Loss of Intercalated Membrane Particles by Treatment with Phorbols (Intramembranous Particles/Phorbol Esters) DOROTHEA ZUCKER-FRANKLIN and ZEENAT F

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6829-6833, September 1986 Cell Biology Loss of intercalated membrane particles by treatment with phorbols (intramembranous particles/phorbol esters) DOROTHEA ZUCKER-FRANKLIN AND ZEENAT F. NABI Department of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016 Communicated by Michael Heidelberger, May 20, 1986

ABSTRACT Because brief exposure to phorbol esters types before and after incubation with PMA. In every type of renders normal cells vulnerable to deformation and cytolysis by cell with the exception of platelets, PMA caused a remark- lymphocytes, it was postulated that these tumor promoters able reduction in intramembranous particles (IMP) associat- might cause a hitherto unrecognized physical alteration in ed with the external leaflet (E face) of the plasma membrane membrane architecture. To investigate this possibility, four while the size and distribution of IMP of the protoplasmic tissue culture cell lines (K-562 erythroleukemia cells, melano- leaflet (P face) were not affected. This observation is partic- ma cells, N1121 adult fibroblasts, and normal fetal fibroblasts) ularly intriguing because it has been recognized in several and three blood cell types (lymphocytes, monocytes, and laboratories that the partition coefficient of the platelet platelets) were subjected to freeze-fracture analysis before and membrane IMP is the reverse of that in other cells, i.e., the after brief treatment with phorbol myristate acetate. Phorbol platelet plasma membrane appears to be turned "inside out" myristate acetate caused a 50% reduction of intramembranous (9, 10). We mention this because the disparity in the PMA particles associated with the external leaflet (E face) of the response ofplatelet IMP when compared with other cells may plasma membrane ofevery cell except platelets. In contrast, no provide new insights into the biochemical makeup of these change in size or number of intramembranous particles asso- structures and membrane organization in general. ciated with the protoplasmic membrane leaflet (P face) was evident. Since the platelet membrane is known to be turned AND "inside out," as regards the partition coefficient of the intra- MATERIALS METHODS membranous particles, the disparity between the results ob- Cells. Neoplastic cells were (i) K-562 erythroleukemia cells tained with platelets and other cells may serve to determine the (used routinely as a standard for natural killer cell cytolytic nature of intramembranous particles affected by phorbols. activity) grown in suspension culture in RPMI 1640, supple- Also, since phorbols affect primarily glycolipids and/or glyco- mented with 10% (vol/vol) heat-inactivated fetal calf serum; proteins anchored in the external membrane leaflet, these (ii) a human melanoma cell line (Rob) studied extensively by findings may provide a useful tool for future exploration of us and described in detail elsewhere (11). Normal cells were membrane structure. (iii) an adult fibroblast line (N1121), derived from normal human skin (and obtained from American Type Culture Numerous studies have been conducted on the effects of Collection); (iv) a fetal fibroblast line prepared in our labo- tumor-promoting phorbol esters on mammalian cells. The ratory from a 15-week-old human abortus as reported (7, 8). most interesting and far-reaching observations have dealt Freshly prepared blood cells consisted of purified (v) lym- with the finding that these agents cause a protracted activa- phocytes, (vi) monocytes, and (vii) platelets, which were tion of protein kinase C, presumably the basis for manifold obtained from heparinized peripheral blood ofvolunteers and alterations in cell physiology (1-3). Morphologic studies of were isolated by routine procedures. cells treated with phorbols have been less numerous, perhaps Treatment with PMA and its Analogs. PMA and inactive because the observed changes have been largely nonspecific, analogs of PMA, 4a-phorbol 12,13-didecanoate and 4p- i.e., degranulation, vacuolization, clumping, blebbing, en- phorbol (Sigma), were dissolved in 100% ethanol (2 mg/ml). hanced pinocytosis, and lateral redistribution of membrane Dilutions were made with RPMI to a final concentration of glycoproteins (4-6). In our laboratory, phorbol myristate 0.01%. Control cells were incubated in ethanol diluent acetate (PMA) has been employed primarily to render the without PMA. The cells were washed at least twice before minority oftumor cells resistant to lysis by natural killer cells incubation with the phorbols (200 ng/ml) in serum-free RPMI vulnerable to attack (7, 8). Although the low concentrations for 1 hr at 37TC. The cells were washed again prior to fixation and short exposures used did not appear to alter the ultra- in 3% (vol/vol) glutaraldehyde in phosphate buffer, after structure or which they were processed for freeze-fracture or thin section proliferative capacity of the target cells per se, electron microscopy. Target cells consisted either of mela- addition of natural killer cells caused massive conjugation, noma cells or K-562 cells and were incubated with lympho- deformation, and emperipolesis. Moreover, PMA-treated cytes in a ratio of 1:100 for 2 hr or overnight. Cytotoxicity target cells became subject to lysis not only by natural killer assays are not described here because they were not relevant cells, but also by T8 lymphocytes, which as a rule, require to the present study and have been reported (7, 8). prior sensitization, i.e., antibody and complement, to be- Freeze-fracture and Electron Microscopy. Following fixa- come cytotoxic for tumor cells. This suggested that phorbol tion for a minimum of 2 hr, the cells were thoroughly washed esters could have a major effect on the structural integrity of and resuspended in 25% (vol/vol) glycerol for 2 hr at room plasma membranes that may not yet have been recognized. temperature. The glycerinated cells were quick frozen with Freeze-fracture analyses were carried out on two tumor cell Freon 22 and further cooled in liquid N2- as described (10). lines, two normal cell lines, and three peripheral blood cell Membrane cleavage was carried out in a Balzer high vacuum

The publication costs of this article were defrayed in part by page charge Abbreviations: PMA, phorbol myristate acetate; IMP, intramem- payment. This article must therefore be hereby marked "advertisement" branous particles; E face, external leaflet of the plasma membrane; in accordance with 18 U.S.C. §1734 solely to indicate this fact. P face, protoplasmic leaflet of the plasma membrane. 6829 Downloaded by guest on October 2, 2021 6830 Cell Biology: Zucker-Franklin and Nabi Proc. Natl. Acad. Sci. USA 83 (1986)

FIG. 1. (A) Melanoma cell treated with PMA in suspension, washed, and incubated with lymphocytes that adhered to the target cell, penetrated, and emperipolesed. N, nucleus of melanoma cell. (x2300.) (B) Melanoma cell from a sample cultured in a monolayer, exposed to PMA for 1 hr, washed three times, after which lymphocytes were added in situ. The specimen was flat-embedded to show the remarkable deformation of target cell membrane by effector cells. Note that a lymphocyte has invaginated the nucleus (arrow). (x 1200.)

freeze etch unit BAF-300 (Hudson, NH) in a vacuum of 10-6 90°, respectively. After thawing, the replicas were cleared by torr (1 torr = 1.333 x 102 Pa) at -1000C. The cleaved surfaces soaking with Clorox overnight, followed by multiple washes were shadowed with platinum and carbon at angles of430 and in diluted Clorox, acetic acid, and distilled water, as de-

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FIG. 2. (A and B) Freeze-fracture replicas of the P faces of melanoma cell plasma membranes before (A) and after (B) treatment with PMA. (C and D) Replicas of E faces of melanoma cells from the same sample as A and B, before (C) and after (D) exposure to PMA, respectively. In D, large areas of membrane are devoid of particles. When comparing C with D, the impression may be gained that it is the smaller particles that have been lost. (x90,000.) Downloaded by guest on October 2, 2021 Cell Biology: Zucker-Franklin and Nabi Proc. Natl. Acad. Sci. USA 83 (1986) 6831 scribed (10). A Siemens Elmiskop 1A electron microscope was used to view the replicas. All electron micrographs were obtained by an uninformed observer at original magnification of x 15,000 with an accelerating voltage of 60 kV. Membrane areas, which because of their relationship to the whole cell could be clearly identified as belonging either to the P face or E face of the plasma membrane, were enlarged photograph- ically to a final magnification of x 150,000 to facilitate counting of IMP. Each experiment was performed in tripli- cate. Particles of each suitable replica were counted in a double blind manner by two individuals. The paired Student's t test was used to evaluate statistical significance. An aliquot of each specimen was also dehydrated and embedded in Poly/Bed 812 for thin sectioning. The sections were stained with uranyl acetate and lead citrate.

RESULTS eAPfaoe Control. Morphology of Cells. When thin sections of PMA-treated and untreated specimens were examined blindly, no obvious ultrastructural difference was detected in any ofthe cells with the exception of platelets. Changes in shape displayed by platelets as a consequence of exposure to PMA have been published (12). Despite the fact that no morphological changes were seen when any of the other cells were treated with PMA, incubation of such cells with lymphocytes result- ed in a remarkable deformation of their surface membrane .1>~~~ ~ ~ ~ ~ ~ ~ ~ ., 5" and even emperipolesis into their cytoplasm (Fig. 1 A and B). Lymphocytes did not interact with or change the shape of untreated control target cells. The phenomenon occurred before any lytic event became detectable morphologically or by isotope release from labeled cells. Fig. 1 is presented to lend significance to the freeze-fracture studies reported below. Freeze-fracture. Representative replicas of melanoma cell plasma membranes before and after exposure to PMA are N.'-~~~~~~~~~~~p shown in Fig. 2 A-D. There was no obvious change in the number or size of IMP associated with the P face. As expected, the E face of control melanoma cells had fewer IMP than the P face. The remarkable reduction of IMP associated with the E face that followed exposure to PMA came as a surprise and was readily noted, even on cursory inspection. Illustrations of representative replicas of K-562 cell membranes before and after exposure to PMA are shown anlye o MAteaePMA ae o encare u in Fig. 3 A-D. A similar reduction ofparticles associated with the E face of PMA-treated cells is apparent. Precise quanti- fication of the particles confirmed that PMA treatment caused roughly a 50% decrease in the number of E face- associated IMP of every cell with the exception of platelets (Table 1). (In the interests of space, no replicas of platelet membranes have been illustrated because control and exper- imental samples were indistinguishable). It is noteworthy that the results obtained with the physiologically inactive phorbol analogs 4a-phorbol 12,13-didecanoate and 4p-phorbol were similar to those obtained with PMA. The ethanol diluent had analysoteinsofnPMA tread cs have in bereni cadmoutl no effect on IMP in the absence of the phorbols. Because of based on Fearly-freuezre-etchsotehnqusm(13m14)ancraeso in56 the heterogeneity in the size and irregularity in the circum- thels(APnumber oftrIMPhave bee noeadrngtecnrl C ae fllocycen(1 ference of the IMP, accurate size measurements of the 16)eatmnd folwingP stimreulationwintheligands orMtoxns(16e17 population of particles affected by PMA treatment have not AggegatidonheretuAtionemayiofoe Tealfreeisazeartchofrthcesofuther patile9pparourtunderstandingfcknowledgtoo co,4)ncrnedsetheseccrrditalstructureswith yet been possible. However, on gross inspection of electron the nubicemfIcal makeuanbehaioofteduingtraembranouse(15 photomicrographs by three uninformed observers, it was uneretfaepatileand,vaerietythereforesiuatconswistenexpewlmentadatof responseocondtosuchasdtoxany manip7)lwit concluded that PMA treatment did not affect particle size. glycoroenvaithean exeismarelyifeetalIMP andiinssc mostlyw DISCUSSION Although the scientific literature is replete with reports temperature (18), low pH (19), and treatment with glycerol or detailing the effects of phorbol esters on mammalian cell dimethyl sulfoxide without pinor fixation (20). On the other membranes, as far as we could ascertain, freeze-fracture hand, such an important physiologic event as the "capping" Downloaded by guest on October 2, 2021 6832 Cell Biology: Zucker-Franklin and Nabi Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Number of IMPs before and after PMA treatment IMPs, no. per jZm2 of membrane Cell type Control P face PMA P face Control E face PMA E face Melanoma 1019.5 ± 20.2 1047.4 ± 22.0 310.1 ± 9.0 162.1 ± 7.8 K-562 1229.7 ± 32.0 1177.2 ± 29.7 669.7 ± 29.4 310.7 ± 14.2 N-1121 fibroblasts 770.0 ± 34.4 652.0 ± 16.4 454.0 ± 12.0 249.5 ± 13.1 Fetal fibroblasts 524.5 ± 21.5 557.0 ± 27.6 368.8 ± 27.1 223.0 ± 29.3 Lymphocytes 511.2 ± 37.0 574.1 ± 31.4 232.7 ± 22.5 123.5 ± 11.8 Monocytes 982.6 ± 25.1 980.0 ± 30.0 456.5 ± 19.7 271.6 ± 14.0 Platelets* 527.5 ± 8.6 553.4 ± 13.2 880.1 ± 13.0 882.1 ± 14.4 *It should be noted that human blood platelets are the only mammalian cells described to date in which the partition coefficient ofthe IMP is reversed, i.e., more IMP are associated with the E face than the Pface ofthe plasma membrane. The statistical difference between the number ofIMP on Pfaces before and after treatment with PMA was not significant, whereas the difference ofIMP on the E faces before and after treatment with PMA was significant with a P value <0.001 in all instances.

phenomenon, which involves lateral relocation of peripheral peripheral membrane components. Indeed, in preliminary membrane components, or platelet aggregation, which is experiments with N-[3H]acetylmannosamine-labeled K-562 accompanied by extreme membrane deformation, is not and melanoma cells (26, 27), we have found that there is a associated with any changes in the number and distribution 60% reduction in surface sialic acid that neuraminidase can of the IMP (9, 10, 18). Nevertheless, it is generally held that release after the cells were treated with PMA (unpublished the IMP represent structures ofproteinaceous nature that are data). Obviously, several approaches will have to be used to intercalated into the hydrophobic matrix of the membrane identify the membrane moieties that have been lost. The bilayer and either anchor or constitute transmembrane pro- label-fracture method ofPinto da Silva (28) seems especially teins that respond to cytoplasmic and/or extracellular stim- applicable to this problem. Since the effect ofPMA is known uli. The asymmetry of the IMP distribution in most mamma- to be reversible, it will also be ofinterest to examine whether lian cells, i.e., the fact that the P face has roughly twice as the physical state of the membrane that permits release of many particles as the E face, is taken to reflect the disposition some of its components can be exploited for the insertion of of various glycoproteins, phospholipids, and other mem- others. brane components localized, respectively, in the external or cytoplasmic aspect ofthe membrane by other means (21, 22). The authors thank Ms. Susan Dittmar for her unfaltering patience The discrepancy of the results obtained with platelets could in the tedious collection of the freeze-fracture data. This research be attributable to a reversed "sidedness" of their plasma was supported by Grants CA 34378 and AM 12274 from the National membrane as reflected by the reversed partition coefficient of Institutes of Health. the IMP reported by several investigators (9, 10). In this regard, it should be mentioned that the aminophospholipids 1. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, V. like phosphatidylserine and phosphatidylethanolamine are & Nishizuka, Y. (1982) J. Biol. Chem. 275, 7847-7851. not from the outside ofintact 2. Niedel, J. E., Kuhn, L. J. & Vandenbark, G. R. (1983) Proc. readily accessible platelets (23). Natl. Acad. Sci. USA 80, 36-40. If short exposure to PMA were to affect primarily phospho- 3. Nishizuka, Y. (1984) Nature (London) 308, 693-698. lipids or proteins in the outer lipid bilayer, a different 4. Kielian, M. C. & Cohn, Z. A. (1981) J. Exp. Med. 154, behavior of platelet IMP is perhaps to be expected. The 101-111. question at hand, however, is how the loss of IMP from the 5. Patarroyo, M., Yogeeswaran, G., Bieberfeld, P., Klein, E. & E face of the cells following treatment with PMA is to be Klein, G. (1982) Int. J. Cancer 30, 707-717. interpreted. First, it should be reiterated that the loss appears 6. Kwong, C. H. & Mueller, G. C. (1982) Cancer Res. 42, to be real. The number of particles on the P face did not 2115-2120. change nor was there an obvious increase in the size of the 7. Nabi, Z. F. & Zucker-Franklin, D. (1984) Fed. Proc. Fed. Am. individual particles. Thus, a movement of particles from the Soc. Exp. Biol. 43, 598 (abstr.). E to the P face was not likely. Since the phorbol esters are 8. Nabi, Z. F. & Zucker-Franklin, D. (1986) Cell. Immunol. 100, very lipophilic and may partition among membrane lipids, 485-500. changes in the physical properties of the lipid bilayer could 9. Chevalier, J., Nurden, A. T., Thiery, J. M., Savariau, E. & affect the distribution and orientation of proteins therein. It Caen, J. P. (1979) J. Lab. Clin. Med. 94, 232-245. is, for instance, conceivable that a decreased membrane 10. Zucker-Franklin, D. (1981) J. Cell Biol. 91, 706-715. viscosity would lessen the stability of a molecule whose 11. Fujinami, N., Zucker-Franklin, D. & Valentine, F. (1981) Lab. major portion extends to the exterior. Small peptides that Invest. 45, 28-37. may not traverse both bilayers would be most vulnerable to 12. Estensen, R. D. & White, J. G. (1974) Am. J. Path. 74, 441- an increase in membrane fluidity of this nature. If this 452. were the nature the or 13. Branton, D. (1966) Proc. Natl. Acad. Sci. USA 55, 1048-1056. assumption correct, of glycoprotein 14. Marchesi, V. T., Tillack, T. W. & Scott, R. E. (1971) in lipid lost from the membrane might differfrom cell to cell. For Glycoproteins ofBlood Cells and Plasma, American National instance, the "free" portion of band 3 of the erythrocyte Red Cross, 4th Annual Scientific Symposium, eds. Jamieson, membrane that copurifies with lipid moieties (24) would be a G. A. & Greenwalt, T. J. (Lippincott, Philadelphia), pp. candidate for such release from PMA-treated erythrocytes. 94-105. This is subject to experimental verification. No loss of 15. Hasty, D. L. & Hay, E. D. (1977) J. Cell Biol. 72, 667-686. glycoprotein bands was seen when membrane extracts of 16. Scott, R. E. & Marchesi, V. T. (1972) Cell Immunol. 3, PMA-treated cells were subjected to NaDodSO4/PAGE. 301-317. However, a decrease in surface sialic acid has been noted by 17. Douglas, S. D., Ooka, M. P. & Zuckerman, S. H. (1976) Exp. others when lymphocytes were treated with PMA (5), and a Cell Res. 101, 111-121. loss of fibronectin occurs when fibroblasts are exposed to 18. Zucker-Franklin, D., Liebes, L. F. & Silber, R. (1979) J. phorbol esters (25). Sialic acid and fibronectin are likely to be Immunol. 122, 97-107. Downloaded by guest on October 2, 2021 Cell Biology: Zucker-Franklin and Nabi Proc. Natl. Acad. Sci. USA 83 (1986) 6833

19. Pinto da Silva, P. (1972) J. Cell Biol. 53, 777-787. 25. Zerlauth, G. & Wolf, G. (1984) Carcinogenesis 5, 863-868. 20. McIntyre, J. A., Gilula, N. B. & Karnovsky, M. J. (1974) J. 26. Yogeeswaran, G., Groberg, A., Hansson, M., Dalianis, T., Cell Biol. 60, 192-203. Keissling, R. & Welsh, R. M. (1981) Int. J. Cancer 28, 21. Bretscher, M. S. (1972) J. Mol. Biol. 71, 523-528. 517-526. 22. Steck, T. L. (1974) J. Cell Biol. 62, 1-19. 27. Kemp, S. F. & Stoolmiller, A. C. (1976) J. Biol. Chem. 251, 23. Schick, P. K., Kurica, K. B. & Chacko, G. K. (1976) J. Clin. 7626-7631. Invest. 57, 1221-1226. 28. Pinto da Silva, P. & Kan, F. W. K. (1984) J. Cell Biol. 99, 24. Bennett, V. (1982) Biochim. Biophys. Acta 689, 475-484. 1156-1161. Downloaded by guest on October 2, 2021