Visualization of actin fibers associated with the cell membrane in amoebae of Dictyostelium discoideum.

Abstract

Amoebae of Dictyostelium discoideum were attached to a surface coated with polylysine, and the upper portion of the cells was sheared off with a stream of buffer. Scanning and transmission electron microscopy showed that the cytoplasmic surface of the exposed membrane was covered with fibers consisting of actin-containing filaments. The actin was identified by its solubility properties and its ability to interact with muscle myosin.In preceding work (1-3) proteins from amoebae of Dictyostelium discoideum that closely resemble the muscle proteins actin and myosin were l)urified and characterized, and it was shown biochemically that some of the Dictyostelium actin is associated with the cell membrane (2). In the present study, membrane-associated actin filaments were visualized by electron microscopy, utilizing a new technique in which cells were attached to surfaces coated with polylysine, and then disrupted to expose the cytoplasmic face of the cell membrane. The use of polylysine as an adhesive for electron microscopy was recently described by Mazia and coworkers (4); protamille sulfate was similarly employed by Vacquier (5) in his examination of the inner membrane surfaces in sea urchin eggs. MATERIALS AND METHODSBuffers and Reagents. The phosphate buffer (Buffer P) used to resuspend D. discoideurn cells contained 20 mM potassium phosphate, pH 6.4, 20 mM KCl, 5 mM MgCl2, and 0.5 mg/ml of streptomycin sulfate (6). The buffer used for cell disrultion (Buffer D) contained 10 mM Tris-HCl, pH 7.5, 0.1 M KCl, and 1 mM MgCl2. Poly(Llysine) (molecular weight 80,000-100,000) was obtained from New England Nuclear, Boston.Growth Scanning Electron Microscopy. Clean glass slides were broken into 1 cm2 plates, and were coated with an aqueous solution of polylysine (1 mg/ml). They were rinsed with distilled H20 and then with Buffer P, and the sample was applied. Samples were fixed by immersing the plates in a solution containing 2.5% glutaraldehyde and 1% acrolein made up in Buffer 1), and were post-fixedl in osmium tetroxide. They were then prel)ared for microscopy by freon 13 critical point drying andlplatinum shadowing, and were examined in a Coates & Welter field emission scanning electron microscope.Transmission Electron Microscopy. Grids were coated with a Parlodion film over which a layer of carbon was evaporated. Polylysine (1 mg/ml in H20) was applied to this surface, which was then rinsed with distilled H20 and Buffer P. The cell suspension was applied, and the cells were disrupted as described above, then negatively stained with 1% uranyl acetate. In some cases, the disrupted cells were treated with the myosin subfragment 1 (S5) according to the procedures described by Moore et al. (7) Other Materials. Actin was purified from amoebae of D. discoideum by column chromatography, as previously described (2). The myosin subfragment SI was prepared from rabbit muscle according to the procedure of Cooke (8). RESULTSWhen a Dictyostelium discoideun amoeba adhered to a polylvsine-coated surface, its attachiment was...

Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

“…Adherent fragments of plasma membrane were recognized as cell-sized areas that were not detectable with phase contrast optics (Figure 1 (Boyles and Bainton, 1979;Boyles and Bainton, 1981). This pattern also looks like the actin-based membrane skeletons visualized by similar techniques in erythrocytes, platelets, macrophages, and Dictyostelium amoebae (Clarke et al, 1975;Hartwig et al, 1989;Stout and Axelrod, 1994), indicating that the neutrophil contains a population of actin filaments in close association with the plasma membrane.…”

Actin-like filaments amd membrane rearrangement in oxyntic cells.

“…sarcoma virus (14). Studies carried out on Dictyostelium discoideum have shown that actin filaments become apparent after contact of the ameba with a substratum (15). Thus, two conditions which are known to affect cell surface molecular architecture (16)(17)(18) …”

Motive force of the culminating mass of cells in the developing fruiting body of Dictyostelium discoideum.

“…Since the contractile proteins are existent in the motile D. discoideum cell (Clarke et al 1975, Eckert et al 1977, in cases of both the migrating slug and the developing fruiting body, these motive forces are probably produced by the con stituent cells. During morphogenesis of D. discoideum from the slug to the fruiting body, the total number of cells composing these multicellulars (for the fruiting body, the sum of cells in the culminating mass and the stalk) is generally accepted to remain almost unchanged (the present study also shows that the "mass" and volume of the two are almost the same).…”