Analysis of lateral redistribution of a plasma membrane glycoprotein- monoclonal antibody complex [corrected] [published erratum appears in J Cell Biol 1988 Apr;106(4):following 1403]

Abstract

Abstract. The lateral redistribution of a major murine glycoprotein, GP80, was studied on locomoting fibroblasts, using rhodamine-conjugated mAbs and ultralow light level digitized fluorescence microscopy. Confirming an earlier study (Jacobson, K., D. O'Dell, B. Holifield, T. L. Murphy, and J. T. August. 1984. J. Cell Biol. 99:1613-1623, the distribution of GP80 was coupled with cell locomotion; motile cells exhibited a gradated distribution of the GP80-mAb complex over the cell surface, increasing from the front to the rear, whereas stationary cells exhibited a nearly uniform GP80 distribution. By monitoring locomoting single cells, we found the gradated fluorescence distribution to be maintained as an approximate steady state. Newly extended leading edges were almost devoid of the fluorescence labeling. This was strikingly demonstrated in prechilled cells in which the extension of fluorescence-free leading edges caused a pronounced boundary between fluorescent and nonfluorescent zones. Subsequently this boundary eroded gradually in a manner consistent with diffusional relaxation. Evidence indicated that the GP80 redistribution was primarily caused by the lateral motion of GP80 in the plasma membrane and not via intracellular membrane traffic. Two cell locomotion models which, in principle, could account for the GP80 redistribution were tested: the retrograde lipid flow (RLF) model (Bretscher, M. S., 1984. Science (Wash. DC).224:681-686) and an alternative hypothesis, the retraction-induced spreading (RIS) model. The predictions of these models were simulated by computer and compared with experiment to assess which model was more appropriate. Whereas both models predicted steady-state gradients similar to the experimental result, only the RIS model predicted the lack of retrograde movement of the fluorescent boundary.

New physical concepts for cell amoeboid motion

“…This aspect ofmotility alone is complicated and not understood at present. Analysis of experimental evidence ( 19) indicates that passive diffusion superposed on forward flow ofthe lipid bilayer can provide sufficient transport. The subsequent reattachment ofreceptors to the actin network remains a mystery.…”

Direct observation of brownian motion of lipids in a membrane.

“…Computer simulations of twodimensional Brownian motion were produced based on microscopic theory of diffusion (12,13). The simulated random walk ofa particle consisted ofa series ofjumps with eachjump consisting of 100 steps.…”

Comparative behavior of membrane protein-antibody complexes on motile fibroblasts: implications for a mechanism of capping.

“…The selective cappingresults also suggest that the retraction induced spreading model to explain the capping of Pgp-1-mAb complex (Ishihara et al, 1988) is not correct. In this model, the low diffusion coefficient of membrane proteins, such as Pgp-1, is explained by transient anchorage of the proteins to immobilized peripheral structures, i.e., membraneassociated cytoskeleton or extracellular matrix.…”

“…When tagged with their respective primary antibodies, both Pgp-1 and HA showed similar diffusion coefficients, the values of which are typical of many plasma membrane proteins tagged with antibodies (reviewed in Peters, 1981;Jacobson et al,, 1987). According to the retrograde lipid flow hypothesis, both HA and Pgp-1 immune complexes should have been capped, since proteins with low diffusion coefficients of '~, 3 × 10 -l° cm2/s would not be able to overcome the proposed rearward lipid flow by diffusion (Bretscher, 1981(Bretscher, , 1984Ishihara et al, 1988). Thus the failure of rh-anfi-HA to redistribute into a gradient, whereas Pgp-1-mAb complexes do form a gradieat, cannot be explained by this mechanism.…”