Abstract
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 degrees 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-Ig complexes, requires two antibodies, and is inhibited at high toxin concentrations. Cholera toxin-GM1, like surface-Ig 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 alpha-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 alpha-actinin, and 57% of the cells that had a cholera toxin-GM1 cap displayed a co-cap of alpha-actinin. Time course studies revealed similar kinetics of external ligand cap formation and the formation of alpha-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.
Highlights
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
To establish that this was true for normal mouse lymphocytes, cell proteins were resolved in SDS polyacrylamide gels, and the gels were overlaid with ~25I-labeledcholera toxin
The result is consistent with the conclusion that the cholera toxin receptor in mouse lymphocytes is exclusively glycolipid in nature
Summary
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. The alternative hypothesis proposes that the cross-linking of cell surface receptors triggers a transmembrane linkage of the ligand-receptor complex to the microfilament system, which directs it to one pole of the cell [12] This latter model is supported by experiments that show that capping of a number of surface proteins is associated with co-capping of components of the microfilament system such as actin (1315), myosin [15,16,17], and a-actinin [18,19]. A detailed analysis of ligand-induced redistribution of surface Ig, thymus leukemia antigen, and Fc receptors on mouse lymphocytes showed that the kinetics of the capping process was essentially similar to that of the co-capping of myosin [17] These results appear to provide strong, though circumstantial evidence for a direct involvement of the microfilament system in the capping of membrane proteins. The finding that Thyl capping in mouse lymphocytes induces conversion of G to F actin is consistent with the above interpretation [20]
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