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
MATERIALS AND METHODS Mouse splenic T lymphocytes were obtained from C57BL/6J mice and were prepared by passing the spleen cells over a nylon wool column as described (5). HeLa cells grown in Eagle's minimal essential suspension medium supplemented with 10% fetal calf serum at 370 were obtained from M. Goulian. Mouse antisera to the H2b haplotype were the gift of Robert Hyman, and rabbit antisera to the antigen T-25 (otherwise known as Thy-i or 0) were generously provided by Ian Trowbridge. The whole sera were used as primary reagents. Goat antibodies to rabbit IgG and to mouse IgG-were affinity-purified for use as the secondary reagents and were conjugated with fluorescein isothiocyanate by standard procedures. The methods used to double stain a surface receptor together with either actin or myosin inside the cell will be described in detail elsewhere.J In outline, the procedure was as follows. Cells were first treated in suspension to fluorescent-label particular surface receptors, by using either: fluorescein-conjugated concanavalin A (F-Con A) in the case of HeLa cells; or mouse anti-H2b antibodies followed by fluorescein-conjugated goat antibodies to mouse immunoglobulins or rabbit anti-T-25 antibodies followed by fluorescein-conjugated goat antibodies to rabbit immunoglobulins in the case of T cells. Incubation of these reagents with the cells was carried out under conditions specified in the figure legends, in either the presence or absence of 10 mM NaN3. After such surface labeling, the cells were lightly fixed with formaldehyde, infused with 1.2 M sucrose, frozen, and sectioned in the frozen state to a thickness of about 1 gm. The thawed sections were then stained either for actin, by using a rhodamine fluorescence method based on heavy meromyosin binding (6), or for myosin, by using a rhodamine indirect immunofluorescence procedure that did not interfere with the antibodies used for surface labeling. The stained sections were then examined in a Zeiss photomicroscope with a X63 oil-immersion lens and an epi-illuminator, with appropriate filter combinations. Photography was on Kodak Plus X film.
The mechanism of capping of cell surface reABSTRACT ceptors has been examined by a double fluorescence staining procedure that permitted simultaneous observations of the distribution of a surface-bound ligand together with intracellular actin or myosi At an early stage in the capping of the T-25 antigen or the H2 histocompati i ity antigens on mouse splenic T lymphocytes, or of concanavalin A receptors on HeLa cells, when the specific receptors in question were collected into patches that were distributed over the entire cell surface, the intracellular membrane-associated actin or myosin was also accumulated into patches that were located directly under the receptor patches. These and other results have led us to propose a general molecular mechanism for the process of capping, in which actin and myosin are directly involved. It is suggested that membrane-associated actin is directly or indirectly bound to an integral protein or class of proteins, X, in the plasma membranes of eukaryotic cells. When any receptor in the membrane is agrated by an external multivalent ligand, the aggregate binds effectively to X, whereas unaggregated receptors do not bind to XX The receptor aggregates, linked to actin (and myosin) through X, are then actively collected into a cap by an analogue of the actin-myosin sliding filament mechanism o muscle contraction. When any of a number of multivalent ligands (such as antibodies or lectins) are bound to their specific receptors on the surfaces of various cells, there often occurs, at 370, a remarkable succession of changes in the membrane. After a rapid initial clustering of the bound receptors into small patches (a process that is an apparently spontaneous crosslinking in the fluid membrane and is energy-independent), the small patches are collected into a few large patches or a single "cap" on the cell surface in a process that requires energy. During and after the process of capping, the bound receptors are internalized by endocytosis of the capped regions of the membrane. These phenomena have been well recognized with lymphocytes for some time (1-3), but the molecular mechanisms involved are not yet understood (4). We have developed methods for the simultaneous fluorescence staining of a surface-bound ligand and one of several intracellular mechanochemical proteins on sections of lymphocytes and other cells in suspension. With these methods, wet have found that, with mouse splenic T and B lymphocytes and mouse fibroblasts in suspension, the capping produced by several different lectins and specific antibody r intrareagents in every case resulted in the concent. cellular myosin and actin immediately under the ca. the experiments reported in this paper, we have examined by the same techniques the earlier stages in the capping process in several systems. From these and other results, an outline of a general molecular mechanism for capping and related phenomena is developed.
RESULTS The efficient capping of the T-25 antigen and the H2 antigen on lymphocytes requires a second antibody (1). The fluorescent caps have been found to be associated with accumulations of actin and myosin immediately under the caps* (not shown). If NaN3 was present (1-3), the antibody-induced redistributions Abbreviations: Con A, concanavalin A; F-Con A, fluorescein-conjugated Con A. Present address: Department of Biology, Wayne State University, Detroit, MI 48202. t To whom reprint requests should be addressed. t L. Y. W. Bourguignon, K. T. Tokuyasu, and S. J. Singer, unpublished data.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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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 over the entire cell surface (Fig. 1 C and G), and at this stage both actin (Fig. 1D) and myosin (Fig. 1H) were present in a corresponding patchy distribution. If NaN3 was absent, the same results as shown in Fig. 1 C, D, C, and H could be obtained by a shorter incubation with the second antibody than was required to produce capping. Experiments were carried out on the capping of HeLa cells with F-Con A. In the unperturbed cell, the intracellular actin was clearly present in two states, one membrane-associated and the other in the interior cytoplasm (Fig. 2B). For the present purposes, it is the distribution of membrane-associated actin (and myosin) that is of primary concern. In the presence of 10 mM NaN3, F-Con A induced a patching (Fig. 2C) of its originally uniform distribution of surface receptors (Fig. 2A). In the process, the membrane-associated actin was converted from its originally uniform distribution on the cytoplasmic face of
the membrane (Fig. 2B) into a patchy distribution (Fig. 2D) that corresponded precisely to the patches of Con A. Similar patching of the membrane-associated myosin was also observed (not shown). If the NaN3 was washed out before the cells were fixed and further incubation was carried out at 250, the Con A receptors became capped (Fig. 2 E and G), and corresponding concentrations (subcaps) of membrane-associated myosin (Fig. 2F) and actin (Fig. 2H) were found. Our results do not permit a quantitative analysis of the effect of capping on that part of the actin and myosin that was originally cytoplasmic, except that it is clear from Fig. 2H that a substantial amount of the intracellular actin remained in the cytoplasm after Con .A caps were formed. DISCUSSION In other workt it was found that the capping of several different 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). The remaining cells were then incubated at 250 for 30 min in the presence of 10 mM NaN:3 (C and D). A portion of these cells was then washed free of NaN.3 and incubated in phosphate-buffered saline containing 0.2% bovine serum albumin at 25° for 20 min to achieve capping (E-H). The differently treated cells were then fixed, frozen, sectioned, and stained for either actin or myosin. A and B, C and D, E and F, and G and H, respectively, are of the same cell. (X1000.) (A) Initial uniform surface labeling of Con A receptors; (B) initial cytoplasmic distribution of actin. (C) Patchy redistribution of Con A receptors; (D) redistributed actin in the same cell. (E) Capped Con A receptors; (F) redistributed myosin in the same cell. (G) Capped Con A receptors; (H) redistributed actin in the same cell. rhat portion of the actin that is membrane-associated is relatively concentrated under the Con A cap compared to the uncapped regions of the membrane.
Cell Biology: Bourguignon and Singer
Proc. Natl. Acad. Sci. USA 74 (1977)
5033
phocytes always resulted in the formation of intracellular accap? In what follows, we outline a coherent general mechanism cumulations beneath the caps (subcaps) containing both actin of capping that accounts for these and 6ther observations. This and myosin. A trivial explanation for these subcaps is that they mechanism consists of the following elements. merely reflect the displacement of much of the cytoplasm of 1. Actin is a peripheral protein that is attached to the the small lymphocytes into the region under.the cap (the uromembrane by direct or indirect linkage to a specific integral pod). In the present study, the formation of similar subcaps protein (or proteins), X. Some fraction of the actin inside under Con A-induced caps on the much larger HeLa cells (Fig. nonmuscle cells is thought to be -bound to the cytoplasmic sur2 E-H) proves that this result cannot be due simply to mass face of the plasma membrane (9). This is evident in the actin cytoplasmic displacement. Another question about the subcaps distributions seen in resting HeLa cells (Fig. 2B) and T lymis whether they represent a secondary accumulation of actin phocytes (Fig. 1B). The nature of this actin-membrane linkage and myosin (and perhaps other proteins) after the process of is not known. Soluble proteins such as actin are very likely ascap formation or reflect a more direct association of actin and sociated with membranes as peripheral proteins (10), attached myosin with the capping process. In this paper we have shown, directly or indirectly to other proteins that are integral to the with three different combinations of ligand, receptor, and cell, membrane. We therefore propose that there exists some integral that at an early stage in the capping process, when the receptors protein, or class of proteins, X, in the plasma membranes of all in question were collected into small patches over the entire cell eukaryotic cells, that protrudes from the cytoplasmic face of surface, accumulations of actin and myosin beneath the patches the membrane to provide the specific attachment site for (subpatches) were already present (Fig. 1 C, D, G, and H and membrane-bound actin. Alternatively, actin might be attached Fig. 2 C and D). These results, together with similar observato another peripheral protein that is in turn bound to the intetions in two additional systemst indicate that quite generally gral protein, X. It is presumed that membrane-associated myactin and myosin are associated with the patches prior to caposin and perhaps other mechanochemical proteins are attached ping, and therefore these mechanochemical proteins most likely to actin. participate directly in the capping of each individual surface 2. The crosslinking of a membrane receptor by an external receptor. ligand leads to the spontaneous formation of a receptor patch, Several mechanisms of capping have been proposed that are in the course of which, linkage of the receptor to X occurs. In inconsistent with these observations. For example, de Petris and order that patches and caps contain only those receptor moleRaff (7) suggested that capping is the result of a "countercurcules that are specifically crosslinked by the ligand, and for rent" process in which patches of crosslinked receptors are capping to be directly mediated by actin and myosin, individual collected passively into the trailing edge of a cell when the fluid isolated receptor molecules in the membrane must generally membrane moves forward around the patches. This mechanism not be linked to actin or myosin. Only after a particular receptor was considered because-capping of Ig receptors on splenic B is specifically crosslinked into a suitable-sized aggregate must lymphocytes seemed to be associated with cell movement and that receptor become linked to actin or myosin, while all other occurred over the uropod that formed as the rest of the cell receptor molecules in the membrane remain unlinked. moved forward. Another proposed mechanism for capping (8) Direct evidence conforming to this proposal has been obsuggested that there is a continuous directed lipid flow in tained in our laboratory (ref. 11; J. F. Ash, D. Louvard, and S. membranes that drags patches of receptors along into a cap. J. Singer, unpublished data). Fibroblasts in monolayer culture Neither the countercurrent nor the lipid flow mechanism, have their intracellular actin and myosin organized largely into however, can account for the appearance of subpatches and extended fiber bundles, the so-called stress fibers (12-14). On subcaps containing myosin and actin associated with the patches the surfaces of these cells, we have found that receptors are and caps, respectively. initially freely mobile but, when any one kind of receptor is Schreiner and Unanue (4) proposed that the capping of Ig crosslinked into an aggregate by its specific lectin or antibody receptors on B lymphocytes by anti-Ig antibodies is mediated ligand, the aggregates become attached to the actin-myosin directly by mechanochemical proteins, but the capping of Con stress fibers located immediately under the membrane and are A receptors by Con A may occur by a different mechanism such thereby immobilized. We suggest that, in lymphocytes and as the countercurrent one (7). This suggestion was based on the other cells in suspension, a similar attachment occurs but, befacts that the capping of Ig and Con A receptors showed certain cause the actin and myosin are not organized into stress fibers, different characteristics, the former being insensitive to cytothe receptor aggregates that become linked to actin or myosin chalasin B and occurring about 10 times more rapidly than the remain mobile in the plane of the membrane, in contrast to the latter. We have foundt however, that both Ig and Con A caps case with the fibroblasts. were associated with actin- and myosin-containing subcaps. Our How does this linkage of receptor aggregates to actin or conclusion is that, although differences exist in different capmyosin, or both, occur? We propose that it occurs indirectlyping processes, these differences are in degree rather than in that aggregates of any such receptor, but not the isolated rekind and all of them occur by a basically similar mechanism. ceptor itself, can bind effectively to the integral protein(s) X We propose that this general mechanism involves the active in the plane of the membrane. It should be noted that, for this collection of receptor patches into caps, with the actin and mechanism to operate, a receptor molecule does not necessarily myosin components associated with the patches performing the have to span the membrane, which would be the case if i recollection process by an analogue of the actin-myosin sliding ceptor aggregate were required to bind directly to actin or filament mechanism of muscle contraction. This i$ similar to myosin. This would accommodate the fact that the Ig receptor the mechanism that Schreiner and Unanue (4) suggested for on B lymphocytes can be readily capped yet probably does not the special case of Ig capping on B lymphocytes, but the genspan the surface membrane (15). We further suggest that diferality of the mechanism to all capping phenomena carries ferent receptors may have some as yet unsuspected structural important new implications about the initiation of the process. features in common, so as to allow any one of them, when agThe question immediately arises-How can such a mechanism gregated, to bind to X. The binding of the complement comallow for any receptor to be capped, so that in every case only ponent Clq to aggregates of several different classes of immolecules of that receptor and' no other are collected into the munoglobulins, but not to their monomeric or subunit forms
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Cell Biology: Bourguignon and Singer
(16), may be roughly analogous to this proposed interaction between X and aggregates of receptor molecules. We have no basis for speculation about the precise stage of receptor aggregation at which binding to X occurs nor about the stoichiometry of receptor-X binding. The patches and subpatches seen in fluorescence microscopy must represent a relatively advanced stage of aggregation; from their sizes, each patch may contain 103-104 molecules of the receptor. It seems likely that binding to X occurs with much smaller receptor aggregates. § 3. Patches of any given receptor, linked to actin and myosin through X, are collected into a cap by a sliding filament mechanism and associated processes. The actin and myosin linked to the patches are presumed to collect the patches into the cap. X-Linked patches may diffuse into proximity in the plane of the membrane, thereby allowing bipolar myosin molecules to bridge F-actin filaments on adjacent patches. An actin-myosin sliding filament mechanism similar to that involved in muscle contraction (17) may then be activated and pull the patches into the cap. In muscle, the sliding filament mechanism is activated by an appropriate increase in the local Ca2+ concentration around the actomyosin fibrils. The formation of crosslinked aggregates, or their binding to X, may therefore be a mechanism to increase the intracellular Ca2+ concentration in the vicinity of the aggregate. This would have to occur indirectlyl rather than through a change of the local membrane permeability to external Ca2+. because it is known that capping does not require Ca2+ in the medium (1, 4). Because the sliding-filament mechanism operates by the hydrolysis of ATP, this hypothesis would in this manner account for the known energy requirement for the collection of patches into a cap. A two-dimensional type of sliding filament mechanism occurring on the cytoplasmic surface of the membrane cannot alone account for the formation of a subcap. Electron microscopic studies of the Ig receptor caps on lymphocytes (18) and of Con A-capped ovarian granulosa cells (19), for example, have shown that there is a massive accumulation of many layers of intracellular filaments under the caps. These accumulations probably are equivalent to the subcaps that we have observed by fluorescence microscopy which, from their sizes, must extend a considerable distance (ca 100-500 nm) into the cytoplasm from the surface of the capped region of the membrane. It therefore appears that, in the course of the capping process, large amounts of actin, myosin, and perhaps other mechanochemical proteins are recruited from the cytoplasm to associate with the actin and myosin originally attached to the patches. The recruited actin and myosin probably also participate in the collection of the patches into the cap, in the process forming the subcap. The details of these events may be complicated and, § This raises the interesting possibility that the visible patches (Figs.
1 C, D, G, and H and 2 C and D) form as a result of an active collection of smaller, invisible aggregates to which X had become linked. This is contrary to the current view of the patching process, which, because it occurs in the presence of NaN3 and other energy inhibitors, is presumed to be driven solely and spontaneously by the ligandinduced crosslinking of receptors. On the contrary, however, an active collection of small aggregates into visible patches might occur by an actin-myosin sliding filament mechanism (see next section) using the small amount of ATP present in NaNs-treated cells. This collection would then stop at the visible patch stage when the ATP was exhausted but would continue to the cap stage when the NaN3 was removed and the normal ATP concentration was recovered. ¶ Such indirect effects could include the local release of Ca2+ from binding sites on the cytoplasmic face of the plasma membrane or from nearby Ca2+ -sequestering vesicles (4).
Proc. Natl. Acad. Sci. USA 74 (1977)
for our present purposes, need not be further dwelt upon. 1 These three elements together provide an outline of a general mechanism of capping that can rationalize many of the known facts concerning the phenomenon. Space limitations prevent an extended treatment here, and a few examples must suffice. An important feature of capping phenomena is that different receptors do not cap equally readily. The Ig receptor on mouse splenic B lymphocytes, for example, is rapidly capped (within 5 min) at 370 with a single ligand, anti-Ig antibody. The H2 receptor, however, is extensively capped only after a second ligand, an anti-antibody, is used, and even then capping occurs over a period of about 30 min. Such differences in capping efficiency have usually been attributed to differences in receptor structure in the membrane-e.g., to steric hindrance to the antibody-induced crosslinking of different receptors. On the other hand, the mechanism of capping we propose suggests some additional factors that might affect capping rates and efficiencies. For example, the stoichiometry of receptor binding to X might be different for structurally different receptors; a second antibody might be required to provide the aggregate size necessary for that receptor to bind X effectively. Another source of variability could be the capacity of a particular receptor aggregate to trigger the Ca2+ activation of the actinmyosin collection mechanism. In some cases, capping appears to be associated with cell motility. For example, capping of- the Ig receptor on mouse splenic B lymphocytes is followed by motility of the cell on a solid substrate, whereas the uncapped cell is nonmotile under the same conditions (4). It is the uncapped portion of the cell that is rendered motile. It may be that this motility is a result of the sequestration of much of the intracellular myosin into the subcap during the capping process. There is evidence (22) that, in certain more motile portions (ruffles) of fibroblast cells in monolayer culture, myosin is severely depleted relative to actin. Myosin redistribution by the capping process may therefore promote ruffling and motility of the myosin-depleted regions of the lymphocyte cytoplasm. The proposed mechanism also provides a direct connection between capping and the endocytosis of the receptors that often accompanies and follows capping. The linkage of actin and myosin with the patches and caps provides the contractile machinery required to invaginate and pinch off regions of the capped membrane. Other mechanisms of capping do not connect the two phenomena. The hypotheses we have advanced are subject to a number of experimental-tests. The more direct of these involve a search for the putative integral protein X. It may be possible to dissociate the integral proteins of lymphocyte or other cell plasma membranes under conditions that do not disrupt the association of X with actin and to recover X along with actin on some suitable anti-actin antibody or heavy meromyosin affinity column. Furthermore, X would be expected to be present, although perhaps at relatively small mol fractions, in caps formed by different ligand-receptor combinations; a method of dissolving membranes without disrupting the caps might allow the detection of X in the fractionated caps. In another direction, cell variants or mutants that showed a defect in the ability to 1I No explicit role for microtubules is invoked in the proposed mechanism of capping. When microtubules are intact, they inhibit the capping induced by large concentrations of Con A but not that induced by any other ligand (20). Also, very large concentrations of cytochalasin B and colchicine combined inhibit cap formation (21) but the significance of this is not clear. We know of no unambiguous evidence that implicates microtubules directly and generally in the generation of caps.
Cell Biology: Bourguignon and Singer cap all of various different receptors might in some cases be depleted of X in their plasma membranes (and hence be depleted of membrane-associated actin). After these studies were largely completed, a brief report was published (23) on the patching and capping of Ig receptors on mouse splenic B cells that showed the accumulation of myosin in subpatches and subcaps. No other ligand-receptor system was studied, however. We are grateful to Mr. Michael H. Heggeness for providing the biotinated heavy meromyosin used for the actin staining procedure. These studies were supported by U.S. Public Health Service Grants AI-06659 and GM-15971. S.J.S. is an American Cancer Society Research Professor. 1. Taylor, R. B., Duffus, P. H., Raff, M. C. & de Petris, S. (1971) Nature New Biol. 233, 225-229. 2. Loor, F., Forni, L. & Pernis, B. (1972) Eur. J. Immunol. 2, 203-212. 3. Unanue, E. R., Perkins, W. D. & Karnovsky, M. J. (1972) J. Exp. Med. 136, 885-906. 4. Schreiner, G. F. & Unanue, E. R. (1976) Adv. Immunol. 24, 37-165. 5. Julius, M. H., Simpson, E. & Herzenberg, L. A. (1973) Eur. J. Immunol. 3, 645-664. 6. Heggeness, M. H. & Ash, J. F. (1977) J. Cell Biol. 73, 783788. 7. de Petris, S. & Raff, M. C. (1972) Eur. J. Immunol. 2, 523535.
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8. Bretscher, M. S. (1976) Nature 260,21-23. 9. Pollard, T. D. & Weihing, R. R. (1974) C.R.C. Crit. Rev. Biochem. 2, 1-65. 10. Singer, S. J. (1974) Annu. rev. Biochem. 43, 806-833. 11. Ash, J. F. & Singer, S. J. (1976) Proc. Natl. Acad. Sci. USA 73, 4575-4579. 12. Weber, K. & Groeschel-Steward, U. (1974) Proc. Natl. Acad. Sci. USA 71, 4561-4564. 13. Lazarides, E. & Weber, K. (1974) Proc. Natl. Acad. Sci. USA 71, 2268-2272. 14. Wang, K., Ash, J. F. & Singer, S. J. (1975) Proc. Natl. Acad. Sci. USA 72,4483-4486. 15. Vitetta, E. S. & Uhr, J. W. (1975) Biochim. Biophys. Acta 415, 253-271. 16. Muiller-Eberhard, H. J. (1972) Harvey Lect. 66, 75-104. 17. Huxley, H. E. (1976) in Cell Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. A., pp. 115-126. 18. de Petris, S. & Raff, M. C. (1973) Locomotion Tissue Cells, CIBA Found. Symp. No. 14, pp. 27-41. 19. Albertini, D. F. & Anderson, E. (1977) J. Cell Biol. 73, 111127. 20. Edelman, G. M., Yahara, L. & Wang, J. L. (1973) Proc. Natl. Acad. Sci. USA 70, 1442-1446. 21. de Petris, S. (1975) J. Cell Biol. 65, 123-146. 22. Heggeness, M. H., Wang, K. & Singer, S. J. (1977) Proc. Natl. Acad. Sci. USA, 74, 3883-3887. 23. Schreiner, G. F., Fujiwara, K., Pollard, T. D. & Unanue, E. R. (1977) J. Exp. Med. 145, 1393-1398.
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
MATERIALS AND METHODS Mouse splenic T lymphocytes were obtained from C57BL/6J mice and were prepared by passing the spleen cells over a nylon wool column as described (5). HeLa cells grown in Eagle's minimal essential suspension medium supplemented with 10% fetal calf serum at 370 were obtained from M. Goulian. Mouse antisera to the H2b haplotype were the gift of Robert Hyman, and rabbit antisera to the antigen T-25 (otherwise known as Thy-i or 0) were generously provided by Ian Trowbridge. The whole sera were used as primary reagents. Goat antibodies to rabbit IgG and to mouse IgG-were affinity-purified for use as the secondary reagents and were conjugated with fluorescein isothiocyanate by standard procedures. The methods used to double stain a surface receptor together with either actin or myosin inside the cell will be described in detail elsewhere.J In outline, the procedure was as follows. Cells were first treated in suspension to fluorescent-label particular surface receptors, by using either: fluorescein-conjugated concanavalin A (F-Con A) in the case of HeLa cells; or mouse anti-H2b antibodies followed by fluorescein-conjugated goat antibodies to mouse immunoglobulins or rabbit anti-T-25 antibodies followed by fluorescein-conjugated goat antibodies to rabbit immunoglobulins in the case of T cells. Incubation of these reagents with the cells was carried out under conditions specified in the figure legends, in either the presence or absence of 10 mM NaN3. After such surface labeling, the cells were lightly fixed with formaldehyde, infused with 1.2 M sucrose, frozen, and sectioned in the frozen state to a thickness of about 1 gm. The thawed sections were then stained either for actin, by using a rhodamine fluorescence method based on heavy meromyosin binding (6), or for myosin, by using a rhodamine indirect immunofluorescence procedure that did not interfere with the antibodies used for surface labeling. The stained sections were then examined in a Zeiss photomicroscope with a X63 oil-immersion lens and an epi-illuminator, with appropriate filter combinations. Photography was on Kodak Plus X film.
The mechanism of capping of cell surface reABSTRACT ceptors has been examined by a double fluorescence staining procedure that permitted simultaneous observations of the distribution of a surface-bound ligand together with intracellular actin or myosi At an early stage in the capping of the T-25 antigen or the H2 histocompati i ity antigens on mouse splenic T lymphocytes, or of concanavalin A receptors on HeLa cells, when the specific receptors in question were collected into patches that were distributed over the entire cell surface, the intracellular membrane-associated actin or myosin was also accumulated into patches that were located directly under the receptor patches. These and other results have led us to propose a general molecular mechanism for the process of capping, in which actin and myosin are directly involved. It is suggested that membrane-associated actin is directly or indirectly bound to an integral protein or class of proteins, X, in the plasma membranes of eukaryotic cells. When any receptor in the membrane is agrated by an external multivalent ligand, the aggregate binds effectively to X, whereas unaggregated receptors do not bind to XX The receptor aggregates, linked to actin (and myosin) through X, are then actively collected into a cap by an analogue of the actin-myosin sliding filament mechanism o muscle contraction. When any of a number of multivalent ligands (such as antibodies or lectins) are bound to their specific receptors on the surfaces of various cells, there often occurs, at 370, a remarkable succession of changes in the membrane. After a rapid initial clustering of the bound receptors into small patches (a process that is an apparently spontaneous crosslinking in the fluid membrane and is energy-independent), the small patches are collected into a few large patches or a single "cap" on the cell surface in a process that requires energy. During and after the process of capping, the bound receptors are internalized by endocytosis of the capped regions of the membrane. These phenomena have been well recognized with lymphocytes for some time (1-3), but the molecular mechanisms involved are not yet understood (4). We have developed methods for the simultaneous fluorescence staining of a surface-bound ligand and one of several intracellular mechanochemical proteins on sections of lymphocytes and other cells in suspension. With these methods, wet have found that, with mouse splenic T and B lymphocytes and mouse fibroblasts in suspension, the capping produced by several different lectins and specific antibody r intrareagents in every case resulted in the concent. cellular myosin and actin immediately under the ca. the experiments reported in this paper, we have examined by the same techniques the earlier stages in the capping process in several systems. From these and other results, an outline of a general molecular mechanism for capping and related phenomena is developed.
RESULTS The efficient capping of the T-25 antigen and the H2 antigen on lymphocytes requires a second antibody (1). The fluorescent caps have been found to be associated with accumulations of actin and myosin immediately under the caps* (not shown). If NaN3 was present (1-3), the antibody-induced redistributions Abbreviations: Con A, concanavalin A; F-Con A, fluorescein-conjugated Con A. Present address: Department of Biology, Wayne State University, Detroit, MI 48202. t To whom reprint requests should be addressed. t L. Y. W. Bourguignon, K. T. Tokuyasu, and S. J. Singer, unpublished data.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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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 over the entire cell surface (Fig. 1 C and G), and at this stage both actin (Fig. 1D) and myosin (Fig. 1H) were present in a corresponding patchy distribution. If NaN3 was absent, the same results as shown in Fig. 1 C, D, C, and H could be obtained by a shorter incubation with the second antibody than was required to produce capping. Experiments were carried out on the capping of HeLa cells with F-Con A. In the unperturbed cell, the intracellular actin was clearly present in two states, one membrane-associated and the other in the interior cytoplasm (Fig. 2B). For the present purposes, it is the distribution of membrane-associated actin (and myosin) that is of primary concern. In the presence of 10 mM NaN3, F-Con A induced a patching (Fig. 2C) of its originally uniform distribution of surface receptors (Fig. 2A). In the process, the membrane-associated actin was converted from its originally uniform distribution on the cytoplasmic face of
the membrane (Fig. 2B) into a patchy distribution (Fig. 2D) that corresponded precisely to the patches of Con A. Similar patching of the membrane-associated myosin was also observed (not shown). If the NaN3 was washed out before the cells were fixed and further incubation was carried out at 250, the Con A receptors became capped (Fig. 2 E and G), and corresponding concentrations (subcaps) of membrane-associated myosin (Fig. 2F) and actin (Fig. 2H) were found. Our results do not permit a quantitative analysis of the effect of capping on that part of the actin and myosin that was originally cytoplasmic, except that it is clear from Fig. 2H that a substantial amount of the intracellular actin remained in the cytoplasm after Con .A caps were formed. DISCUSSION In other workt it was found that the capping of several different 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). The remaining cells were then incubated at 250 for 30 min in the presence of 10 mM NaN:3 (C and D). A portion of these cells was then washed free of NaN.3 and incubated in phosphate-buffered saline containing 0.2% bovine serum albumin at 25° for 20 min to achieve capping (E-H). The differently treated cells were then fixed, frozen, sectioned, and stained for either actin or myosin. A and B, C and D, E and F, and G and H, respectively, are of the same cell. (X1000.) (A) Initial uniform surface labeling of Con A receptors; (B) initial cytoplasmic distribution of actin. (C) Patchy redistribution of Con A receptors; (D) redistributed actin in the same cell. (E) Capped Con A receptors; (F) redistributed myosin in the same cell. (G) Capped Con A receptors; (H) redistributed actin in the same cell. rhat portion of the actin that is membrane-associated is relatively concentrated under the Con A cap compared to the uncapped regions of the membrane.
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phocytes always resulted in the formation of intracellular accap? In what follows, we outline a coherent general mechanism cumulations beneath the caps (subcaps) containing both actin of capping that accounts for these and 6ther observations. This and myosin. A trivial explanation for these subcaps is that they mechanism consists of the following elements. merely reflect the displacement of much of the cytoplasm of 1. Actin is a peripheral protein that is attached to the the small lymphocytes into the region under.the cap (the uromembrane by direct or indirect linkage to a specific integral pod). In the present study, the formation of similar subcaps protein (or proteins), X. Some fraction of the actin inside under Con A-induced caps on the much larger HeLa cells (Fig. nonmuscle cells is thought to be -bound to the cytoplasmic sur2 E-H) proves that this result cannot be due simply to mass face of the plasma membrane (9). This is evident in the actin cytoplasmic displacement. Another question about the subcaps distributions seen in resting HeLa cells (Fig. 2B) and T lymis whether they represent a secondary accumulation of actin phocytes (Fig. 1B). The nature of this actin-membrane linkage and myosin (and perhaps other proteins) after the process of is not known. Soluble proteins such as actin are very likely ascap formation or reflect a more direct association of actin and sociated with membranes as peripheral proteins (10), attached myosin with the capping process. In this paper we have shown, directly or indirectly to other proteins that are integral to the with three different combinations of ligand, receptor, and cell, membrane. We therefore propose that there exists some integral that at an early stage in the capping process, when the receptors protein, or class of proteins, X, in the plasma membranes of all in question were collected into small patches over the entire cell eukaryotic cells, that protrudes from the cytoplasmic face of surface, accumulations of actin and myosin beneath the patches the membrane to provide the specific attachment site for (subpatches) were already present (Fig. 1 C, D, G, and H and membrane-bound actin. Alternatively, actin might be attached Fig. 2 C and D). These results, together with similar observato another peripheral protein that is in turn bound to the intetions in two additional systemst indicate that quite generally gral protein, X. It is presumed that membrane-associated myactin and myosin are associated with the patches prior to caposin and perhaps other mechanochemical proteins are attached ping, and therefore these mechanochemical proteins most likely to actin. participate directly in the capping of each individual surface 2. The crosslinking of a membrane receptor by an external receptor. ligand leads to the spontaneous formation of a receptor patch, Several mechanisms of capping have been proposed that are in the course of which, linkage of the receptor to X occurs. In inconsistent with these observations. For example, de Petris and order that patches and caps contain only those receptor moleRaff (7) suggested that capping is the result of a "countercurcules that are specifically crosslinked by the ligand, and for rent" process in which patches of crosslinked receptors are capping to be directly mediated by actin and myosin, individual collected passively into the trailing edge of a cell when the fluid isolated receptor molecules in the membrane must generally membrane moves forward around the patches. This mechanism not be linked to actin or myosin. Only after a particular receptor was considered because-capping of Ig receptors on splenic B is specifically crosslinked into a suitable-sized aggregate must lymphocytes seemed to be associated with cell movement and that receptor become linked to actin or myosin, while all other occurred over the uropod that formed as the rest of the cell receptor molecules in the membrane remain unlinked. moved forward. Another proposed mechanism for capping (8) Direct evidence conforming to this proposal has been obsuggested that there is a continuous directed lipid flow in tained in our laboratory (ref. 11; J. F. Ash, D. Louvard, and S. membranes that drags patches of receptors along into a cap. J. Singer, unpublished data). Fibroblasts in monolayer culture Neither the countercurrent nor the lipid flow mechanism, have their intracellular actin and myosin organized largely into however, can account for the appearance of subpatches and extended fiber bundles, the so-called stress fibers (12-14). On subcaps containing myosin and actin associated with the patches the surfaces of these cells, we have found that receptors are and caps, respectively. initially freely mobile but, when any one kind of receptor is Schreiner and Unanue (4) proposed that the capping of Ig crosslinked into an aggregate by its specific lectin or antibody receptors on B lymphocytes by anti-Ig antibodies is mediated ligand, the aggregates become attached to the actin-myosin directly by mechanochemical proteins, but the capping of Con stress fibers located immediately under the membrane and are A receptors by Con A may occur by a different mechanism such thereby immobilized. We suggest that, in lymphocytes and as the countercurrent one (7). This suggestion was based on the other cells in suspension, a similar attachment occurs but, befacts that the capping of Ig and Con A receptors showed certain cause the actin and myosin are not organized into stress fibers, different characteristics, the former being insensitive to cytothe receptor aggregates that become linked to actin or myosin chalasin B and occurring about 10 times more rapidly than the remain mobile in the plane of the membrane, in contrast to the latter. We have foundt however, that both Ig and Con A caps case with the fibroblasts. were associated with actin- and myosin-containing subcaps. Our How does this linkage of receptor aggregates to actin or conclusion is that, although differences exist in different capmyosin, or both, occur? We propose that it occurs indirectlyping processes, these differences are in degree rather than in that aggregates of any such receptor, but not the isolated rekind and all of them occur by a basically similar mechanism. ceptor itself, can bind effectively to the integral protein(s) X We propose that this general mechanism involves the active in the plane of the membrane. It should be noted that, for this collection of receptor patches into caps, with the actin and mechanism to operate, a receptor molecule does not necessarily myosin components associated with the patches performing the have to span the membrane, which would be the case if i recollection process by an analogue of the actin-myosin sliding ceptor aggregate were required to bind directly to actin or filament mechanism of muscle contraction. This i$ similar to myosin. This would accommodate the fact that the Ig receptor the mechanism that Schreiner and Unanue (4) suggested for on B lymphocytes can be readily capped yet probably does not the special case of Ig capping on B lymphocytes, but the genspan the surface membrane (15). We further suggest that diferality of the mechanism to all capping phenomena carries ferent receptors may have some as yet unsuspected structural important new implications about the initiation of the process. features in common, so as to allow any one of them, when agThe question immediately arises-How can such a mechanism gregated, to bind to X. The binding of the complement comallow for any receptor to be capped, so that in every case only ponent Clq to aggregates of several different classes of immolecules of that receptor and' no other are collected into the munoglobulins, but not to their monomeric or subunit forms
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(16), may be roughly analogous to this proposed interaction between X and aggregates of receptor molecules. We have no basis for speculation about the precise stage of receptor aggregation at which binding to X occurs nor about the stoichiometry of receptor-X binding. The patches and subpatches seen in fluorescence microscopy must represent a relatively advanced stage of aggregation; from their sizes, each patch may contain 103-104 molecules of the receptor. It seems likely that binding to X occurs with much smaller receptor aggregates. § 3. Patches of any given receptor, linked to actin and myosin through X, are collected into a cap by a sliding filament mechanism and associated processes. The actin and myosin linked to the patches are presumed to collect the patches into the cap. X-Linked patches may diffuse into proximity in the plane of the membrane, thereby allowing bipolar myosin molecules to bridge F-actin filaments on adjacent patches. An actin-myosin sliding filament mechanism similar to that involved in muscle contraction (17) may then be activated and pull the patches into the cap. In muscle, the sliding filament mechanism is activated by an appropriate increase in the local Ca2+ concentration around the actomyosin fibrils. The formation of crosslinked aggregates, or their binding to X, may therefore be a mechanism to increase the intracellular Ca2+ concentration in the vicinity of the aggregate. This would have to occur indirectlyl rather than through a change of the local membrane permeability to external Ca2+. because it is known that capping does not require Ca2+ in the medium (1, 4). Because the sliding-filament mechanism operates by the hydrolysis of ATP, this hypothesis would in this manner account for the known energy requirement for the collection of patches into a cap. A two-dimensional type of sliding filament mechanism occurring on the cytoplasmic surface of the membrane cannot alone account for the formation of a subcap. Electron microscopic studies of the Ig receptor caps on lymphocytes (18) and of Con A-capped ovarian granulosa cells (19), for example, have shown that there is a massive accumulation of many layers of intracellular filaments under the caps. These accumulations probably are equivalent to the subcaps that we have observed by fluorescence microscopy which, from their sizes, must extend a considerable distance (ca 100-500 nm) into the cytoplasm from the surface of the capped region of the membrane. It therefore appears that, in the course of the capping process, large amounts of actin, myosin, and perhaps other mechanochemical proteins are recruited from the cytoplasm to associate with the actin and myosin originally attached to the patches. The recruited actin and myosin probably also participate in the collection of the patches into the cap, in the process forming the subcap. The details of these events may be complicated and, § This raises the interesting possibility that the visible patches (Figs.
1 C, D, G, and H and 2 C and D) form as a result of an active collection of smaller, invisible aggregates to which X had become linked. This is contrary to the current view of the patching process, which, because it occurs in the presence of NaN3 and other energy inhibitors, is presumed to be driven solely and spontaneously by the ligandinduced crosslinking of receptors. On the contrary, however, an active collection of small aggregates into visible patches might occur by an actin-myosin sliding filament mechanism (see next section) using the small amount of ATP present in NaNs-treated cells. This collection would then stop at the visible patch stage when the ATP was exhausted but would continue to the cap stage when the NaN3 was removed and the normal ATP concentration was recovered. ¶ Such indirect effects could include the local release of Ca2+ from binding sites on the cytoplasmic face of the plasma membrane or from nearby Ca2+ -sequestering vesicles (4).
Proc. Natl. Acad. Sci. USA 74 (1977)
for our present purposes, need not be further dwelt upon. 1 These three elements together provide an outline of a general mechanism of capping that can rationalize many of the known facts concerning the phenomenon. Space limitations prevent an extended treatment here, and a few examples must suffice. An important feature of capping phenomena is that different receptors do not cap equally readily. The Ig receptor on mouse splenic B lymphocytes, for example, is rapidly capped (within 5 min) at 370 with a single ligand, anti-Ig antibody. The H2 receptor, however, is extensively capped only after a second ligand, an anti-antibody, is used, and even then capping occurs over a period of about 30 min. Such differences in capping efficiency have usually been attributed to differences in receptor structure in the membrane-e.g., to steric hindrance to the antibody-induced crosslinking of different receptors. On the other hand, the mechanism of capping we propose suggests some additional factors that might affect capping rates and efficiencies. For example, the stoichiometry of receptor binding to X might be different for structurally different receptors; a second antibody might be required to provide the aggregate size necessary for that receptor to bind X effectively. Another source of variability could be the capacity of a particular receptor aggregate to trigger the Ca2+ activation of the actinmyosin collection mechanism. In some cases, capping appears to be associated with cell motility. For example, capping of- the Ig receptor on mouse splenic B lymphocytes is followed by motility of the cell on a solid substrate, whereas the uncapped cell is nonmotile under the same conditions (4). It is the uncapped portion of the cell that is rendered motile. It may be that this motility is a result of the sequestration of much of the intracellular myosin into the subcap during the capping process. There is evidence (22) that, in certain more motile portions (ruffles) of fibroblast cells in monolayer culture, myosin is severely depleted relative to actin. Myosin redistribution by the capping process may therefore promote ruffling and motility of the myosin-depleted regions of the lymphocyte cytoplasm. The proposed mechanism also provides a direct connection between capping and the endocytosis of the receptors that often accompanies and follows capping. The linkage of actin and myosin with the patches and caps provides the contractile machinery required to invaginate and pinch off regions of the capped membrane. Other mechanisms of capping do not connect the two phenomena. The hypotheses we have advanced are subject to a number of experimental-tests. The more direct of these involve a search for the putative integral protein X. It may be possible to dissociate the integral proteins of lymphocyte or other cell plasma membranes under conditions that do not disrupt the association of X with actin and to recover X along with actin on some suitable anti-actin antibody or heavy meromyosin affinity column. Furthermore, X would be expected to be present, although perhaps at relatively small mol fractions, in caps formed by different ligand-receptor combinations; a method of dissolving membranes without disrupting the caps might allow the detection of X in the fractionated caps. In another direction, cell variants or mutants that showed a defect in the ability to 1I No explicit role for microtubules is invoked in the proposed mechanism of capping. When microtubules are intact, they inhibit the capping induced by large concentrations of Con A but not that induced by any other ligand (20). Also, very large concentrations of cytochalasin B and colchicine combined inhibit cap formation (21) but the significance of this is not clear. We know of no unambiguous evidence that implicates microtubules directly and generally in the generation of caps.
Cell Biology: Bourguignon and Singer cap all of various different receptors might in some cases be depleted of X in their plasma membranes (and hence be depleted of membrane-associated actin). After these studies were largely completed, a brief report was published (23) on the patching and capping of Ig receptors on mouse splenic B cells that showed the accumulation of myosin in subpatches and subcaps. No other ligand-receptor system was studied, however. We are grateful to Mr. Michael H. Heggeness for providing the biotinated heavy meromyosin used for the actin staining procedure. These studies were supported by U.S. Public Health Service Grants AI-06659 and GM-15971. S.J.S. is an American Cancer Society Research Professor. 1. Taylor, R. B., Duffus, P. H., Raff, M. C. & de Petris, S. (1971) Nature New Biol. 233, 225-229. 2. Loor, F., Forni, L. & Pernis, B. (1972) Eur. J. Immunol. 2, 203-212. 3. Unanue, E. R., Perkins, W. D. & Karnovsky, M. J. (1972) J. Exp. Med. 136, 885-906. 4. Schreiner, G. F. & Unanue, E. R. (1976) Adv. Immunol. 24, 37-165. 5. Julius, M. H., Simpson, E. & Herzenberg, L. A. (1973) Eur. J. Immunol. 3, 645-664. 6. Heggeness, M. H. & Ash, J. F. (1977) J. Cell Biol. 73, 783788. 7. de Petris, S. & Raff, M. C. (1972) Eur. J. Immunol. 2, 523535.
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8. Bretscher, M. S. (1976) Nature 260,21-23. 9. Pollard, T. D. & Weihing, R. R. (1974) C.R.C. Crit. Rev. Biochem. 2, 1-65. 10. Singer, S. J. (1974) Annu. rev. Biochem. 43, 806-833. 11. Ash, J. F. & Singer, S. J. (1976) Proc. Natl. Acad. Sci. USA 73, 4575-4579. 12. Weber, K. & Groeschel-Steward, U. (1974) Proc. Natl. Acad. Sci. USA 71, 4561-4564. 13. Lazarides, E. & Weber, K. (1974) Proc. Natl. Acad. Sci. USA 71, 2268-2272. 14. Wang, K., Ash, J. F. & Singer, S. J. (1975) Proc. Natl. Acad. Sci. USA 72,4483-4486. 15. Vitetta, E. S. & Uhr, J. W. (1975) Biochim. Biophys. Acta 415, 253-271. 16. Muiller-Eberhard, H. J. (1972) Harvey Lect. 66, 75-104. 17. Huxley, H. E. (1976) in Cell Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. A., pp. 115-126. 18. de Petris, S. & Raff, M. C. (1973) Locomotion Tissue Cells, CIBA Found. Symp. No. 14, pp. 27-41. 19. Albertini, D. F. & Anderson, E. (1977) J. Cell Biol. 73, 111127. 20. Edelman, G. M., Yahara, L. & Wang, J. L. (1973) Proc. Natl. Acad. Sci. USA 70, 1442-1446. 21. de Petris, S. (1975) J. Cell Biol. 65, 123-146. 22. Heggeness, M. H., Wang, K. & Singer, S. J. (1977) Proc. Natl. Acad. Sci. USA, 74, 3883-3887. 23. Schreiner, G. F., Fujiwara, K., Pollard, T. D. & Unanue, E. R. (1977) J. Exp. Med. 145, 1393-1398.
