Department of Cell Biology
Actins; Binding, Competitive; Cell Membrane; Chromatography, Affinity; Cytoskeleton; Dictyostelium; Electrophoresis, Polyacrylamide Gel; Membrane Proteins; Microscopy, Electron; Molecular Weight
Cell Biology | Life Sciences | Medicine and Health Sciences
In novel, low-speed sedimentation assays, highly purified, sonicated Dictyostelium discoideum plasma membrane fragments bind to F-actin beads (fluorescein-labeled F-actin on antifluorescein IgG-Sephacryl S-1000 beads). Binding was found to be (a) specific, since beads containing bound fluorescein-labeled ovalbumin or beads without bound fluorescein-labeled protein do not bind membranes, (b) saturable at approximately 0.6 microgram of membrane protein per microgram of bead-bound F-actin, (c) rapid with a t1/2 of 4-20 min, and (d) apparently of reasonable affinity since the off rate is too slow to be measured by present techniques. Using low-speed sedimentation assays, we found that sonicated plasma membrane fragments, after extraction with chaotropes, still bind F-actin beads. Heat-denatured membranes, proteolyzed membranes, and D. discoideum lipid vesicles did not bind F-actin beads. These results indicate that integral membrane proteins are responsible for the binding between sonicated membrane fragments and F-actin on beads. This finding agrees with the previous observation that integral proteins mediate interactions between D. discoideum plasma membranes and F-actin in solution (Luna, E.J., V. M. Fowler, J. Swanson, D. Branton, and D. L. Taylor, 1981, J. Cell Biol., 88:396-409). We conclude that low-speed sedimentation assays using F-actin beads are a reliable method for monitoring the associations between F-actin and membranes. Since these assays are relatively quantitative and require only micrograms of membranes and F-actin, they are a significant improvement over other existing techniques for exploring the biochemical details of F-actin-membrane interactions. Using F-actin beads as an affinity column for actin-binding proteins, we show that at least 12 integral polypeptides in D. discoideum plasma membranes bind to F-actin directly or indirectly. At least four of these polypeptides appear to span the membrane and are thus candidates for direct transmembrane links between the cytoskeleton and the cell surface.
DOI of Published Version
J Cell Biol. 1984 Jul;99(1 Pt 1):58-70. Link to article on publisher's website
The Journal of cell biology
Luna, Elizabeth J.; Goodloe-Holland, C. M.; and Ingalls, H. M., "A membrane cytoskeleton from Dictyostelium discoideum. II. Integral proteins mediate the binding of plasma membranes to F-actin affinity beads" (1984). Women’s Health Research Faculty Publications. 285.