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Visualisation of the actin cytoskeleton by cryo-electron microscopy

¹ Institute of Molecular Biology, Department Cell Biology, Billrothstrasse 11, A-5020 Salzburg, Austria
² European Molecular Biology Laboratory, Structure Programme, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

View larger version (88K): [in a new window]   Fig. 1. Time lapse series of lamellipodia crossing the holes of a perforated film. (A) GFP-actin-transfected B16F1 melanoma cell; the holes in the lamellipodium region in this sequence are 1-2 µm in diameter. (B,C) GFP-VASP-expressing B16 cells. The central 2 µm (B) and 4 µm (C) holes are indicated by arrowheads; the 4 µm hole causes a temporary delay in local protrusion. Each sequence covers approximately 10 minutes. Bar, 10 µm.

View larger version (175K): [in a new window]   Fig. 2. Images of the actin cytoskeleton in frozen hydrated samples; the cytoskeletons were produced by 1 minute extraction in the Triton/GA mixture. (A) A filopodium protruding at the cell's edge; in the top right corner, another thick filopodium is seen. (B) A lamellipodium showing a relatively sparse network, together with filaments arranged parallel to the edge, probably corresponding to a non-protruding region (see text). (C) Sparse actin networks found in regions deeper inside the lamella; the globular structures of approximately 20x30 nm seen frequently are interpreted as ribosomes. (D) Another lamella region showing F-actin, microtubules and ribosomes. Bar, 200 nm.

View larger version (203K): [in a new window]   Fig. 3. Lamellipodia regions in mouse melanoma cells, extracted either with the Triton/glutaraldehyde mixture (1 minute; A,C) or with Triton/PEG (B). The grainy aggregates seen in (B) are typical of this extraction procedure. In C, the very front of the lamellipodium is 0.2 µm from the top edge, in the direction indicated by the arrowhead. Bar, 200 nm (A,B); 200 nm (C).

View larger version (64K): [in a new window]   Fig. 4. Correlation of light microscope and cryo-EM images. (A) Phalloidin-actin stain merged with a filtered phase contrast image of the holey film. (B,C) Details from the labelled holes in (A), showing actin filaments, microtubules and ribosomes. Bar, 20 µm (A); 200 nm (B,C).