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Transmembrane Interactions and Themechanism of Capping Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 5031-5035, November 1977 Cell Biology Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands (non-muscle-cell myosin and actin/T lymphocytes/HeLa cells/sliding filament mechanism) LILLY Y. W. BOURGUIGNON* AND S. J. SINGERt Department of Biology, University of California, San Diego, La Jolla, California 92093 Contributed by S. J. Singer, August 19, 1977 ABSTRACT The mechanism of capping of cell surface re- MATERIALS AND METHODS ceptors has been examined by a double fluorescence staining procedure that permitted simultaneous observations of the Mouse splenic T lymphocytes were obtained from C57BL/6J distribution of a surface-bound ligand together with intracel- mice and were prepared by passing the spleen cells over a nylon lular actin or myosi At an early stage in the capping of the T-25 wool column as described (5). HeLa cells grown in Eagle's antigen or the H2 histocompati i ity antigens on mouse splenic minimal essential suspension medium supplemented with 10% T lymphocytes, or of concanavalin A receptors on HeLa cells, fetal calf serum at 370 were obtained from M. Goulian. Mouse when the specific receptors in question were collected into patches that were distributed over the entire cell surface, the antisera to the H2b haplotype were the gift of Robert Hyman, intracellular membrane-associated actin or myosin was also and rabbit antisera to the antigen T-25 (otherwise known as accumulated into patches that were located directly under the Thy-i or 0) were generously provided by Ian Trowbridge. The receptor patches. These and other results have led us to propose whole sera were used as primary reagents. Goat antibodies to a general molecular mechanism for the process of capping, in rabbit IgG and to mouse IgG-were affinity-purified for use as which actin and myosin are directly involved. It is suggested the secondary reagents and were conjugated with fluorescein that membrane-associated actin is directly or indirectly bound to an integral protein or class of proteins, X, in the plasma isothiocyanate by standard procedures. membranes of eukaryotic cells. When any receptor in the The methods used to double stain a surface receptor together membrane is agrated by an external multivalent ligand, the with either actin or myosin inside the cell will be described in aggregate binds effectively to X, whereas unaggregated re- detail elsewhere.J In outline, the procedure was as follows. Cells ceptors do not bind to XX The receptor aggregates, linked to actin were first treated in suspension to fluorescent-label particular (and myosin) through X, are then actively collected into a cap surface receptors, by using either: fluorescein-conjugated by an analogue of the actin-myosin sliding filament mechanism concanavalin A (F-Con A) in the case of HeLa cells; or mouse o muscle contraction. anti-H2b antibodies followed by fluorescein-conjugated goat When any of a number of multivalent ligands (such as anti- antibodies to mouse immunoglobulins or rabbit anti-T-25 an- bodies or lectins) are bound to their specific receptors on the tibodies followed by fluorescein-conjugated goat antibodies to surfaces of various cells, there often occurs, at 370, a remarkable rabbit immunoglobulins in the case of T cells. Incubation of succession of changes in the membrane. After a rapid initial these reagents with the cells was carried out under conditions clustering of the bound receptors into small patches (a process specified in the figure legends, in either the presence or absence that is an apparently spontaneous crosslinking in the fluid of 10 mM NaN3. After such surface labeling, the cells were membrane and is energy-independent), the small patches are lightly fixed with formaldehyde, infused with 1.2 M sucrose, collected into a few large patches or a single "cap" on the cell frozen, and sectioned in the frozen state to a thickness of about surface in a process that requires energy. During and after the 1 gm. The thawed sections were then stained either for actin, process of capping, the bound receptors are internalized by by using a rhodamine fluorescence method based on heavy endocytosis of the capped regions of the membrane. These meromyosin binding (6), or for myosin, by using a rhodamine phenomena have been well recognized with lymphocytes for indirect immunofluorescence procedure that did not interfere some time (1-3), but the molecular mechanisms involved are with the antibodies used for surface labeling. The stained sec- not yet understood (4). We have developed methods for the tions were then examined in a Zeiss photomicroscope with a simultaneous fluorescence staining of a surface-bound ligand X63 oil-immersion lens and an epi-illuminator, with appro- and one of several intracellular mechanochemical proteins on priate filter combinations. Photography was on Kodak Plus X sections of lymphocytes and other cells in suspension. With these film. methods, wet have found that, with mouse splenic T and B lymphocytes and mouse fibroblasts in suspension, the capping RESULTS produced by several different lectins and specific antibody The efficient capping of the T-25 antigen and the H2 antigen reagents in every case resulted in the concent. r intra- on lymphocytes requires a second antibody (1). The fluorescent cellular myosin and actin immediately under the ca. the caps have been found to be associated with accumulations of experiments reported in this paper, we have examined by the and same techniques the earlier stages in the capping process in actin myosin immediately under the caps* (not shown). If several systems. From these and other results, an outline of a NaN3 was present (1-3), the antibody-induced redistributions general molecular mechanism for capping and related phe- Abbreviations: Con A, concanavalin A; F-Con A, fluorescein-conju- nomena is developed. gated Con A. Present address: Department of Biology, Wayne State University, The costs of publication of this article were defrayed in part by the Detroit, MI 48202. payment of page charges. This article must therefore be hereby marked t To whom reprint requests should be addressed. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate t L. Y. W. Bourguignon, K. T. Tokuyasu, and S. J. Singer, unpublished this fact. data. 5031 Downloaded by guest on September 26, 2021 5032 Cell Biology: Bourguignon and Singer Proc. Natl. Acad. Sci. USA 74 (1977) I FIG. 1. Mouse splenic T cells were treated in suspension with either rabbit antisera to the T-25 antigen (A-D) or mouse antisera to the H2 histocompatibility antigens (E-H), followed by fluorescein-conjugated goat antibodies (100 pg/ml) to rabbit IgG (A-D) or to mouse IgG (E-H) at 00 for 30 min in the presence of 10 mM NaN:1. After the antibody binding reaction? were complete, the cells were washed and incubated either at 00 (A, B, E, and F) or at 370 (C, D, G, and H) for 30 min in the presence of NaNI. The cells were then fixed, frozen, sectioned, and stained for either actin or myosin. A and B, C and F, and G and H, respectively, are of the same cell. (X1000.) (A) Initial uniform surface labeling for T-25; (B) initial cytoplasmic distribution of actin. (C) Patchy redistribution of T-25 antigen; (D) redistributed actin in the same cells. (E) Initial uniform surface labeling of H2 antigens; (F) initial cytoplasmic distribution of myosin. (G) Patchy redistribution of H2 antigens; (H) redistributed myosin in the same cells. of receptors stopped at the stage in which patches were formed the membrane (Fig. 2B) into a patchy distribution (Fig. 2D) over the entire cell surface (Fig. 1 C and G), and at this stage that corresponded precisely to the patches of Con A. Similar both actin (Fig. 1D) and myosin (Fig. 1H) were present in a patching of the membrane-associated myosin was also observed corresponding patchy distribution. If NaN3 was absent, the (not shown). If the NaN3 was washed out before the cells were same results as shown in Fig. 1 C, D, C, and H could be ob- fixed and further incubation was carried out at 250, the Con tained by a shorter incubation with the second antibody than A receptors became capped (Fig. 2 E and G), and corre- was required to produce capping. sponding concentrations (subcaps) of membrane-associated Experiments were carried out on the capping of HeLa cells myosin (Fig. 2F) and actin (Fig. 2H) were found. Our results with F-Con A. In the unperturbed cell, the intracellular actin do not permit a quantitative analysis of the effect of capping was clearly present in two states, one membrane-associated and on that part of the actin and myosin that was originally cyto- the other in the interior cytoplasm (Fig. 2B). For the present plasmic, except that it is clear from Fig. 2H that a substantial purposes, it is the distribution of membrane-associated actin amount of the intracellular actin remained in the cytoplasm (and myosin) that is of primary concern. In the presence of 10 after Con .A caps were formed. mM NaN3, F-Con A induced a patching (Fig. 2C) of its origi- nally uniform distribution of surface receptors (Fig. 2A). In the DISCUSSION process, the membrane-associated actin was converted from In other workt it was found that the capping of several different its originally uniform distribution on the cytoplasmic face of receptors in the surface membranes of mouse splenic lym- FIG. 2. HeLa cells were treated in suspension with F-Con A (30 mg/ml) at 00 for 30 min in the presence of 10 mM NaN-. A sample of these ,.ells was examined (A and B).
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