Reversibility of cell surface label rearrangement.

Abstract

Cell surface labeling can cause rearrangements of randomly distributed membrane components. Removal of the label bound to the cell surface allows the membrane components to return to their original random distribution, demonstrating that label is necessary to maintain as well as to induce rearrangements. With scanning electron microscopy, the rearrangement of concanavalin A (con A) and ricin binding sites on LA-9 cells has been followed by means of hemocyanin, a visual label. The removal of con A from its binding sites at the cell surface with alpha-methyl mannoside, and the return of these sites to their original distribution are also followed in this manner.There are labeling differences with con A and ricin. Under some conditions, however, the same rearrangements are seen with both lectins. The disappearance of labeled sites from areas of ruffling activity is a major feature of the rearrangements seen. Both this ruffling activity and the rearrangement of label are sensitive to cytochalasin B, and ruffling activity, perhaps along with other cytochalasin-sensitire structure, may play a role in the rearrangements of labeled sites.Evidence from a number of laboratories indicates that the application of label to cell surfaces causes a rearrangement of randomly distributed membrane molecules (12,15,20,30,35,43,51,53). Rearrangement is not a general membrane phenomenon, but involves only the labeled sites; surface molecules which do not interact with the label remain in a random, homogeneous distribution (29). It is not known how the interaction between label and receptor causes rearrangement.Two components can be recognized during label-induced rearrangement (12,35,53,55): the formation of clusters of label; and the preferential relocation of label to certain areas on the cell and away from others, e.g., capping. It has been proposed that the first component, clustering, results from crosslinking of surface sites by multivalent label molecules, since univalent antibody fragments appear uniformly distributed on the cell surface (16,35,53). Stackpole et al. (50), however, have demonstrated that clustering can occur even with strictly univalent reagents. They suggest that interaction of label with its binding sites may cause an alteration which thermodynamically favors aggregation. The second component of rearrangement must involve more than crosslinking or aggregation of labeled molecules in the plane of the membrane; it requires energy (12,15,20,35,53,57) and must therefore be directed by some activity of the cell.In this communication we follow the rearrangements at the cell surface obtained with two different lectins and show that the label-receptor interaction is necessary for the maintenance as well as the induction of rearrangements. Mechanisms consistent with our findings whereby label-receptor THE JOURNAL OF CELL BIOLOGY 9 VOLUME 68, 1976 9 pages 629-641 629

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

“…Again lanes were washed sequentially in incubation buffer and BSA (2 x 1 h each). Incubation with l"I-protein A, prepared by the chloramine-T procedure (12), approximately 5 x 106 cpm in 5 ml of BSA, proceeded for a period of 6 to 18 h. Lanes were washed as described above, dried on filter paper, and exposed to Kodak No-Screen X-ray film for 1 to 6 days.…”

“…Some progress has been made by labeling membrane components. The specificity of lectin binding, for example, has allowed investigators to localize glycoprotein in cell membranes and to study mechanisms of lateral mobility of membrane components (6,34). Immunological approaches have been successful in identifying receptor sites on membranes (29), but studies with antisera to intact membranes have failed to produce much useful information concerning molecular mechanisms of membrane-associated phenomena.…”

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

“…WGA-microspheres, however, are maintained in a dense uniform array on the cell surface. Brown and Revel [6] have also reported that when cultured mouse L cells were continuously labeled with ricin-hemocyanin markers, a homogeneous pattern of label was observed; cells continuously labeled with Con A-hemocyanin displayed a heterogeneous labeling pattern. This difference in the pattern of redistribution of Con A sites and WGA or RCA…”

“…Con A possesses mitogenic responses toward some classes of lymphocytes [34] and alters the morphology and differentiation pattern o f 3 number of cell types. In particular, it has been reported that Con A reverses differentiation in neuroblastoma cells [35], causes a rounding up and clustering of microvilli on Dictyostelium cells [5] , and inhibits ruffling activity on L cells [6] . WGA and RCA.…”

“…Light and electron microscope studies on a variety of cell types indicate that many membrane components which normally are displayed randomly over the cell surface redistribute t o form small clusters or patches when labeled with multivalent ligands such as plant lectins or antibodies [1][2][3][4][5][6][7] . These clusters of labeled components can subsequently rearrange in an energy-dependent process t o form a cap on the surface of the cell or they can become internalized by endocytosis.…”