreous ice sectioning (cryo-sectioning) has enabled us to panoramically view the membrane cytoskeleton in its native state with extremely high contrast. Grids pre-treated with Alcian blue were placed on cells, and a portion of the dorsal plasma membrane was transferred onto the grid, which was then floated in buffer solution. These membrane fragments contained sufficient cytoskeleton and were of suitable thickness for observation by cryo-EM. Many actin filaments and microtubules were clearly observed on the cytoplasmic surface of the plasma membrane with extremely high contrast because the soluble components of the cytoplasm flowed out and broke away from the cells. Actin filaments extended in all directions in a smooth contour with little branching. Microtubules spread out as far as 3 µm or more while winding gently in their native state. Upon fixation with 1% glutaraldehyde, however, the microtubules became straight and fragmented. Cryo-EM revealed for the first time a smooth endoplasmic reticulum network beneath the cell membrane in native cells. Clathrin coats and caveolae were also observed on the cytoplasmic surface of the plasma membrane, similar to those seen using freeze-etching replica EM (freeze-etching EM). Unroofing was also useful for immuno-labelling in cryo-EM. Antibody-labelled IQGAP1, one of the effector proteins facilitating the formation of actin filament networks, was localized alongside actin filaments. Freeze-etching EM confirmed the morphological findings of cryo-EM.

microscopy

A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy