Capping of Cholera Toxin-Ganglioside GM1 on Mouse Lymphocytes Is Accompanied by Co-capping of o -Actinin Complexes

We used cholera toxin, which binds exclusively and with a high affinity to the ganglioside GM1, as a probe to investigate the distribution of this glycolipid on the surface of mouse lymphocytes. When lymphocytes are incubated with cholera toxin (or its B subunit) and then sequentially with horse anti-toxin and FITC-swine anti-horse Ig at 37°C, the cholera toxin-ganglioside GM1 complex is redistributed to a cap at one pole of the cell. The capping of cholera toxin-GM1 complexes is slower than the capping of surface-lg complexes, requires two antibodies, and is inhibited at high toxin concentrations. Cholera toxin-GM, like surfaceIg capping, is an energy-dependent process and is inhibited by sodium azide, low temperatures, or cytochalasin B, but is unaffected by demecolcine. An affinity-purified antibody against ~-actinin was used to examine the distribution of this cytoskeletal component during the capping process. 88% of the cells that had a surface Ig cap displayed a co-cap of c~-actinin, and 57% of the cells that had a cholera toxin-GM~ cap displayed a co-cap of c~-actinin. Time course studies revealed similar kinetics of external ligand cap formation and the formation of ~-actinin co-caps. We conclude that capping of a cell-surface glycolipid is associated with a reorganization of the underlying cytoskeleton. The implications of such an association are discussed in the context of current models of the mechanism of capping. Although improvements in analytical procedures have led to the characterization of a large number of new glycosphingolipids in animal cells in recent years, surprisingly little is known about the function and organization of glycosphingolipids in membranes (1). The complexity of membrane glycolipid profiles suggest that they are likely to fulfill other than a purely structural role, and evidence that glycolipids can act as cell surface receptors for Escherichia coli and cholera enterotoxins, tetanus toxin, the glycoprotein hormones, interferon, macrophage migration inhibition factor, fibronectin, and Sendai virus has been presented (reviewed in reference 1), although in some cases this has been disputed (2, 3). The conclusion that the high-affinity binding of cholera toxin to the cell surface is mediated by the sialic acid-containing glycolipid ganglioside GM~ l is supported by a wide variety of experimental approaches, including the isolation of the cholera toxin-ganglioside GMI complex from the surface of Ganglioside nomenclature is according to L. Svennerholm, 1963, £ Neurochem. 10:613-623. several different cell types by immune precipitation (4-6). Because of this, cholera toxin has been widely used as a probe to investigate the levels and organization of ganglioside GM~ in membranes. Interestingly, binding of cholera toxin to lymphocytes leads to the subsequent redistribution of the toxinganglioside GM~ complex to one pole of the cell, the so-called capping phenomenon (7, 8). A similar response can be induced with anti-glycolipid antibodies (9). Capping of the toxin-ganglioside GMt complex is temperature dependent, requires energy, and is inhibited by cytochalasin B (7, 8). The results are analogous to those obtained in experiments on capping of surface Ig in lymphocytes (reviewed in reference 10). Two main models have been proposed to explain the capping of cell surface receptors. Bretscher (11) suggested that there was a directed flow of membrane lipids towards one pole of the cell, and that monomeric proteins could resist this flow by Brownian motion, whereas large cross-linked aggregates would he swept along to one pole of the cell. The alternative hypothesis proposes that the cross-linking of cell THE JOURNAL OF CELL BIOLOGY • VOLUME 97 AUGUST 1983 447-454 © The Rockefeller University Press 0021-9525/83 /08 /0447/08 $1.00 447 on M arch 3, 2016 jcb.rress.org D ow nladed fom Published August 1, 1983