Redistribution of a Cell Movement Major Cell Surface Glycoprotein during

The distribution in living cells of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been studied by use of monoclonal antibodies. The presence of the molecule throughout the plasma membrane and on the substrate attached surface of the cell was demonstrated by immunofluorescence. Cell growth kinetics were not altered and the cells remained motile in the presence of the antibody. The uniform distribution of the direct immunofluorescence stain persisted for long periods (>100 h), which indicates that the fluorescent monoclonal antibodies may be used to trace antigen surface distribution during cell functions. In motile cells, but not Go or confluent cells, the degree of fluorescent staining decreased toward the leading edge; this gradient increased markedly during the time that the antibody was bound to the cells. However, the gradation was not seen with the lipid probe, dihexadecylindocarbocyanine. The antigen was "patched" only by the application of a second antibody directed to the rat monoclonal antibody and the relationships of these patches to the underlying cytoskeleton were characterized. Redistributions of plasma membrane components and movement of the membrane itself are intimately involved in cellular motility (for recent reviews, see references l and 2). Glycoprotein redistributions during motility have been studied using multivalent ligands. For example, surface immunoglobulins have been observed to redistribute in motile B lymphocytes (3) and fluoresceinated concanavalin A bound to locomoting polymorphonuclear leukocytes preferentially accumulated in the uropod as opposed to the lamellapodium (4). Gradients in the distribution of unspecified surface antigens toward the trailing edge of motile fibroblasts were also observed in the photographs of Heath (5; Fig. 1 i). Gradations in the concentrations of polymorphonuclear leukocyte surface proteins were also seen when fluorescein was used as a monovalent ligand (6). A major cell surface protein of mouse embryo 3T3 cells as well as macrophages and epithelial cells of the mouse is a polymorphic glycoprotein of -80 ,000 daltons (7, 8), designated Ly-24 (Pgp-l)L This glycoprotein is the predominant iodinated component of 3T3 cells labeled by lactoperoxidase J Morse, H. C., I. F. C. McKenzie, U. Hammenburg, and F.-W. Shen, personal communication. and constitutes -0.1% of total cell protein, with o v e r 10 6 antigenic sites distributed throughout the surface of the NIH/ 3T3 cell (9). Trypsin treatment of intact cells releases a 65,000dalton fragment (10). The molecule is an integral membrane component and is likely to have the topology ofa transmembrane glycoprotein, with extracellular, hydrophobic, and cytoplasmic domains. The gene-controlling expression of the antigen is on chromosome 2, close to the locus encoding the cell surface markers H-3, Ir-2, ~2 microglobulin, and Ly-23 (11). Trowbridge et al. (12, 13) have independently identified the same glycoprotein of mouse lymphoid tissues. Expression of the antigen is differentiation-related. It is present in much higher concentrations in NIH/3T3 cells, G8-1 myoblasts, and IC-21 macrophages than in many other tissue culture cells. In vivo, the antigen is prominent on macrophages and lymphocytes and strong surface staining is observed on most epithelial cells. 2 The present studies have utilized a monoclonal antibody to describe the cell surface distribution and lateral mobility of this 80,000-dalton glycoprotein. The results provide evidence of its redistribution during cell motility. Such experiments 2 Murphy, T. L., T. W. Bauer, and J. T. August, unpublished data. THE JOURNAL OF CELL BIOLOGY • VOLUME 99 NOVEMBER 1984 1613-1623 © The Rockefeller University Press • 0021-9525/84/11/1613/11 $1.00 1 61 3 on Jne 7, 2017 D ow nladed fom Published November 1, 1984