Junctional plasma membrane domains isolated from aggregating Dictyostelium discoideum amebae.

Regions of plasma membrane involved in Dictyostelium discoideum intercellular adhesion resist solubilization with the nonionic detergent Triton X-100. Electron microscopy shows that these regions of the plasma membrane adhere to each other, forming many bi- and multilamellar structures. NaDodSO4/polyacrylamide gels of these regions contain major polypeptides at 225 kDa (residual myosin), 105 kDa, 88 kDa, 84 kDa, 47 kDa (residual actin), and 34 kDa. These membranes contain a subset of the total plasma membrane proteins, as analyzed by labeling of electrophoretically fractionated and blotted membrane proteins with radioiodinated Con A and by electrophoresis of membrane proteins from surface-labeled cells. Antibodies specific for gp80, a glycoprotein implicated in intercellular adhesion, intensely stain the 88-kDa and 84-kDa bands. Since these membrane regions resist Triton extraction, they appear to be stabilized by protein-protein interactions. Such stabilizing interactions may involve multivalent linkages with adjacent cells, or associations with intracellular actin and myosin, or both. Since these membranes appear to represent regions of intercellular contact, we call them "contact regions."

A membrane cytoskeleton from Dictyostelium discoideum. II. Integral proteins mediate the binding of plasma membranes to F-actin affinity beads.

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.

A membrane cytoskeleton from Dictyostelium discoideum. III. Plasma membrane fragments bind predominantly to the sides of actin filaments.

The binding between sonicated Dictyostelium discoideum plasma membrane fragments and F-actin on Sephacryl S-1000 beads was found to be competitively inhibited by myosin subfragment-1. This inhibition is MgATP-sensitive, exhibits a Ki of approximately 5 X 10(-8) M, and is reciprocal, since membranes inhibit the binding of 125I-heavy meromyosin to F-actin on beads. These experiments demonstrate that membrane binding and S-1 binding to F-actin on beads are mutually exclusive and, therefore, that the membrane fragments bind predominantly to the sides, rather than to the ends, of the actin filaments. This conclusion is supported by electron micrographs that show many lateral associations between membrane fragments and bead-associated actin filaments. Such lateral associations could play an important role in the organization and lateral movement of membrane proteins by the cytomusculature.