Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin.
Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell's actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.
D000199:Actins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000648:ultrastructure; D003208:Concanavalin A / Q000378:metabolism; D004023:Dictyostelium / Q000276:immunology / Q000648:ultrastructure*; D007152:Immunologic Capping; D009218:Myosins / Q000302:isolation & purification* / Q000378:metabolism; D011952:Receptors, Concanavalin A / Q000302:isolation & purification* / Q000378:metabolism; D011955:Receptors, Drug / Q000302:isolation & purification*
D000199:Actins; D011952:Receptors, Concanavalin A; D011955:Receptors, Drug; D003208:Concanavalin A; D009218:Myosins