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Cytochemical demonstration of negative surface charges in central myelin
Brain Research, 262 (1983) 225-232 Elsevier Biomedical Press
225
Cytochemical Demonstration of Negative Surface Charges in Central Myelin R. DERMIETZEL, N. TH1]RAUF and D. SCHLINKE
lnstitut fiir Anatomie, Universitiitsklinikum der GHS Essen, Hufelandstrasse 55, 4300 Essen 1 (F.R.G.) (Accepted August 10th, 1982)
Key words: myelin cytochemistry - - negative charges - - labelling - - cationic ferritin
Homogenates of central myelin were treated with ferritin derivatives having different isoelectric points. It was found that considerable amounts of cationic ferritin (pI 8.5-9.5) had access to the extracellular space, but that anionic ferritin (pl 4.0) and native ferritin (pI 4.5) did not. The electrostatic nature of the binding of cationic ferritin was demonstrated by treating the homogenates with poly-L-lysineand 1 M NaC1: both reagents led to a complete displacement of the bound cationic ferritin. Neither extensive trypsination nor neuraminidase treatment showed a significant effect on the intralamellar distribution of the bound cationic ferritin molecules. This suggests that the net negative charge on the extracellular myelin face stems primarily from acidic lipid groups in the membrane. INTRODUCTION The myelin sheath is composed of a multilamellar system of bilayers. Myelin in the central nervous system (CNS) is formed b3 the oligodendrocytes and involves the wrapping of glial cell membrane spirally around a segment of axon. The maintenance of this arrangement of membranes requires specific molecular interactions between the electrostatic repulsive forces of the acidic lipid layers and long range electrodynamic attraction6, t4. We have attempted to examine in detail the nature of the electrostatic forces using a cytochemical probe. A particularly useful probe for visualizing the distribution of negatively charged groups on the surface of membranes is polycationic ferritin 7. This substance has been found to be effective in demonstrating the distribution of anionic charges on the surface of living cells at physiological pH10,13,16,24. The present study uses cationized ferritin (CF), anionized ferritin (AF) and native ferritin (NF) and the results suggest that the electrostatic interaction between pairs of myelin membranes depends upon the net negative charge from acidic lipid groups in the membranes. MATERIALS AND METHODS
Animal and tissue preparation Anaesthetized Wistar rats (Hannover
strain)
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
weighing 300-350 g were used. The entire brain was removed as quickly as possible and myelin prepared immediately according to the standard procedure as described by Norton 15.
Anionized ferritin (AF) and cationized ferritin ( CF) AF was prepared from native ferritin (NF) (Sigma, cadmium content < 1.0 ~ ) by the procedure by Burry and WoodL An LKB multiphore system was used for isoelectric focusing, which gave a pI of between 3.7 and 4.0 for A F as compared to a value of 4.5 for NF. CF was prepared by a modification of the method of D a n o n et al. 7, described by Th/irauf and Dermietze127. The isoelectric point of CF ranged from 8.5 to 9.2. Unstained A F and N F were spread on carbon-coated grids and examined in the electron microscope to check whether or not the particles were completely dispersed and were only used when free of particle clusters. Labelling procedure A solution o f C F , A F or N F dissolved in 0.15 M NaC1 and containing 10 mg ferritin/ml was added to 2 ml of myelin homogenate suspended in Tris-HC1 buffer. The final p H of the different ferritin/buffer solutions was: CF/buffer p H 7.4; AF/buffer p H 7.2; NF/buffer p H 7.2. In some instances the homogenates were incubated in CF dissolved in 0.15 M
226 NaCI at pH 2.0. After preparation the suspensions were immediately placed on ice in a probe sonicator for 5 min. Since sonication of the suspension did not affect the integrity of the lamellar complexes it was routinely used. To check whether treatment with a polycation leads to an inhibition of the CF labelling, the homogenates were incubated with poly-L-lysine (5 mg/ml, Sigma, type VI).
Trypsin treatment and control of protein removal To test the effect of trypsination, aliquots of each homogenate (containing about 100 mg myelin wet weight) were incubated in a 0.01 M Tris-HCl buffer (pH 7.4) with or without the addition of 1% trypsin (Sigma type XII). In each case the final volume was adjusted to 2.0 ml and the samples incubated for 24 h at room temperature. At the end of the incubation period the samples were placed in an ice bath and then centrifuged at 9000g for 10 min at 4 °C. Part of the trypsin treated material was incubated with CF as described above. The rest was delipidated in chloroform-methanol (2:l, v/v), then acetone and solubilized in 3 % SDS and 5 % mercaptoethanol. SDS gel electrophoresis and staining with Coomassie blue were performed according to the method of Laemmlila. Neuraminidase incubation and blocking of N-acetylneuraminic acid ( NANA) Samples of the homogenates (I00 mg wet weight) were treated with neuraminidase from either Vibrio cholera (Behring, Marburg) or Clostridium perfringens (Sigma, type V). The activity of both enzymes was 1 U/ml. The enzymes were dissolved in a sodium acetate buffer (pH 5.5) and 0.5 ml of this solution was added at the beginning of incubation period with a subsequent addition of 0.5 ml after 6 h. The samples were incubated for a total of 12 h at 21 °C. The amount of NANA released into the supernatant was determined by the colorimetric method of Warren 29 which had a sensitivity of 0.1/ /tg as calibrated by standard curves. Blocking of negative charges of NANA is based on the introduction of positive charges into the carbohydrate chains of glycoconjugates via Schiff base formation with N-N-dimethyl-m-phenylene diamine (PD) 25,28. We followed the original method ofVeh et al. 28 which includes the following steps: (1)
mild oxidation of the samples with periodic acid (5 mM, 25 °C, 10 min); (2) Schiff base formation with PD (0.2%, pH 5.0, 25 °C, 60 min) in a sodium acetate buffer. Each step was followed by a thorough rinse in the appropriate buffer and subsequent centrifugation. The myelin fractions were then incubated with CF as described above. The myelin pellets were washed 3 times with the same buffer solution and then fixed with buffered 2.5% glutaraldehyde and postfixed with 2% osmium tetroxide. The samples were dehydrated in an ethanol series, embedded in Epon 812 and sectioned on a Reichert Ultracut. Sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 400 fitted with a goniometer stage. Part of the homogenates treated with CF and AF were freeze-fractured according to standard techniques 17. RESULTS
CF, AF and NF binding to myelin membranes CF at pH 7.4 binds strongly to the double intraperiod lines. The ferritin particles were clearly observed as strands of regularly spaced, electrondense globules within the intraperiod space (the former extracellular space). Such labelling of the extracellular membrane leaflets could be found regularly in the successive layers of myelin lamellae (Fig. 1A). The incorporation of the CF molecules into the myelin complex led to an expansion of the intraperiod space to about 10 nm: the intraperiod space in myelin homogenates not treated with CF was about 1-1.5 nm. Oblique sections across myelin membranes showed a crystalline arrangement of the CF particles (Fig. 1B). The centre-to-centre spacing of the particle arrays was about 11 nm, this being very close to the dimension of the ferritin molecule. Where splitting of the lamellar complex along the intraperiod line occurred, labelling of both the exposed extracellular surfaces of the membranes was evident. In such cases, double rows of CF particles were found within the intraperiod space (Fig. 1C). In purified myelin labelled with CF at pH 2.0 (final pH of the myelin/ferritin solution) the binding was less prominent than at pH 7.4. The occurrence of ferritin molecules within the intraperiod space after treatment at this low pH indicates the presence of
Fig. 1. A: a fraction of myelin treated with CF (pI 8.5). The ferritin molecules are aligned in the intraperiod space between the consecutive layers of the lamellar complexes. B: grazing sections reveal a crystalline arrangement of the ferritin molecules (arrows). C: splitting of the lamellar complex along the intraperiod space reveals that both extracellular leaflets are labelled with CF. D: labelling of strong acidic groups in myelin vesicles at pH 2.0. E and F: no labelling effect after using anionic ferritin derivatives (native ferritin 4.5 and anionized ferritin 4.0). pH of the incubation media, if not indicated, was 7.4. All micrographs are reproduced to a final magnification of × 90,000.
228
E Fig. 2. A and B : post-treatment with poly-L-lysine (A) and a high concentration of sodium chloride (B) led to a displacement of CF. C and D: enzymatic digestion: proteolysis and nemaminidase treatment did not have any effect on the intralamellar CF distribution. E: there are signs of CF within the intraperiodic space despite treatment with neuraminidaseat pH 2.0, indicating the presence of strongly acidic groups other than the carboxyl groups of sialoglycoproteins.
strongly acidic groups associated with the external leaflets o f the m e m b r a n e s (Fig. 1D). A F a n d N F did n o t enter the lamellar complex. Occasionally, small
aggregates or single A F particles were seen o n the outer surfaces o f the myelin vesicles (Fig. 1E, F).
229
Fig. 3. A: freeze-fracture view of myelin E-face from CF-treated material. Note the crystalline arrangement of the ferritin particles. B : freeze-fracture view of myelin treated with A F showing the absence of any crystalline arrangements of ferritin particles.
230
Poly-L-lysine and sodium chloride treatment Pretreatment of homogenates with poly-L-lysine was found not to inhibit CF labelling completely. Treatment of the homogenates with poly-L-lysine (21 h at 21 °C) after incubation was necessary to displace the CF molecules from their binding sites (Fig. 2A). Similarly, treatment with a solution of high ionic strength (1 M sodium chloride) effectively displaced the CF particles (Fig. 2B).
Enzyme treatment These experiments were intended to provide a rough characterization of the electrostatic binding sites. The incubation of the pure myelin homogenates with 1 ~o trypsin led to a complete loss of the basic protein and the Wolfgram protein. Furthermore, the proteolipid protein was removed from the membranes, as indicated by SDS gel electrophoresis. This finding is in accordance with the results of Raghavan et al. 2° and Wood et al. z0. However, intralamellar binding of CF was not prevented by treatment with trypsin prior to the ferritin incubation and the distribution of the CF molecules was identical to that of untreated specimens (Fig. 2C), although the major dense line could no longer be discerned. Similar results were obtained after treatment with neuraminidase. Both enzymes used in this study showed no effect on the distribution of CF: even the outer layers of the myelin vesicles, which are readily accessible to the enzyme, were consistently labelled by CF. However, the amount of NANA in the supernatant as measured by the colorimetric method of Warren 29 was only about 0.1~).2/zm per 100 mg myelin (wet weight). So to further test whether or not NANA is a major membrane constituent responsible for binding CF we performed blocking experiments using the PD technique (see Materials and Methods). Mild oxidation with periodic acid and subsequent treatment with PD has been shown to block the staining of sialic acid by certain dyesz5 but does not influence the binding of CF to even the outermost myelin lamella of our material. Incubation of the material treated with neuraminidase and then with CF at pH 2.0 did not appear to affect the distribution of ferritin within the lamellae (Fig. 2D) compared with material treated at the same low pH but without neuraminidase (Fig.
2E). However, the preservation of myelin at this low pH was poor, making the identification of individual myelin lamellae difficult.
Freeze-fracture preparation Replicas prepared from material treated with CF showed a striking crystalline arrangement of the ferritin particles associated apparently with the entire membrane surface (Fig. 3A). The ferritin particles were evident only by the impression they made on the E-face that covered them, indicating that the membranes had probably split at the level of the hydrophobic interior of the membrane (along the major dense line). In samples treated with AF, a comparable phenomenon was not evident: the membrane faces appeared smooth with intramembranous particles distributed randomly over them. DISCUSSION
Accessibility of the intraperiod space The binding of CF to the external surfaces of the lamellar complexes showed a regular, well-ordered distribution similar to that previously described in crude brain homogenates by Bittinger and Heid 2 and by Linington and RumsbylL Our results indicate further that the observed binding of CF is specific for anionic sites on the surfaces on the membranes and that, although the intraperiod space was accessible to considerable amounts of CF, AF and NF did not gain access. The deposition of the CF particles within the intraperiod space was undoubtedly due to the strong electrostatic attraction of this polycation by the anionic sites, to the extent that it probably forced the external surfaces apart. From the results of tracer experiments using in vivo techniques it seems likely that the accessibility of different molecules to the intraperiod space depends on their dimensiona,9,2z,zz. In contrast, the results presented here of in vitro experiments using 3 types of ferritin which differ principally in their charge (and not in their size) show that the net charge of the tracer is a decisive factor with respect to its ability to gain access to the intraperiod space. The accessibility of the intraperiod space may therefore depend on the charge of not only exogenous tracers but also endogenous proteins.
231
The surface charge of myelin CF has been used at a physiological level of pH as a marker for anionic surface charges7,16, whereas at low pH it appears to act as a reliable indicator of strong acidic groups such as the carboxyl groups of membrane sialoglycoproteinsa, 17. The intense and consistent labelling of the intraperiod space by CF at pH 7.4 and pH 2.0 indicates the presence of numerous anionic and acidic sites, respectively. Treatment of homogenates with trypsin led to a dissolution of the principal myelin proteins, as shown by the results of SDS electrophoresis, but did not alter the distribution of intralamellar CF, suggesting that the major proteins are not involved in binding CF. Similarly, neuraminidase treatment did not influence the binding of CF, although the amount of N A N A released was at the lower limit of that which can be detected by the colorimetric method. Considering the results of Suzuki et al. 26 and Quarles and Everlyt9 on isolated glycoproteins and gangliosides, this amount is considerably less than the estimated total concentration of N A N A in myelin. This may be in part be due to a heterogeneous distribution of N A N A in the myelin fraction18,19, ~6 so that our use of neuraminidase led only to an enzymatic cleaving of the freely accessible N A N A molecules. Stronger evidence that the carboxyl groups of N A N A contribute little to the net negative charge on the intraperiod surfaces and thus the binding of CF is provided by the results of the blocking experiments. The selective blocking with PD of carboxyl groups of N A N A resulted in no change in the distribution of CF. It is, however, possible that the enzyme treatment uncovers anionic sites that, despite the extraction of protein and the presence of blocking agent, lead to a persistent binding of CF to the intraperiod surfaces, although this possibility has yet to be tested. At the moment it
REFERENCES Ben-Ishay, Z., Reichert, F. and Gallily, R., Crystallinelike surface charge array of rnurine macrophages and lymphocytes: visualization with cationized ferritin, J. ultrastruct. Res., 53 (1975) 119-127. 2 Bittinger, H. and Heid, J., The subcellular distribution of particle-bound negative charges in rat brain, J. Neurochem., 28 (1977) 917-922. 3 Braun, P. E., Molecular architecture of myelin. In P. 1
appears that proteins are not responsible for CF binding but that some other membrane components must be involved, as suggested by the observation of Burry and Wood 5 that CF can label pure phospholipid vesicles. Moreover, the polar sulphate groups of galactolipids may also bind CF electrostatically3. Accordingly, one expects there to exist an 'electric bilayer' between adjacent lamellar complexes as suggested by Bittinger and Heid 2 and demonstrated by Rand et al. 21 for peripheral myelin.
Crystalline orientation of the CF particles The crystalline arrangement of the CF particles is particularly noteworthy. Ben-Ishay et al. 1 found similar crystalline arrangements of CF in restricted areas of the exterior surface of macrophages and lymphocytes and suggested that these assemblies might reflect the crystalline arrangement of some surface component such as an antigenic determinant. This explanation is not tenable in our case where CF particles appeared to have a crystalline arrangement over the entire surface of the outer leaflets of the lamellar complexes. It is probable that the negative charges responsible for binding CF are distributed randomly over the membrane, but that the ferritin complex is too large to resolve this and consequently arrange themselves according to their own molecular and electrostatic geometry. ACKNOWLEDGEMENTS Dr. J.-P. Revel provided stimulating discussions and helpful advice. Drs. C. S. Raine and H. deF. Webster are thanked for their comments on reading the manuscript. This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (SFB 114).
Morell (Ed.), Myelin, Plenum Press, New York, 1977, pp. 91-115. Brightman, M. W., The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. II. Parenchymal distribution, Amer. J. Anat., 117 (1965) 193-219. Burry, R. W. and Wood, J. D., Contribution of lipids and proteins to the surface charges of membranes: an electron microscopy study with cationized and anionized ferritin, J. CellBioL, 82 (1979) 726-741.
232 6 Caspar, D. L. D., Melchior, V., HoUingshead, C. J. and Kirschner, D. A., Dynamics of myelin membrane contacts. In N. B. Gilula (Ed.), Membrane-Membrane Interactions, Raven Press, New York, 1980, pp. 195-211. 7 Danon, D., Goldstein, L., Marikovsky, Y. and Skutelsky, E., Use of cationized ferritin as a label of negative charges on cell surfaces, J. ultrastruct. Res., 38 (1972) 500-510. 8 De Bruyn, P. P. H. and Michelson, S., Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae, J. Cell Biol., 82 (1979) 708-714. 9 Feder, N., Microperoxidase: an ultrastructure tracer of low molecular weight, J. Cell BioL, 51 (1971) 339-343. 10 Jersild, R. A. and Crawford, R. W., The distribution and mobility of anionic sites on the brush border of intestinal absorptive cells, Amer. J. Anat., 152 (1978) 287-306. 11 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage, Nature (Lond.), 227 (1970) 680-685. 12 Linington, C. and Rumsby, M. G., Accessibility of galactosyl ceramides to probe reagents in central nervous system myelin, J. Neurochem., 35 (1980) 983-992. 13 Matoska, J. and Siracky, J., Negative surface charge of epithelial cells from benign and malignant human breast tissues in organ culture, BioL Cellulaire, 39 (1980) 221-224. 14 Melchior, V., Hollingshead, C. D. and Caspar, D. L. D., Divalent cations cooperatively stabilized close membrane contacts in myelin, Biochim. biophys. Acta, 554 (1979) 204-226. 15 Norton, W. T., Isolation of myelin from nerve tissue. In S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. 31, Academic Press, New York, 1974, pp. 435-444. 16 Pinto da Silva, P., Moss, P. S. and Fudenberg, H. H., Anionic sites on the membrane intercalated particles human erythrocyte ghost membranes: freeze-etch localization, Exp. Cell Res., 81 (1973) 127-138. 17 Pinto da Silva, P., Parkinson, S. and Dwyer, N., Fracturelabel: cytochemistry of freeze-fracture faces in the erythrocyte membrane, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 343-347. 18 Quarles, R. H., Glycoproteins in myelin and myelin-
19
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related membranes, In R. U. Margolis and R. K. Margolis (Eds.), Complex Carbohydrates of Nervoas Tissue, Plenum Press, New York, 1979, pp. 209-233. Quarles, R. H. and Everly, J. L., Glycopeptide fractions prepared from purified central and peripheral rat myelin, Biochim. biophys. Acta, 466 (1977) 176-186. Raghavan, S. S., Rhoads, D. B. and Kanfer, J. N., The effect of trypsin on purified myelin, Biochim. biophys. Acta, 328 (1973) 205-212. Rand, R. P., Fuller, N. L. and Lis, L. J., Myelin swelling and measurement of forces between myelin membranes, Nature (Lond.), 279)(1979) 258-260. Reier, P. J., Tabira, R. and Webster, H. DEF., q he penetration of fluorescein-conjugated and electron-dense tracer protein into Xenopus tadpole optic nerves following perineural injection, Brain Research, 102 (1976) 229-244. Revel, J.-P., and Hamilton, D. W., The double nature of the intermediate dense line in peripheral nerve myelin, Anat. Rec., 163 (1969) 7-16. Skutelsky, E. and Hardy, B., Regneration of plasmalemma and surface properties in macrophages, Exp. Cell. Res., 101 (1976) 337-345. Spicer, S. S., Diamine methods for differentiating mucosubstances histochemically, J. Histochern. Cytochem., 13 (1963) 211-234. Suzuki, K. S., Poduslo, J. F. and Poduslo, S. E., Further evidence for a specific ganglioside fraction closely associated with myelin, Biochim. biophys. Acta, 152 (1968) 576-586. ThiJrauf, N. and Dermietzel, R., An improved technique for the preparation of polycationic ferritin-derivatives, Stain TechnoL, in press. Veh, R. W., Meessen, D., Kuntz, H. D. and May, B., Histochemical demonstration of side-chain substituted sialic acids. In R. A. Malt and R. C. N. Williamson (Eds.), Colonic Carcinogenesis, MTP Press, Lancaster, 1981, pp. 355-365. Warren, L., Thiobarbituric assay of sialic acids. In S. P. Colowick and N. O. Kaplan (Eds.), Methods in Enzymology VI, Academic Press, New York, 1963, pp. 463-465. Wood, J. G., Dawson, R. M. C. and Hauser, H., Effect of proteolytic attack on the structure of CNS myelin membrane, J. Neurochem., 22 (1974) 637-643.
225
Cytochemical Demonstration of Negative Surface Charges in Central Myelin R. DERMIETZEL, N. TH1]RAUF and D. SCHLINKE
lnstitut fiir Anatomie, Universitiitsklinikum der GHS Essen, Hufelandstrasse 55, 4300 Essen 1 (F.R.G.) (Accepted August 10th, 1982)
Key words: myelin cytochemistry - - negative charges - - labelling - - cationic ferritin
Homogenates of central myelin were treated with ferritin derivatives having different isoelectric points. It was found that considerable amounts of cationic ferritin (pI 8.5-9.5) had access to the extracellular space, but that anionic ferritin (pl 4.0) and native ferritin (pI 4.5) did not. The electrostatic nature of the binding of cationic ferritin was demonstrated by treating the homogenates with poly-L-lysineand 1 M NaC1: both reagents led to a complete displacement of the bound cationic ferritin. Neither extensive trypsination nor neuraminidase treatment showed a significant effect on the intralamellar distribution of the bound cationic ferritin molecules. This suggests that the net negative charge on the extracellular myelin face stems primarily from acidic lipid groups in the membrane. INTRODUCTION The myelin sheath is composed of a multilamellar system of bilayers. Myelin in the central nervous system (CNS) is formed b3 the oligodendrocytes and involves the wrapping of glial cell membrane spirally around a segment of axon. The maintenance of this arrangement of membranes requires specific molecular interactions between the electrostatic repulsive forces of the acidic lipid layers and long range electrodynamic attraction6, t4. We have attempted to examine in detail the nature of the electrostatic forces using a cytochemical probe. A particularly useful probe for visualizing the distribution of negatively charged groups on the surface of membranes is polycationic ferritin 7. This substance has been found to be effective in demonstrating the distribution of anionic charges on the surface of living cells at physiological pH10,13,16,24. The present study uses cationized ferritin (CF), anionized ferritin (AF) and native ferritin (NF) and the results suggest that the electrostatic interaction between pairs of myelin membranes depends upon the net negative charge from acidic lipid groups in the membranes. MATERIALS AND METHODS
Animal and tissue preparation Anaesthetized Wistar rats (Hannover
strain)
0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
weighing 300-350 g were used. The entire brain was removed as quickly as possible and myelin prepared immediately according to the standard procedure as described by Norton 15.
Anionized ferritin (AF) and cationized ferritin ( CF) AF was prepared from native ferritin (NF) (Sigma, cadmium content < 1.0 ~ ) by the procedure by Burry and WoodL An LKB multiphore system was used for isoelectric focusing, which gave a pI of between 3.7 and 4.0 for A F as compared to a value of 4.5 for NF. CF was prepared by a modification of the method of D a n o n et al. 7, described by Th/irauf and Dermietze127. The isoelectric point of CF ranged from 8.5 to 9.2. Unstained A F and N F were spread on carbon-coated grids and examined in the electron microscope to check whether or not the particles were completely dispersed and were only used when free of particle clusters. Labelling procedure A solution o f C F , A F or N F dissolved in 0.15 M NaC1 and containing 10 mg ferritin/ml was added to 2 ml of myelin homogenate suspended in Tris-HC1 buffer. The final p H of the different ferritin/buffer solutions was: CF/buffer p H 7.4; AF/buffer p H 7.2; NF/buffer p H 7.2. In some instances the homogenates were incubated in CF dissolved in 0.15 M
226 NaCI at pH 2.0. After preparation the suspensions were immediately placed on ice in a probe sonicator for 5 min. Since sonication of the suspension did not affect the integrity of the lamellar complexes it was routinely used. To check whether treatment with a polycation leads to an inhibition of the CF labelling, the homogenates were incubated with poly-L-lysine (5 mg/ml, Sigma, type VI).
Trypsin treatment and control of protein removal To test the effect of trypsination, aliquots of each homogenate (containing about 100 mg myelin wet weight) were incubated in a 0.01 M Tris-HCl buffer (pH 7.4) with or without the addition of 1% trypsin (Sigma type XII). In each case the final volume was adjusted to 2.0 ml and the samples incubated for 24 h at room temperature. At the end of the incubation period the samples were placed in an ice bath and then centrifuged at 9000g for 10 min at 4 °C. Part of the trypsin treated material was incubated with CF as described above. The rest was delipidated in chloroform-methanol (2:l, v/v), then acetone and solubilized in 3 % SDS and 5 % mercaptoethanol. SDS gel electrophoresis and staining with Coomassie blue were performed according to the method of Laemmlila. Neuraminidase incubation and blocking of N-acetylneuraminic acid ( NANA) Samples of the homogenates (I00 mg wet weight) were treated with neuraminidase from either Vibrio cholera (Behring, Marburg) or Clostridium perfringens (Sigma, type V). The activity of both enzymes was 1 U/ml. The enzymes were dissolved in a sodium acetate buffer (pH 5.5) and 0.5 ml of this solution was added at the beginning of incubation period with a subsequent addition of 0.5 ml after 6 h. The samples were incubated for a total of 12 h at 21 °C. The amount of NANA released into the supernatant was determined by the colorimetric method of Warren 29 which had a sensitivity of 0.1/ /tg as calibrated by standard curves. Blocking of negative charges of NANA is based on the introduction of positive charges into the carbohydrate chains of glycoconjugates via Schiff base formation with N-N-dimethyl-m-phenylene diamine (PD) 25,28. We followed the original method ofVeh et al. 28 which includes the following steps: (1)
mild oxidation of the samples with periodic acid (5 mM, 25 °C, 10 min); (2) Schiff base formation with PD (0.2%, pH 5.0, 25 °C, 60 min) in a sodium acetate buffer. Each step was followed by a thorough rinse in the appropriate buffer and subsequent centrifugation. The myelin fractions were then incubated with CF as described above. The myelin pellets were washed 3 times with the same buffer solution and then fixed with buffered 2.5% glutaraldehyde and postfixed with 2% osmium tetroxide. The samples were dehydrated in an ethanol series, embedded in Epon 812 and sectioned on a Reichert Ultracut. Sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 400 fitted with a goniometer stage. Part of the homogenates treated with CF and AF were freeze-fractured according to standard techniques 17. RESULTS
CF, AF and NF binding to myelin membranes CF at pH 7.4 binds strongly to the double intraperiod lines. The ferritin particles were clearly observed as strands of regularly spaced, electrondense globules within the intraperiod space (the former extracellular space). Such labelling of the extracellular membrane leaflets could be found regularly in the successive layers of myelin lamellae (Fig. 1A). The incorporation of the CF molecules into the myelin complex led to an expansion of the intraperiod space to about 10 nm: the intraperiod space in myelin homogenates not treated with CF was about 1-1.5 nm. Oblique sections across myelin membranes showed a crystalline arrangement of the CF particles (Fig. 1B). The centre-to-centre spacing of the particle arrays was about 11 nm, this being very close to the dimension of the ferritin molecule. Where splitting of the lamellar complex along the intraperiod line occurred, labelling of both the exposed extracellular surfaces of the membranes was evident. In such cases, double rows of CF particles were found within the intraperiod space (Fig. 1C). In purified myelin labelled with CF at pH 2.0 (final pH of the myelin/ferritin solution) the binding was less prominent than at pH 7.4. The occurrence of ferritin molecules within the intraperiod space after treatment at this low pH indicates the presence of
Fig. 1. A: a fraction of myelin treated with CF (pI 8.5). The ferritin molecules are aligned in the intraperiod space between the consecutive layers of the lamellar complexes. B: grazing sections reveal a crystalline arrangement of the ferritin molecules (arrows). C: splitting of the lamellar complex along the intraperiod space reveals that both extracellular leaflets are labelled with CF. D: labelling of strong acidic groups in myelin vesicles at pH 2.0. E and F: no labelling effect after using anionic ferritin derivatives (native ferritin 4.5 and anionized ferritin 4.0). pH of the incubation media, if not indicated, was 7.4. All micrographs are reproduced to a final magnification of × 90,000.
228
E Fig. 2. A and B : post-treatment with poly-L-lysine (A) and a high concentration of sodium chloride (B) led to a displacement of CF. C and D: enzymatic digestion: proteolysis and nemaminidase treatment did not have any effect on the intralamellar CF distribution. E: there are signs of CF within the intraperiodic space despite treatment with neuraminidaseat pH 2.0, indicating the presence of strongly acidic groups other than the carboxyl groups of sialoglycoproteins.
strongly acidic groups associated with the external leaflets o f the m e m b r a n e s (Fig. 1D). A F a n d N F did n o t enter the lamellar complex. Occasionally, small
aggregates or single A F particles were seen o n the outer surfaces o f the myelin vesicles (Fig. 1E, F).
229
Fig. 3. A: freeze-fracture view of myelin E-face from CF-treated material. Note the crystalline arrangement of the ferritin particles. B : freeze-fracture view of myelin treated with A F showing the absence of any crystalline arrangements of ferritin particles.
230
Poly-L-lysine and sodium chloride treatment Pretreatment of homogenates with poly-L-lysine was found not to inhibit CF labelling completely. Treatment of the homogenates with poly-L-lysine (21 h at 21 °C) after incubation was necessary to displace the CF molecules from their binding sites (Fig. 2A). Similarly, treatment with a solution of high ionic strength (1 M sodium chloride) effectively displaced the CF particles (Fig. 2B).
Enzyme treatment These experiments were intended to provide a rough characterization of the electrostatic binding sites. The incubation of the pure myelin homogenates with 1 ~o trypsin led to a complete loss of the basic protein and the Wolfgram protein. Furthermore, the proteolipid protein was removed from the membranes, as indicated by SDS gel electrophoresis. This finding is in accordance with the results of Raghavan et al. 2° and Wood et al. z0. However, intralamellar binding of CF was not prevented by treatment with trypsin prior to the ferritin incubation and the distribution of the CF molecules was identical to that of untreated specimens (Fig. 2C), although the major dense line could no longer be discerned. Similar results were obtained after treatment with neuraminidase. Both enzymes used in this study showed no effect on the distribution of CF: even the outer layers of the myelin vesicles, which are readily accessible to the enzyme, were consistently labelled by CF. However, the amount of NANA in the supernatant as measured by the colorimetric method of Warren 29 was only about 0.1~).2/zm per 100 mg myelin (wet weight). So to further test whether or not NANA is a major membrane constituent responsible for binding CF we performed blocking experiments using the PD technique (see Materials and Methods). Mild oxidation with periodic acid and subsequent treatment with PD has been shown to block the staining of sialic acid by certain dyesz5 but does not influence the binding of CF to even the outermost myelin lamella of our material. Incubation of the material treated with neuraminidase and then with CF at pH 2.0 did not appear to affect the distribution of ferritin within the lamellae (Fig. 2D) compared with material treated at the same low pH but without neuraminidase (Fig.
2E). However, the preservation of myelin at this low pH was poor, making the identification of individual myelin lamellae difficult.
Freeze-fracture preparation Replicas prepared from material treated with CF showed a striking crystalline arrangement of the ferritin particles associated apparently with the entire membrane surface (Fig. 3A). The ferritin particles were evident only by the impression they made on the E-face that covered them, indicating that the membranes had probably split at the level of the hydrophobic interior of the membrane (along the major dense line). In samples treated with AF, a comparable phenomenon was not evident: the membrane faces appeared smooth with intramembranous particles distributed randomly over them. DISCUSSION
Accessibility of the intraperiod space The binding of CF to the external surfaces of the lamellar complexes showed a regular, well-ordered distribution similar to that previously described in crude brain homogenates by Bittinger and Heid 2 and by Linington and RumsbylL Our results indicate further that the observed binding of CF is specific for anionic sites on the surfaces on the membranes and that, although the intraperiod space was accessible to considerable amounts of CF, AF and NF did not gain access. The deposition of the CF particles within the intraperiod space was undoubtedly due to the strong electrostatic attraction of this polycation by the anionic sites, to the extent that it probably forced the external surfaces apart. From the results of tracer experiments using in vivo techniques it seems likely that the accessibility of different molecules to the intraperiod space depends on their dimensiona,9,2z,zz. In contrast, the results presented here of in vitro experiments using 3 types of ferritin which differ principally in their charge (and not in their size) show that the net charge of the tracer is a decisive factor with respect to its ability to gain access to the intraperiod space. The accessibility of the intraperiod space may therefore depend on the charge of not only exogenous tracers but also endogenous proteins.
231
The surface charge of myelin CF has been used at a physiological level of pH as a marker for anionic surface charges7,16, whereas at low pH it appears to act as a reliable indicator of strong acidic groups such as the carboxyl groups of membrane sialoglycoproteinsa, 17. The intense and consistent labelling of the intraperiod space by CF at pH 7.4 and pH 2.0 indicates the presence of numerous anionic and acidic sites, respectively. Treatment of homogenates with trypsin led to a dissolution of the principal myelin proteins, as shown by the results of SDS electrophoresis, but did not alter the distribution of intralamellar CF, suggesting that the major proteins are not involved in binding CF. Similarly, neuraminidase treatment did not influence the binding of CF, although the amount of N A N A released was at the lower limit of that which can be detected by the colorimetric method. Considering the results of Suzuki et al. 26 and Quarles and Everlyt9 on isolated glycoproteins and gangliosides, this amount is considerably less than the estimated total concentration of N A N A in myelin. This may be in part be due to a heterogeneous distribution of N A N A in the myelin fraction18,19, ~6 so that our use of neuraminidase led only to an enzymatic cleaving of the freely accessible N A N A molecules. Stronger evidence that the carboxyl groups of N A N A contribute little to the net negative charge on the intraperiod surfaces and thus the binding of CF is provided by the results of the blocking experiments. The selective blocking with PD of carboxyl groups of N A N A resulted in no change in the distribution of CF. It is, however, possible that the enzyme treatment uncovers anionic sites that, despite the extraction of protein and the presence of blocking agent, lead to a persistent binding of CF to the intraperiod surfaces, although this possibility has yet to be tested. At the moment it
REFERENCES Ben-Ishay, Z., Reichert, F. and Gallily, R., Crystallinelike surface charge array of rnurine macrophages and lymphocytes: visualization with cationized ferritin, J. ultrastruct. Res., 53 (1975) 119-127. 2 Bittinger, H. and Heid, J., The subcellular distribution of particle-bound negative charges in rat brain, J. Neurochem., 28 (1977) 917-922. 3 Braun, P. E., Molecular architecture of myelin. In P. 1
appears that proteins are not responsible for CF binding but that some other membrane components must be involved, as suggested by the observation of Burry and Wood 5 that CF can label pure phospholipid vesicles. Moreover, the polar sulphate groups of galactolipids may also bind CF electrostatically3. Accordingly, one expects there to exist an 'electric bilayer' between adjacent lamellar complexes as suggested by Bittinger and Heid 2 and demonstrated by Rand et al. 21 for peripheral myelin.
Crystalline orientation of the CF particles The crystalline arrangement of the CF particles is particularly noteworthy. Ben-Ishay et al. 1 found similar crystalline arrangements of CF in restricted areas of the exterior surface of macrophages and lymphocytes and suggested that these assemblies might reflect the crystalline arrangement of some surface component such as an antigenic determinant. This explanation is not tenable in our case where CF particles appeared to have a crystalline arrangement over the entire surface of the outer leaflets of the lamellar complexes. It is probable that the negative charges responsible for binding CF are distributed randomly over the membrane, but that the ferritin complex is too large to resolve this and consequently arrange themselves according to their own molecular and electrostatic geometry. ACKNOWLEDGEMENTS Dr. J.-P. Revel provided stimulating discussions and helpful advice. Drs. C. S. Raine and H. deF. Webster are thanked for their comments on reading the manuscript. This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (SFB 114).
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