Techniques for the visualisation of cytoskeletal components in Dictyostelium discoideum.
A general description is given of the various techniques that may be used in ultrastructural studies of the cytoskeleton. Electron microscopy of the cytoskeleton of Dictyostelium discoideum serves as a source of examples illustrating the general effects of detergent treatment and fixation techniques. A concise review is given of the structure and function of the actin microfilament system and the cytoplasmic microtubules in Dictyostelium, based on electron microscopical, light microscopical and biochemical studies. Special attention is paid to their involvement in cell movement and chemotaxis. Conventional thin sectioning, fast freezing freeze substitution, whole mounts, freeze fracturing and freeze etching and negative staining techniques are discussed and their respective advantages and limitations are mentioned. A recently developed technique, wet-cleaving, is described which gives promising results in experiments in which the inside of the plasma membrane with the adhering cortical cytoskeleton is studied. This technique may turn out to be useful in high-resolution scanning electron microscopy. A description is given of protocols that proved to be successful in the author's and other laboratories. In a few cases the feasibility of immunogold labelling (illustrated by anti-tubulin labelling of cytoplasmic microtubules) is also dealt with.
De Priester
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Department of Biology, Leiden University, The Netherlands.
D000818:Animals; D003599:Cytoskeleton / Q000648:ultrastructure*; D004023:Dictyostelium / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods; D013048:Specimen Handling