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 /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 or the H2 histocompati i ity 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 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 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 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)

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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). 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. Downloaded by guest on September 26, 2021 Cell Biology: Bourguignon and Singer Proc. Natl. Acad. Sci. USA 74 (1977) 5033 phocytes always resulted in the formation of intracellular ac- cap? 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 uro- membrane 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 sur- 2 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 lym- is 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 as- cap 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 observa- to another peripheral protein that is in turn bound to the inte- tions in two additional systemst indicate that quite generally gral protein, X. It is presumed that membrane-associated my- actin and myosin are associated with the patches prior to cap- osin 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. Thecrosslinkingofa membrane receptor by an external receptor. ligand leads to the spontaneous formation ofa receptor patch, Several mechanisms of capping have been proposed that are in the course ofwhich, linkage ofthe receptor to X occurs. In inconsistent with these observations. For example, de Petris and order that patches and caps contain only those receptor mole- Raff (7) suggested that capping is the result of a "countercur- cules 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 ob- suggested 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, be- facts 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 cyto- the 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 cap- myosin, or both, occur? We propose that it occurs indirectly- ping processes, these differences are in degree rather than in that aggregates of any such receptor, but not the isolated re- kind 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 re- collection 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 gen- span the surface membrane (15). We further suggest that dif- erality 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 ag- The question immediately arises-How can such a mechanism gregated, to bind to X. The binding of the complement com- allow for any receptor to be capped, so that in every case only ponent Clq to aggregates of several different classes of im- molecules of that receptor and'no other are collected into the munoglobulins, but not to their monomeric or subunit forms Downloaded by guest on September 26, 2021 5034 Cell Biology: Bourguignon and Singer Proc. Natl. Acad. Sci. USA 74 (1977) (16), may be roughly analogous to this proposed interaction for our present purposes, need not be further dwelt upon. 11 between X and aggregates of receptor molecules. These three elements together provide an outline of a general We have no basis for speculation about the precise stage of mechanism of capping that can rationalize many of the known receptor aggregation at which binding to X occurs nor about facts concerning the phenomenon. Space limitations prevent the stoichiometry of receptor-X binding. The patches and an extended treatment here, and a few examples must suffice. subpatches seen in fluorescence microscopy must represent a An important feature of capping phenomena is that different relatively advanced stage of aggregation; from their sizes, each receptors do not cap equally readily. The Ig receptor on mouse patch may contain 103-104 molecules of the receptor. It seems splenic B lymphocytes, for example, is rapidly capped (within likely that binding to X occurs with much smaller receptor 5 min) at 370 with a single ligand, anti-Ig antibody. The H2 aggregates. § receptor, however, is extensively capped only after a second 3. Patches ofany given receptor, linked to actin and myosin ligand, an anti-antibody, is used, and even then capping occurs through X, are collected into a cap by a sliding filament over a period of about 30 min. Such differences in capping ef- mechanism and associated processes. The actin and myosin ficiency have usually been attributed to differences in receptor linked to the patches are presumed to collect the patches into structure in the membrane-e.g., to steric hindrance to the the cap. X-Linked patches may diffuse into proximity in the antibody-induced crosslinking of different receptors. On the plane of the membrane, thereby allowing bipolar myosin other hand, the mechanism of capping we propose suggests molecules to bridge F-actin filaments on adjacent patches. An some additional factors that might affect capping rates and actin-myosin sliding filament mechanism similar to that in- efficiencies. For example, the stoichiometry of receptor binding volved in muscle contraction (17) may then be activated and to X might be different for structurally different receptors; a pull the patches into the cap. In muscle, the sliding filament second antibody might be required to provide the aggregate mechanism is activated by an appropriate increase in the local size necessary for that receptor to bind X effectively. Another Ca2+ concentration around the actomyosin fibrils. The for- source of variability could be the capacity of a particular re- mation of crosslinked aggregates, or their binding to X, may ceptor aggregate to trigger the Ca2+ activation of the actin- therefore be a mechanism to increase the intracellular Ca2+ myosin collection mechanism. concentration in the vicinity of the aggregate. This would have In some cases, capping appears to be associated with cell to occur indirectlyl rather than through a change of the local motility. For example, capping of- the Ig receptor on mouse membrane permeability to external Ca2+. because it is known splenic B lymphocytes is followed by motility of the cell on a that capping does not require Ca2+ in the medium (1, 4). Be- solid substrate, whereas the uncapped cell is nonmotile under cause the sliding-filament mechanism operates by the hydrol- the same conditions (4). It is the uncapped portion of the cell ysis of ATP, this hypothesis would in this manner account for that is rendered motile. It may be that this motility is a result the known energy requirement for the collection of patches into of the sequestration of much of the intracellular myosin into a cap. the subcap during the capping process. There is evidence (22) A two-dimensional type of sliding filament mechanism oc- that, in certain more motile portions (ruffles) of fibroblast cells curring on the cytoplasmic surface of the membrane cannot in monolayer culture, myosin is severely depleted relative to alone account for the formation of a subcap. Electron micro- actin. Myosin redistribution by the capping process may scopic studies of the Ig receptor caps on lymphocytes (18) and therefore promote ruffling and motility of the myosin-depleted of Con A-capped ovarian granulosa cells (19), for example, have regions of the lymphocyte cytoplasm. shown that there is a massive accumulation of many layers of The proposed mechanism also provides a direct connection intracellular filaments under the caps. These accumulations between capping and the endocytosis of the receptors that often probably are equivalent to the subcaps that we have observed accompanies and follows capping. The linkage of actin and by fluorescence microscopy which, from their sizes, must extend myosin with the patches and caps provides the contractile a considerable distance (ca 100-500 nm) into the cytoplasm machinery required to invaginate and pinch off regions of the from the surface of the capped region of the membrane. It capped membrane. Other mechanisms of capping do not therefore appears that, in the course of the capping process, connect the two phenomena. large amounts of actin, myosin, and perhaps other mechano- The hypotheses we have advanced are subject to a number chemical proteins are recruited from the cytoplasm to associate of experimental-tests. The more direct of these involve a search with the actin and myosin originally attached to the patches. for the putative integral protein X. It may be possible to disso- The recruited actin and myosin probably also participate in the ciate the integral proteins of lymphocyte or other cell plasma collection of the patches into the cap, in the process forming the membranes under conditions that do not disrupt the association subcap. The details of these events may be complicated and, of X with actin and to recover X along with actin on some suitable anti-actin antibody or heavy meromyosin affinity § This raises the interesting possibility that the visible patches (Figs. column. Furthermore, X would be expected to be present, al- 1 C, D, G, and H and 2 C and D) form as a result of an active col- though perhaps at relatively small mol fractions, in caps formed lection of smaller, invisible aggregates to which X had become linked. by different ligand-receptor combinations; a method of dis- This is contrary to the current view of the patching process, which, solving membranes without disrupting the caps might allow because it occurs in the presence of NaN3 and other energy inhibitors, of X in the fractionated In another is presumed to be driven solely and spontaneously by the ligand- the detection caps. direction, induced crosslinking of receptors. On the contrary, however, an active cell variants or mutants that showed a defect in the ability to collection of small aggregates into visible patches might occur by an actin-myosin sliding filament mechanism (see next section) using 1I No explicit role for is invoked in the proposed mecha- the small amount of ATP present in NaNs-treated cells. This col- nism of capping. When microtubules are intact, they inhibit the lection would then stop at the visible patch stage when the ATP was capping induced by large concentrations of Con A but not that in- exhausted but would continue to the cap stage when the NaN3 was duced by any other ligand (20). Also, very large concentrations of removed and the normal ATP concentration was recovered. cytochalasin B and colchicine combined inhibit cap formation (21) ¶ Such indirect effects could include the local release of Ca2+ from but the significance of this is not clear. We know of no unambiguous binding sites on the cytoplasmic face of the plasma membrane or evidence that implicates microtubules directly and generally in the from nearby Ca2+ -sequestering vesicles (4). generation of caps. Downloaded by guest on September 26, 2021 Cell Biology: Bourguignon and Singer Proc. Natl. Acad. Sci. USA 74(1977) 5035 cap all of various different receptors might in some cases be 8. Bretscher, M. S. (1976) Nature 260,21-23. depleted of X in their plasma membranes (and hence be de- 9. Pollard, T. D. & Weihing, R. R. (1974) C.R.C. Crit. Rev. Biochem. pleted of membrane-associated actin). 2, 1-65. After these studies were largely completed, a brief report was 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, published (23) on the patching and capping of Ig receptors on 4575-4579. mouse splenic B cells that showed the accumulation of myosin 12. Weber, K. & Groeschel-Steward, U. (1974) Proc. Natl. Acad. Sci. in subpatches and subcaps. No other ligand-receptor system USA 71, 4561-4564. was studied, however. 13. Lazarides, E. & Weber, K. (1974) Proc. Natl. Acad. Sci. USA 71, We are grateful to Mr. Michael H. Heggeness for providing the 2268-2272. biotinated heavy meromyosin used for the actin staining procedure. 14. Wang, K., Ash, J. F. & Singer, S. J. (1975) Proc. Natl. Acad. Sci. These studies were supported by U.S. Public Health Service Grants USA 72,4483-4486. AI-06659 and GM-15971. S.J.S. is an American Cancer Society Re- 15. Vitetta, E. S. & Uhr, J. W. (1975) Biochim. Biophys. Acta 415, search Professor. 253-271. 16. Muiller-Eberhard, H. J. (1972) Harvey Lect. 66, 75-104. 1. Taylor, R. B., Duffus, P. H., Raff, M. C. & de Petris, S. (1971) 17. Huxley, H. E. (1976) in Cell Motility, eds. Goldman, R., Pollard, Nature New Biol. 233, 225-229. T. & Rosenbaum, J. (Cold Spring Harbor Laboratory, Cold Spring 2. Loor, F., Forni, L. & Pernis, B. (1972) Eur. J. Immunol. 2, Harbor, NY), Vol. A., pp. 115-126. 203-212. 18. de Petris, S. & Raff, M. C. (1973) Locomotion Tissue Cells, CIBA 3. Unanue, E. R., Perkins, W. D. & Karnovsky, M. J. (1972) J. Exp. Found. Symp. No. 14, pp. 27-41. Med. 136, 885-906. 19. Albertini, D. F. & Anderson, E. (1977) J. Cell Biol. 73, 111- 4. Schreiner, G. F. & Unanue, E. R. (1976) Adv. Immunol. 24, 127. 37-165. 20. Edelman, G. M., Yahara, L. & Wang, J. L. (1973) Proc. Natl. Acad. 5. Julius, M. H., Simpson, E. & Herzenberg, L. A. (1973) Eur. J. Sci. USA 70, 1442-1446. Immunol. 3, 645-664. 21. de Petris, S. (1975) J. Cell Biol. 65, 123-146. 6. Heggeness, M. H. & Ash, J. F. (1977) J. Cell Biol. 73, 783- 22. Heggeness, M. H., Wang, K. & Singer, S. J. (1977) Proc. Natl. 788. Acad. Sci. USA, 74, 3883-3887. 7. de Petris, S. & Raff, M. C. (1972) Eur. J. Immunol. 2, 523- 23. Schreiner, G. F., Fujiwara, K., Pollard, T. D. & Unanue, E. R. 535. (1977) J. Exp. Med. 145, 1393-1398. Downloaded by guest on September 26, 2021