Proc. Nati. Acad. Scl. USA Vol. 75, No. 8, pp. 3823-3825, August 1978
Cell Biology
Effect of anti-spectrin antibody and ATP on deformability of resealed erythrocyte membranes (ektacytometer/phosphorylation/dephosphorylation)
Koji NAKASHIMA AND ERNEST BEUTLER* Department of Hematology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010
Contrbuted by Ernest Beutler, May 19,1978
ABSTRACT Deformability of resealed erythrocyte membranes was measured by using an ektacytometer. Divalent anti-spectrin antibody, but not monovalent anti-spectrin Fab fragments, decreased membrane deformability. Membranes resealed with MgATP were more deformable than those without MgATP. Exogenous alkaline phosphatase, which dephosphorylates spectrin, decreased membrane deformability. These results suggest that spectrin is an essential component of the system that determines erythrocyte deformability. They are consistent with the view that the role of ATP in membrane deformability is mediated through phosphorylation of the spectrin. The deformability of the erythrocyte is essential for its smooth passage through anatomically restricted regions of the microcirculation (1). The life-span of normal circulating erythrocytes is largely determined by their ability to deform and to change shape, and pathological decreases in cellular deformability lead to premature removal of them from the circulation by splenic sequestration. In fact, decreases in erythrocyte deformability have been reported in various hemolytic disorders (2-4). The human erythrocyte is a relatively simple cell, but the extreme reversible deformability and discocyte-echinocyfe shape change, which occur under various conditions, suggest that an elastomeric protein network is associated with the membrane as an essential component. Indeed, the membrane has been shown to possess an actomyosin-like protein (5) consisting of spectrin (components 1 and 2) (6-8) and actin (component 5) (9). It has been suggested that such a spectrin-actin complex and ATP are important in maintaining cell shape and deformability. However, there have been conflicting reports of the relationship among the spectrin-actin complex, ATP, and cell deformability (7, 10-15). Recent investigations by Sheetz and Singer (16) show that crosslinldng the spectrin network with bivalent anti-spectrin antibody accelerates the ATP effect in restoring erythrocyte membranes to a disc shape. In order to evaluate the role of spectrin and ATP in membrane deformability more directly, we have studied the in vitro deformability of resealed erythrocyte membranes into which we incorporated monospecific anti-spectrin antibody, anti-spectrin Fab fragments, MgATP, or alkaline phosphatase. MATERIALS AND METHODS Heparinized blood was used in the study of deformability. Spectrin was prepared from normal human blood collected less than 24 hr previously into citrate/phosphate/dextrose anticoagulant. An EDTA extract of erythrocyte membranes with a high spectrin content was prepared according to the method of Marchesi et al. (17). Spectrin was purified to homogeneity The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertiement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
by water extraction and Sephadex G-200 gel filtration according to the method described by Nicolson and Painter (18). The purity of spectrin was confirmed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (19). New Zealand White rabbits were injected subcutaneously with 2 mg of the spectrin-rich EDTA extract with Freund's complete adjuvant. Two additional injections were given 2 and 3 weeks later. The animals were bled 10 days after the last immunization, and the serum from three animals was pooled. Anti-spectrin antibody was purified from the sera by affinity chromatography on columns prepared by coupling homogeneous spectrin to CNBr-activated Sepharose 4B. Anti-spectrin Fab fragments were made by papain digestion of anti-spectrin antibody and purified by CM-cellulose column chromatography (20). Anti-spectrin antibody and Fab fragments were sequestered inside resealed ghosts by using the method of Dale et al. (21). Anti-spectrin antibody (0.1 mg) or Fab fragments (0.07 mg) were added to 0.5 ml of washed erythrocytes packed in 0.154 M NaCl to a hematocrit value of 80%. Alkaline phosphatase was diluted in 0.154 M NaCl solution and dialyzed against 2000 vol of 0.154 M NaCl for 2 hr; 0.88sg of the enzyme was added to 0.5 ml of washed erythrocytes. After dialysis for 2 hr against 5 mM phosphate buffer (pH 7.4) (21), the ghosts were resealed by dialysis against 0.12 M KCI/5 mM phosphate, pH 7.4 with and without 2.0 mM MgATP. Resealed ghosts were washed twice with 30 vol of phosphate-buffered saline (0.12 M NaCI/20 mM Na2HPO4/5 mM KH2PO4, pH 7.4) and suspended in 2 vol of the same buffer. Thirty microliters of this suspension was mixed with 5.8 ml of a dextran solution (25 g of dextran 110 dissolved in 100 ml of phosphate-buffered saline). Nonimmunized rabbit gamma globulin and Fab fragments were used as controls. Isotonic saline was used as a control for alkaline phosphatase because of the negligible quantity of enzyme protein used. Resealing was performed in a slightly hypotonic solution, and deformability was measured in a solution of higher osmotic pressure than the resealing buffer in order to avoid apparent rigidity due to decreased surface to volume ratio. Deformability was measured in an ektacytometer, the viscometric-diffraction apparatus recently developed by Bessis and Mohandas (22). The elongation of the cells under shear stress was estimated by measuring the shape change of the diffractometric pattern (22). The morphology of resealed erythrocyte membranes was studied by phase contrast microscopy. CNBr-activated Sepharose 4B and dextran 110 were products of Pharmacia Fine Chemicals (Uppsala, Sweden), and papain, MgATP, and alkaline phosphatase (Escherichia coli, type III) were from Sigma Chemical Co. (St. Louis, MO). *
3823
To whom reprint requests should be addressed.
3824
Cell Biology: Nakashima and Beutler
Proc. Nati.,,Acad. Sci. USA 75 (1978) 9
12.0-
E A
10.0* ~~~~~9~~~
0
-j
8.0 It-
T-
dyne crn
200 400 600 Shear stress, dyne/cm2
FIG. 1. Diffraction patterns, photographed at the indicated ap-
plied shear stress, of resealed ghosts with MgATP and rabbit gamma globulin (Upper) and resealed ghosts with anti-spectrin antibody but no MgATP (Lower).
RESULTS Affinity-purified anti-spectrin antibody contained only light chain and heavy chain bands on sodium dodecyl sulfate/ polyacrylamide gel electrophoresis. Purifed Fab fragments were found to have one band at the position of the light chain and no band in the heavy chain position. Double-immunodiffusion plates showed precipitation lines between anti-spectrin antibody and spectrin. No precipitation was observed between the anti-spectrin Fab fragment and spectrin, although the concentrations of the antibody and Fab fragments were 0.2 and 0.1 mg/ml, respectively, providing similar numbers of antigen combining sites. To determine whether the Fab fragments react with spectrin, Fab fragment solution was mixed with spectrin-coupled CNBr-Sepharose suspension. After incubation at 370 for 1 hr and centrifugation, protein was not detectable in the supernatant by Lowry's method (23). In contast, all of Fab made from normal rabbit gamma globulin was recovered. These results indicated that the Fab fragment is able to react with spectrin but is unable to form a precipitate because of the monovalent active site and loss of the Fc fragment. In order to observe the effect of addition of ATP to the ghosts, washed cells were stored in physiological saline overnight at 40 before study but, for investigating dephosphorylation of spectrin by alkaline phosphatase, freshly prepared membranes were used. Typical diffraction patterns are shown in Fig. 1, and the relationship between shear stress and the deformability is presented in-Fig. 2. It is apparent that MgATP tended to increase the deformability of the resealed ghosts and that anti-
800
FIG. 3. Variation of resealed ghost length as a function of the applied shear stress. 0, Nonimmune rabbit Fab fragment; 0, antispectrin Fab fragment.
spectrin antibody decreased the deformability. Thus, the most deformable membranes were those into which ATP and nonimmune rabbit y-globulin had been incorporated, whereas the most rigid membranes were those that had been resealed without MgATP but with anti-spectrin antibody. In contrast, as shown in Fig. 3, anti-spectrin Fab fragments had no effect on membrane deformability compared with membranes into which nonimmune Fab fragments had been sealed. The deformability of resealed ghosts with and without alkaline phosphatase is shown in Fig. 4. It is evident that such treatment makes the ghost less deformable, presumably by dephosphorylation of spectrin as reported previously (24, 25). Resealed erythrocyte ghosts consisted of mixtures of discocytes and echinocytes, and no difference was observed between the appearance of ghosts resealed with antibody, MgATP, as alkaline phosphatase and the control. DISCUSSION It has been suggested that spectrin associates with actin to form an anastomosing network beneath the erythrocyte membrane (9). This network presumably functions in the control of cell shape and in restricting lateral movement of integral membrane proteins. The effect of exogenous ATP on the shape of erythrocyte membranes was first reported by Nakao et al. (26). Recently, Sheetz and Singer (16) confirmed that the incorporation of MgATP into resealed ghosts resulted in the conversion of echinocytes to discocytes. Moreover, the incorporation of optimal amounts of monospecific bivalent but not of monovalent 12.0
E
E .e 10.0
0
-
U
--
0)
c 0) -J
-J
1t
800
600 400 Shear stress, dyne/cm2
200
200
FIG. 2. Variation of resealed ghost length as a function of applied shear stress. 0, MgATP and rabbit gamma globulin; rabbit gamma globulin but no MgATP; *, MgATP and anti-spectrin antibody; *, anti-spectrin antibody but no MgATP. 0,
400 600 Shear stress, dyne/cm2
800
FIG. 4. Variation of the ghost length as a function of the applied shear stress. 0, Control ghost; 0, ghost resealed with alkaline phosphatase.
Cell Biology: Nakashima and Beutler anti-spectrin Fab fragments into ghosts along with MgATP markedly accelerated this effect. They suggested that -the crosslinking of spectrin by bivalent anti-spectrin antibody was responsible for the synergistic effects of antibody and MgATP. Nicolson and Painter (18) also observed that the crosslinking anti-spectrin antibodies sequestered inside the ghost caused aggregation of colloidal iron hydroxide binding sites on the outer membrane surface and suggested that there might be a structural linkage between spectrin on the inner surface and the integral membrane component glycophorin. These findings seem to support the fluid-mosaic membrane model (27), which proposes that the integral protein is capable of lateral movement in the lipid bilayer and suggest that spectrin might exert direct or indirect control on the external topography of some membrane components. We found that bivalent but not monovalent anti-spectrin antibody caused a decrease in membrane deformability. There seem to be three possible explanations for these results. First, anti-spectrin antibody may crosslink spectrin, inhibiting spectrin movement mechanically and reducing the elasticity of the spectrin-actin network. Second, anti-spectrin antibody may modify the spectrin molecular conformation in such a way as to inhibit the physiological function as a contractile protein, just as antibody to an enzyme inhibits enzyme activity. Third, antibody may act to separate the spectrin from the membrane surface. The last possibility is not likely because of the morphological observation by Nicolson et al. (28). The second possibility is attractive because of increased deformability caused by addition of MgATP, a substrate for spectrin phosphorylation. However, the fact that monospecific anti-spectrin Fab fragments did not effect deformability militates against this possibility. It is most probable that bivalent anti-spectrin antibody forms an antigen-antibody complex on the inner surface of the erythrocyte membrane, crosslinking the spectrin network and preventing its lateral movement mechanically when a shear stress is applied. From another point of view, spectrin may be considered to exist as an anchor for the integral proteins in the lipid bilayer, controlling their topographic arrangement (17). Whenever a shear stress produces cellular deformation, the movement of integral proteins is limited by attachments to spectrin acting as an anchor. This may be instrumental in restoring membrane shape after the removal of stress. In the case of our experiments, spectrin movement, and thus the movement of integral proteins in a lipid bilayer, is restricted mechanically by bivalent antispectrin antibody, resulting in a decrease in deformation of the membrane in response to shear stress. Thus, in contrast to the morphologic observations by Sheetz and Singer (16), in which ATP and bivalent antibody exerted synergistic effects, their effect on deformability is antagonistic: the effect of MgATP is to increase deformability of erythrocyte membranes, and deformability is diminished by bivalent antibody. Weed et al. (10) found that ATP and Ca2+ played an important role in the control of erythrocyte shape and deformability, but Feo and Mohandas (14) reported that the deformability of ATP-depleted erythrocytes is normal, deformability being independent of the intracellular level of ATP and only dependent on the shape of the cells It was shown that the shape changes of ghosts are associated with the phosphorylation of a serine residue on component 2 of spectrin subunits and suggested that phosphorylation/dephosphorylation reactions might control a polymerization/depolymerization process (24, 29).
Proc. Nati. Acad. Sci. USA 75 (1978)
3825
Pinder et al. (25) reported that the phosphorylation of spectrin induced the polymerization of actin. In our experiments, MgATP served to phosphorylate and alkaline phosphatase to dephosphorylate spectrin. The changes of deformability by such agents suggest that phosphorylation/dephosphorylation of spectrin contributes to cell membrane deformability as well as cell shape. it is likely that the effect of ATP on membrane deformability is an indirect one mediated through phosphorylation of spectrin. Thus, when the experimental conditions are such as to allow spectrin dephosphorylation as well as ATP depletion to take place, changes in membrane deformability might be expected. On the other hand, acute cellular ATP depletion might have no such effect before dephosphorylation of spectrin had occurred. The results of our studies are consistent with such a view.
This work was supported, in part, by Grant HL 07449 from the National Institutes of Health. K.N. is a Public Health Service International Research Fellow (1 F05 TW 2503-01). 1. Weed, R. I. (1970) Am. J. Med. 49, 147-150. 2. Jacob, H. S. (1974) in The Red Blood Cell, ed. Surgenor, D. M. (Academic, New York), 2nd Ed., pp. 269-292. 3. LaCelle, P. L. (1970) Semin. Hematol. 7,355-371. 4. Allard, C., Mohandas, N. & Bessis, M. (1977) Blood Cells 3, 209-221. 5. Ohnishi, T. (1962) J. Biochem. 52,307-308. 6. Marchesi, V. T. & Steers, E., Jr. (1968) Science 159, 203-204. 7. Kirkpatrick, F. H. (1976) Life Sci. 19, 1-18. 8. Sheetz, M. P., Painter, R. G. & Singer, S. J. (1976) Biochemistry
15,4486-4492.
9. Tilney, L. G. & Detmers, P. (1975) J. Cell. Biol. 66,508-520. 10. Weed, R. I., LaCelle, P. L. & Merrill, E. W. (1969) J. Clin. Invest.
48,795-809. 11. Leblond, P. (1973) in Red Cell Shape, eds. Bessis, M., Weed, R. I. & Leblond, P. F. (Springler-Verlag, New York), pp. 95-104. 12. Mohandas, N., Greenquist, A. & Shohet, S. B. (1976) Blood, 48, 991a. 13. Heusinkveld, R. S., Goldstein, D. A., Weed, R. I. & LaCelle, P. L. (1977) Blood Cells 3, 175-182. 14. Feo, C. & Mohandas, N. (1977) Blood Cells 3, 153-161. 15. Lux, S. E., John, K. M. & Ukena, T. E. (1978) J. Clin. Invest. 61, 815-827. 16. Sheetz, M. P. & Singer, S. J. (1977) J. Cell Biol. 73, 638-646. 17. Marchesi, S. L., Steers, E., Marchesi, V. T. & Tillack, T. W. (1970)
Biochemistry 9,50-57. 18. Nicolson, G. L. & Painter, R. G. (1973) J. Cell Biol. 59, 395406. 19. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617. 20. Putnam, F. W., Tan, M., Lynn, L. T., Easley, C. W. & Migita, S. (1962) J. Biol. Chem. 237, 717-726. 21. Dale, G. L., Villacorte, D. G. & Beutler, E. (1977) Biochem. Med.
18,220-225. 22. Bessis, M. & Mohandas, N. (1975) Blood Cells 1, 307-313. 23. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193,265-275.
24. Birchmeier, W. & Singer, S. J. (1977) J. Cell Biol. 73, 647659. 25. Pinder, J. C., Bray, D. & Gratzer, W. B. (1977) Nature (London)
270,752-754.
26. Nakao, M., Nakao, T., Tatibana, M. & Yoshikawa, H. (1960) J. Biochem. 47, 694-695. 27. Singer, S. J. & Nicolson, G. L. (1972) Science 175, 720-731. 28. Nicolson, G. L., Marchesi, V. T. & Singer, S. J. (1971) J. Cell Biol.
51,265-272.
29. Wyatt, J. L., Greenquist, A. C. & Shohet, S. B. (1977) Biochem. Biophys. Res. Commun. 79, 1279-1285.
Cell Biology
Effect of anti-spectrin antibody and ATP on deformability of resealed erythrocyte membranes (ektacytometer/phosphorylation/dephosphorylation)
Koji NAKASHIMA AND ERNEST BEUTLER* Department of Hematology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010
Contrbuted by Ernest Beutler, May 19,1978
ABSTRACT Deformability of resealed erythrocyte membranes was measured by using an ektacytometer. Divalent anti-spectrin antibody, but not monovalent anti-spectrin Fab fragments, decreased membrane deformability. Membranes resealed with MgATP were more deformable than those without MgATP. Exogenous alkaline phosphatase, which dephosphorylates spectrin, decreased membrane deformability. These results suggest that spectrin is an essential component of the system that determines erythrocyte deformability. They are consistent with the view that the role of ATP in membrane deformability is mediated through phosphorylation of the spectrin. The deformability of the erythrocyte is essential for its smooth passage through anatomically restricted regions of the microcirculation (1). The life-span of normal circulating erythrocytes is largely determined by their ability to deform and to change shape, and pathological decreases in cellular deformability lead to premature removal of them from the circulation by splenic sequestration. In fact, decreases in erythrocyte deformability have been reported in various hemolytic disorders (2-4). The human erythrocyte is a relatively simple cell, but the extreme reversible deformability and discocyte-echinocyfe shape change, which occur under various conditions, suggest that an elastomeric protein network is associated with the membrane as an essential component. Indeed, the membrane has been shown to possess an actomyosin-like protein (5) consisting of spectrin (components 1 and 2) (6-8) and actin (component 5) (9). It has been suggested that such a spectrin-actin complex and ATP are important in maintaining cell shape and deformability. However, there have been conflicting reports of the relationship among the spectrin-actin complex, ATP, and cell deformability (7, 10-15). Recent investigations by Sheetz and Singer (16) show that crosslinldng the spectrin network with bivalent anti-spectrin antibody accelerates the ATP effect in restoring erythrocyte membranes to a disc shape. In order to evaluate the role of spectrin and ATP in membrane deformability more directly, we have studied the in vitro deformability of resealed erythrocyte membranes into which we incorporated monospecific anti-spectrin antibody, anti-spectrin Fab fragments, MgATP, or alkaline phosphatase. MATERIALS AND METHODS Heparinized blood was used in the study of deformability. Spectrin was prepared from normal human blood collected less than 24 hr previously into citrate/phosphate/dextrose anticoagulant. An EDTA extract of erythrocyte membranes with a high spectrin content was prepared according to the method of Marchesi et al. (17). Spectrin was purified to homogeneity The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertiement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
by water extraction and Sephadex G-200 gel filtration according to the method described by Nicolson and Painter (18). The purity of spectrin was confirmed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (19). New Zealand White rabbits were injected subcutaneously with 2 mg of the spectrin-rich EDTA extract with Freund's complete adjuvant. Two additional injections were given 2 and 3 weeks later. The animals were bled 10 days after the last immunization, and the serum from three animals was pooled. Anti-spectrin antibody was purified from the sera by affinity chromatography on columns prepared by coupling homogeneous spectrin to CNBr-activated Sepharose 4B. Anti-spectrin Fab fragments were made by papain digestion of anti-spectrin antibody and purified by CM-cellulose column chromatography (20). Anti-spectrin antibody and Fab fragments were sequestered inside resealed ghosts by using the method of Dale et al. (21). Anti-spectrin antibody (0.1 mg) or Fab fragments (0.07 mg) were added to 0.5 ml of washed erythrocytes packed in 0.154 M NaCl to a hematocrit value of 80%. Alkaline phosphatase was diluted in 0.154 M NaCl solution and dialyzed against 2000 vol of 0.154 M NaCl for 2 hr; 0.88sg of the enzyme was added to 0.5 ml of washed erythrocytes. After dialysis for 2 hr against 5 mM phosphate buffer (pH 7.4) (21), the ghosts were resealed by dialysis against 0.12 M KCI/5 mM phosphate, pH 7.4 with and without 2.0 mM MgATP. Resealed ghosts were washed twice with 30 vol of phosphate-buffered saline (0.12 M NaCI/20 mM Na2HPO4/5 mM KH2PO4, pH 7.4) and suspended in 2 vol of the same buffer. Thirty microliters of this suspension was mixed with 5.8 ml of a dextran solution (25 g of dextran 110 dissolved in 100 ml of phosphate-buffered saline). Nonimmunized rabbit gamma globulin and Fab fragments were used as controls. Isotonic saline was used as a control for alkaline phosphatase because of the negligible quantity of enzyme protein used. Resealing was performed in a slightly hypotonic solution, and deformability was measured in a solution of higher osmotic pressure than the resealing buffer in order to avoid apparent rigidity due to decreased surface to volume ratio. Deformability was measured in an ektacytometer, the viscometric-diffraction apparatus recently developed by Bessis and Mohandas (22). The elongation of the cells under shear stress was estimated by measuring the shape change of the diffractometric pattern (22). The morphology of resealed erythrocyte membranes was studied by phase contrast microscopy. CNBr-activated Sepharose 4B and dextran 110 were products of Pharmacia Fine Chemicals (Uppsala, Sweden), and papain, MgATP, and alkaline phosphatase (Escherichia coli, type III) were from Sigma Chemical Co. (St. Louis, MO). *
3823
To whom reprint requests should be addressed.
3824
Cell Biology: Nakashima and Beutler
Proc. Nati.,,Acad. Sci. USA 75 (1978) 9
12.0-
E A
10.0* ~~~~~9~~~
0
-j
8.0 It-
T-
dyne crn
200 400 600 Shear stress, dyne/cm2
FIG. 1. Diffraction patterns, photographed at the indicated ap-
plied shear stress, of resealed ghosts with MgATP and rabbit gamma globulin (Upper) and resealed ghosts with anti-spectrin antibody but no MgATP (Lower).
RESULTS Affinity-purified anti-spectrin antibody contained only light chain and heavy chain bands on sodium dodecyl sulfate/ polyacrylamide gel electrophoresis. Purifed Fab fragments were found to have one band at the position of the light chain and no band in the heavy chain position. Double-immunodiffusion plates showed precipitation lines between anti-spectrin antibody and spectrin. No precipitation was observed between the anti-spectrin Fab fragment and spectrin, although the concentrations of the antibody and Fab fragments were 0.2 and 0.1 mg/ml, respectively, providing similar numbers of antigen combining sites. To determine whether the Fab fragments react with spectrin, Fab fragment solution was mixed with spectrin-coupled CNBr-Sepharose suspension. After incubation at 370 for 1 hr and centrifugation, protein was not detectable in the supernatant by Lowry's method (23). In contast, all of Fab made from normal rabbit gamma globulin was recovered. These results indicated that the Fab fragment is able to react with spectrin but is unable to form a precipitate because of the monovalent active site and loss of the Fc fragment. In order to observe the effect of addition of ATP to the ghosts, washed cells were stored in physiological saline overnight at 40 before study but, for investigating dephosphorylation of spectrin by alkaline phosphatase, freshly prepared membranes were used. Typical diffraction patterns are shown in Fig. 1, and the relationship between shear stress and the deformability is presented in-Fig. 2. It is apparent that MgATP tended to increase the deformability of the resealed ghosts and that anti-
800
FIG. 3. Variation of resealed ghost length as a function of the applied shear stress. 0, Nonimmune rabbit Fab fragment; 0, antispectrin Fab fragment.
spectrin antibody decreased the deformability. Thus, the most deformable membranes were those into which ATP and nonimmune rabbit y-globulin had been incorporated, whereas the most rigid membranes were those that had been resealed without MgATP but with anti-spectrin antibody. In contrast, as shown in Fig. 3, anti-spectrin Fab fragments had no effect on membrane deformability compared with membranes into which nonimmune Fab fragments had been sealed. The deformability of resealed ghosts with and without alkaline phosphatase is shown in Fig. 4. It is evident that such treatment makes the ghost less deformable, presumably by dephosphorylation of spectrin as reported previously (24, 25). Resealed erythrocyte ghosts consisted of mixtures of discocytes and echinocytes, and no difference was observed between the appearance of ghosts resealed with antibody, MgATP, as alkaline phosphatase and the control. DISCUSSION It has been suggested that spectrin associates with actin to form an anastomosing network beneath the erythrocyte membrane (9). This network presumably functions in the control of cell shape and in restricting lateral movement of integral membrane proteins. The effect of exogenous ATP on the shape of erythrocyte membranes was first reported by Nakao et al. (26). Recently, Sheetz and Singer (16) confirmed that the incorporation of MgATP into resealed ghosts resulted in the conversion of echinocytes to discocytes. Moreover, the incorporation of optimal amounts of monospecific bivalent but not of monovalent 12.0
E
E .e 10.0
0
-
U
--
0)
c 0) -J
-J
1t
800
600 400 Shear stress, dyne/cm2
200
200
FIG. 2. Variation of resealed ghost length as a function of applied shear stress. 0, MgATP and rabbit gamma globulin; rabbit gamma globulin but no MgATP; *, MgATP and anti-spectrin antibody; *, anti-spectrin antibody but no MgATP. 0,
400 600 Shear stress, dyne/cm2
800
FIG. 4. Variation of the ghost length as a function of the applied shear stress. 0, Control ghost; 0, ghost resealed with alkaline phosphatase.
Cell Biology: Nakashima and Beutler anti-spectrin Fab fragments into ghosts along with MgATP markedly accelerated this effect. They suggested that -the crosslinking of spectrin by bivalent anti-spectrin antibody was responsible for the synergistic effects of antibody and MgATP. Nicolson and Painter (18) also observed that the crosslinking anti-spectrin antibodies sequestered inside the ghost caused aggregation of colloidal iron hydroxide binding sites on the outer membrane surface and suggested that there might be a structural linkage between spectrin on the inner surface and the integral membrane component glycophorin. These findings seem to support the fluid-mosaic membrane model (27), which proposes that the integral protein is capable of lateral movement in the lipid bilayer and suggest that spectrin might exert direct or indirect control on the external topography of some membrane components. We found that bivalent but not monovalent anti-spectrin antibody caused a decrease in membrane deformability. There seem to be three possible explanations for these results. First, anti-spectrin antibody may crosslink spectrin, inhibiting spectrin movement mechanically and reducing the elasticity of the spectrin-actin network. Second, anti-spectrin antibody may modify the spectrin molecular conformation in such a way as to inhibit the physiological function as a contractile protein, just as antibody to an enzyme inhibits enzyme activity. Third, antibody may act to separate the spectrin from the membrane surface. The last possibility is not likely because of the morphological observation by Nicolson et al. (28). The second possibility is attractive because of increased deformability caused by addition of MgATP, a substrate for spectrin phosphorylation. However, the fact that monospecific anti-spectrin Fab fragments did not effect deformability militates against this possibility. It is most probable that bivalent anti-spectrin antibody forms an antigen-antibody complex on the inner surface of the erythrocyte membrane, crosslinking the spectrin network and preventing its lateral movement mechanically when a shear stress is applied. From another point of view, spectrin may be considered to exist as an anchor for the integral proteins in the lipid bilayer, controlling their topographic arrangement (17). Whenever a shear stress produces cellular deformation, the movement of integral proteins is limited by attachments to spectrin acting as an anchor. This may be instrumental in restoring membrane shape after the removal of stress. In the case of our experiments, spectrin movement, and thus the movement of integral proteins in a lipid bilayer, is restricted mechanically by bivalent antispectrin antibody, resulting in a decrease in deformation of the membrane in response to shear stress. Thus, in contrast to the morphologic observations by Sheetz and Singer (16), in which ATP and bivalent antibody exerted synergistic effects, their effect on deformability is antagonistic: the effect of MgATP is to increase deformability of erythrocyte membranes, and deformability is diminished by bivalent antibody. Weed et al. (10) found that ATP and Ca2+ played an important role in the control of erythrocyte shape and deformability, but Feo and Mohandas (14) reported that the deformability of ATP-depleted erythrocytes is normal, deformability being independent of the intracellular level of ATP and only dependent on the shape of the cells It was shown that the shape changes of ghosts are associated with the phosphorylation of a serine residue on component 2 of spectrin subunits and suggested that phosphorylation/dephosphorylation reactions might control a polymerization/depolymerization process (24, 29).
Proc. Nati. Acad. Sci. USA 75 (1978)
3825
Pinder et al. (25) reported that the phosphorylation of spectrin induced the polymerization of actin. In our experiments, MgATP served to phosphorylate and alkaline phosphatase to dephosphorylate spectrin. The changes of deformability by such agents suggest that phosphorylation/dephosphorylation of spectrin contributes to cell membrane deformability as well as cell shape. it is likely that the effect of ATP on membrane deformability is an indirect one mediated through phosphorylation of spectrin. Thus, when the experimental conditions are such as to allow spectrin dephosphorylation as well as ATP depletion to take place, changes in membrane deformability might be expected. On the other hand, acute cellular ATP depletion might have no such effect before dephosphorylation of spectrin had occurred. The results of our studies are consistent with such a view.
This work was supported, in part, by Grant HL 07449 from the National Institutes of Health. K.N. is a Public Health Service International Research Fellow (1 F05 TW 2503-01). 1. Weed, R. I. (1970) Am. J. Med. 49, 147-150. 2. Jacob, H. S. (1974) in The Red Blood Cell, ed. Surgenor, D. M. (Academic, New York), 2nd Ed., pp. 269-292. 3. LaCelle, P. L. (1970) Semin. Hematol. 7,355-371. 4. Allard, C., Mohandas, N. & Bessis, M. (1977) Blood Cells 3, 209-221. 5. Ohnishi, T. (1962) J. Biochem. 52,307-308. 6. Marchesi, V. T. & Steers, E., Jr. (1968) Science 159, 203-204. 7. Kirkpatrick, F. H. (1976) Life Sci. 19, 1-18. 8. Sheetz, M. P., Painter, R. G. & Singer, S. J. (1976) Biochemistry
15,4486-4492.
9. Tilney, L. G. & Detmers, P. (1975) J. Cell. Biol. 66,508-520. 10. Weed, R. I., LaCelle, P. L. & Merrill, E. W. (1969) J. Clin. Invest.
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