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Ultrastructure and Some Properties of the Surface Coat of Chilodonella steini and Chilodonella cucullulus
Arch. Protistenk. 123 (1980): 367 -381
Zoological Institute, Dept. of Cytology, University of 'Warsaw
Ultrastructure and Some Properties of the Surface Coat of Ohilodonella steini and Ohilodonella cucullulus By MALGORZATA GOLEMBIEWSKA and STEFAN RADZIKOWSKI With 16 Figures
Summary Using specific staining and digestion by enzymes it has been shown that hyaluronic acid and other acid mucopolysaccharides are included in the composition of the surface coat in Chilodonella steini and CMlodonella cucullulus. In both species abrupt rejection of the surface coat from the whole cell surface and formation of pseudocysts take place as a result of the action of L. Light ribonuclease 4 X cryst. preparation. Partial destruction of the outer membranes of the pellicular alveoli was observed in electronograms of cells subjected to the action of this preparation. This reversible reaction is not connected with the action of RNAse itself as an enzyme, but probably by admixtures in it of a different character. This property was utilized to examine the role of the surface coat in the conjugation process. The absence of agglutination reaction in cells of opposite mating types deprived of their surface coat indicates that the receptors responsible for this process are present in this particular layer of the cell.
Introduction In animal cells the cell membrane is covered by a surface coat defined as a, ,glycocalyx" and composed chiefly of substances of a carbohydrate character (BENNETT 1963; GASIC and GASIC 1962 ; MARTINEZ-PALOMO 1970; RAMBOURG 1971). Its chemical composition and the functions it carries out differentiates the cells (BENNETT 1969; SZUBINSKA and LUFT 1971; PINTO da SILVA et al. 1975; WYROBA and PRZELECKA 1973; POLLOCK 1978). A considerable amount of data are to be found in literature relating to the general occurrence of the surface coat in representatives of all groups of Protozoa. It is known that in different ciliates it plays an important part in intercellular interactions preceding the process of conjugation, and also in the process of cell union (MIYAKE 1974; HONDA 1976; WOLFE 1974). In dorso-ventrally flattened representatives of the genus Okilodonella, Ok. steini and Ok. cucullulus, an argentophil reticulum was found to occur on both surfaces of the cell (RADZIKOWSKI 1966; DOBRZANSKA-KACZANOWSKA 1965; RADZIKOWSKI and GOLEMBIEWSKA 1977), and observations in vivo pointed to the presence in the cortex of numerous alveole with fluid contents (KAczANOWSKA-KoWALSKA 1969; RADZIKOWSKI and GOLEMBIEWSKA 1977). 24
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Studies III an eleotron mioroscope showed that the argentophil reticulum visible in vivo and on silvered slides, termed by CHATTON and LWOFF 1935 "argyrome", constitutes the sculpture of the surface resulting from the pellicular membrane system. The borders of pellicular alveoli become silvered (PUYTORAO 1959; EHRET and PoWERS 1959; PITELKA 1961, 1965; DIPPEL 1962). There are, however, no data on the surface coat of these ciliates in the abundant literature on the morphology and morphogenetic processes of these two species (RADZIKOWSKI 1966; KAOZANOWSKA and KOWALSKA 1968; KOWALSKA and KAOZANOWSKA 1969; SOLTYNSKA 1971; RADZIKOWSKI and GOLEMBIEWSKA 1977). An attempt has therefore been made at defining the chemical composition, ultrastructure and part played by the surface coat in the conjugation process in these two species.
Material and Methods The studies were made on two species of the genus Okilodonella,' Ok. steini and Ok. cucullulus. The methods used for their culture have been described in the papers by RADZIKOWSKI (1966) for Ok. cucullulus and RADZIKOWSKI and GOl.EMBIEWSKA (1977) for Ok. steini. The following staining methods were used to define the structure and chemical composition of the surface coat: 1. Staining with alcian blue for acid mucopolysaccharides The material was fixed in a mixture of 96 % ethyl alcohol and formalin in parts of 9: 1. Staining with alcian blue 8G E. Gurr Ltd. London SW 14, was carried out after SCOTT and DORLING (1965). 2. Staining with Ruthenium red for acid mucopolysaccharides Standard Ruthenium red stain produced by Fluka AG, Buchs S. G. was used for staining carried out in accordance with the following procedure, used in electron microscopy (LUFT 1971): A - 5 mg Ruthenium red solution was dissolved in 1 ml of distilled water heated to 60 °e, then cooled B - 3.6 % glutaraldehyde solution in 0.2 cacodylic buffer, pH 7.3 e - 3 mg Ruthenium red solution dissolved in 1 ml of distilled water, heated to 60 °e, then cooled, adding 1 ml of osmium tetroxide and 1 ml pure cacodylic buffer. Solutions A and B were mixed immediately before use. Fixing lasted 1 h at a temperature of
o °e, followed by rinsing in three changes of cacodylic buffer, after which the material was trans· ferred to solution C for three hours. 3. PAS reaction to polysaccharides Reactions with periodic acid and Schiff reagent were made after PEARS 1957. Enzymatic reactions were made using the following enzymes:
1. Proteolytic enzymes Tripsin inhibitor ovomucoid salt·free, Koch.Light Laboratories Ltd. eolnbrook, Bucks, England, at a concentration of 0.1 mg/ml in 0.05 M phosphate buffer with pH 6; Pronase ex Streptomyces gri88u8 Koch-Light Laboratories Ltd. Colnbrook, Bucks, England no. 66269, at a concentration of 0.1 mg/ml in 0.05 M phosphate buffer with pH 6. 2. Carbohydrate-specific for hydrolases lX-amylase ex bacteria, produced by Fluke A. G., concentration 1 %. Diastase from malt, produced by BDH, concentration 1 % Hialuronidase, produced by WSiS, Warsaw, strength 3,200 i. u.
mtrastructure and Some Properties of the Surface Coat
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3. Nucleases Ribonuclease salt and protease free 40 k Unit vlmgm, 4 X cryst. ex bovine pancreas, produced by L. Light and Co. Ltd., Colnbrook, England. This preparation was used in three variants: not heated. after incubation at 90°C for 5 min, after incubation at 100°C for 30 min. The preparation was heated to 90°C in order to free it of any possible pollution by other enzymes, while incubation at 100°C caused denaturation of the enzyme it~elf. Ribonuclease ex bovine pancreas salt·free, 4 X cryst., and ribonuclease ex bovine pancreas salt.free, 5 X cryst., produced by Koch-Light Laboratories Ltd., Colnbrook, Bucks, England. Ribonuclease Type IA ex bovine pancreas, 5 X cryst., produced by Serva. RNA-se solutions were used at concentrations from 0.5 to 1 % in a veronal buffer and in a culture watar solution with pH 7.7 and 7.4 respectively. The following methods were used for examination of the structure of the cortex and surface coat: 1. Impregnation with salts of silver a) with nitrate of silver after CHATToN-LwoFF, as modified by CORLISS 1953; b) with protargol, produced by Establissement Rogues, Paris for silvering the surface by Bodian method. 2. Acting on living cells using the above enzymes and also organic and inorganic substances a) the following organic compounds were nsed: Concanavalin A in glucose solution, produced by Calbiochem, concentration 500-100 g/ml; Concanavalin A was used on account of its specific combining with (X-D-glucose and (X-Dmannose. (Concanavalin A administered to Stylonychia mytilu8 causes rejection of the surface coat - personal communication by AMMERMANN.) Formalin in concentrations from 0.01 to 0.1 %; Ethyl alcohol in concentrations from 0.1 to 4 %; Both these compounds are included in the composition of the fixing preparation. b) Inorganic compounds used: sulphate of ammonia, which enters into RNA-se production; calcium, potassium, barium and magnesium chloride in concentrations of 10, 20, 30 mM respectively; acetic, phosphe.te and veronal buffers with pH of 6.6 and 7.7. The techniques used in electron microscopy were employed to define the ultrastructure of the surface coat and cortex. The following fixing preparations were used: 2 % glutaraldehyde solution in a phosphate buffer with pH 7.2 and 1 % osmium tetroxide solution in the same buffer. The fixed ciliates were first embedded in 1 % agar-agar, then standard dehydration was carried out, after which the material was embedded in Epon 812 mixture. The material was sectioned on an LKB III ultramicrotome, and contrasted with uranyl acetate and lead citrate. The section were examined under a Tesla BS 500 and JEM-7A (Joelco) electron microscope. The staining method with Ruthenium red has been described earlier in this paper.
Results By staining ciliates with 1 % alcian blue solution it proved possible to discover the characteristic arrangement of small polygons on both the cell surfaces (Figs. 1 and 2), the position of which coincides with the position of pellicular alveoli. It would seem that alcian blue stains only the outer parts of the surface coat (Fig. 3). 24*
370
M.
GOLEMBIEWSKA
and ST.
RADZIKOWSKI
,
. .,
Fig. 1. Part of the ventral surface of Chilodonella steini stailled with alcian blue .
X
1I';,.'1
• . ·-0j!0, ........
,.;.• . r {
:,~.J;
1,500.
Fig. 2. Part of the dorsal surface of Chilodonella steini. Sta ining - a lcian blue. X 1,500. Fig. 3. Part of a Chilodonella cucullulu8 cell after 1 mho act ion of L. Light RNAse 4 X cryst Arrow points to material trailing b ehind cell. Alcian blue. X 1,500.
Ultrastructure and Some Properties of the Surface Coat
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In order to define the kind or kinds of substances included in the composition of the material stained with alcian blue, the fixed ciliates were digested by enzymes, then stained. The results of these experiments are set out in Table 1. Tabl e I. R esults of staining eells of Chilodonella steini and Chilodonella cucullulus with alcian blue after digestion by enzymes Enzymes
Result of staining
Trypsin Pronase (X· a mylase Diasta se Hyalw'onidase Ribonucleases (va rious)
stains reanily, deformation of pattern stains readily, deformation of pattern no e ffect no e ffect faint st a ining, losses in the staining p attern no e ffect
Analysis of the results given in Table 1 shows that proteolytic enzymes (trypsin and pronase) cause only spatial deformation of the pattern stained with alcian blue, without however affecting intensity of staining, which indicates that they do not digest t.he structures, which are actually stained. Hyaluronidase, on t.he other hand, causes mainly a decrease in intensity of staining and considerable losses in the pattern. In would appear that digestion by the remaining enzymes does not affect the capacity for staining and regularity of the staining pattern. Examination was also made in vivo of the effect on the behaviour of the cells treated by enzymes, organic and inorganic substances, and of the condition of the surface coat of these cells stained with alcian blue. The results of these experiments are given in Table 2. Reactions in v i v 0 due to the effect of factors from column A are given in column B. The state of the surface coat, stained with alcian blue, of cells previously subjected to the action of substances detailed in column A, are shown in column C. + indicates absence of significant differences in the pattern stained with alcian blue in comparison with control cells. - complete absence of any tendency to stain.
Fig. 4. Chilodonella cucullulus cell in the pseudocyst form as the l'esult of the action of RNAse prepara tion. Phase contrast. X 1,500. Fig. 5. Part of Chilodonella cucullulus cell aft er I min action of RNAse preparation. Alcian blue. X 1,500. Fig. 6. Part of Chilodonella cucullulus cell surface after 10 min action of RNAse. Absence of stain. ing re action with alcian blue. X 1,500.
372
M. GOLEMBIEWSKA and ST. RADZIKOWSKI
Table 2. Reactions of cells in vivo, and the state of the surface coat in these cells after treatment by different exogenic factors Kind of substance
Cell reaction
Result of staining
A
B
C
Trypsin Pronase (X·amylase Diastase Hyaluronidase
reversion reversion reversion reversion reversion
+ + + + +
RNAse L. Light 4 X cryst. a) in water and buffer b) after incubation at 90°C c) after incubat.ion at 100°C
reversion and formation of pseudocyst reversion and formation of pseudocyst reversion and formation of pseudocyst
RNAse Koch-Light a) 4 X cryst. in water and buffer b) 5 X cryst. in water and buffer
reversion reversion
+ +
reversion
+
approx. 40 % of cells adhere
+ +
RNAse A Serva a) in water and buffer Organic compounds Concanavalin A Formalin at different concentrations Ethanol at a concentrations of 1- 4 %
reversion with low concentrations reversion and retardation
+
Inorganic compounds Ammonium sulphate Chlorides Buffers
reversion receding reversion
+ +
reversion
+
In ciliates su bj ected in v i v 0 to the effect of 4 X cryst. ribonuclease produced by L. Light we observed, formation of a pseudocyst in which the cell is contained for a certain time (Fig. 5). Incubation of the L. Light ribonuclease preparation at temperatures of 90 and 100 °0 does not cause the preparation to lose its capacity for inducement of pseudocyst formation. Staining of cells with alcian blue after formation of the pseudocyst (Fig. 6) showed that in comparison with the control (Figs. 1 and 2), they are deprived of the characteristic pattern. This pattern occurs in a slightly deformed shape on the walls of the pseudocyst (Fig. 4).
Fig. 7. Part of ciliate surface one hour from time of transfer from RNAse solution to normal culture m edium. Alcian blue. X 1,500. Fig. 8. Part of ciliate surface three hours after transfer tl) cultUl'e medium. Alcia n blue. X 1,500. Fig. 9. Cell sUl'face 7 h from time of transfer to culture medium. Alcian blue. X1,500. Fig. 10. Chilodonclla cuculluZ.us. Electronogram of dorsal surfa ce with spadiciform structure. x 12,000. Fig. 11. Dorsal surface of ciliate silvcred by CHATTON-LwOFF m ethod. Arrows indicate distribution of spadiciform structures.
374
',.
M. GOl'.EMBIEWSKA and ST. RADZIK OW SKI
'w
'"
..
Figs. 12 and 13. Photograph under electron microscope part of cell cross section. Ventral side (12). Ruthenium red. X 12,000. Dorsal side (13). Ruthenium red. X 12,000.
Ultrastructure and Some Properties of thc Surface Coat
375
Figs. 14, 15.and 16. Electronogram of cells after action of RNAse prep a ration. Ruthenium red. x 14,000.
376
M. GOLEMBIEWSKA and ST. RADZIKOWSKI
Fig. 16. Plan of the experiment on the effect of L. Light RNAse 4 X cryst. preparation on conjugation reactions of Ohilodonella steini cells. Control procedure
Experimental procedure
ciliates placed in culture water
ciliates incubated for 10 minutes in 1 % RNAse water solution
8 8 8 8 r
I
n\nj
~
i n
culture
O-fb
Agglutination reaction and formation of first pairs 30 minutes after mixing cells
m e diu m
\/
~ O-fb
Aggulation reaction and formation of first pairs 7 - 8 hours after mixing cells
When cells treated for 10 min in vivo with the L. Light RNAse preparation (Fig. 7) are rinsed in culture fluid in order to free them of RNAse residues and then cultured in a normal medium, formation of the characteristic pattern of small polygons takes place gradually, starting from one hour after rinsing. Considerable parts of a new pattern can be seen in Fig. 8, presenting part of the cell after 3 h regeneration, and its completely re-forms after 7 h. Electronograms of cells stained with ruthenium red, and subjected to the action of the L. Light RNAse preparation, reveal the occurrence of a structure-less pseudocyst round the cells, with fragments of membranes probably originating from damage alveolar membrane (Figs. 14 and 15). In addition, in such cells, immediately below the cortex there are large numbers of vesicles, on the outer surface of which there are numerous ribosomes (Fig. 15), unlike control cells in which structures of this kind were not observed (Figs. 12 and 13). When, however, living ciliates were subjected to the effect of 4 and 5 cryst. RNAse preparations produced by Koch-Light and Serva (Table 2), only intensive the longlasting reversive movement was observed, but without formation of pseudo cysts. The action of Concanavalina A on living cells of Chiloilonella 8teini produced agglutination reactions. About 40 % of the ciliates agglutinate with any given part of the cell surface, forming a compact pellet on the bottom of the dish. The reaction of rejection of surface coat, which takes place in Stylonychia mytilu8 (AMMERMANN
Ultrastructure and Some Properties of the Surface Coat
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1979 - personal communication) was not, however, observed neither was this lectin observed to effect the result of staining the surface coat with alcian blue. Other enzymes and organic and inorganic substances used in vivo caused only the reversion reaction, without exerting any visible effect on the result of staining with alcian blue (Table 2). In order to obtain additional information as to the character of the surface coat PAS reaction was also carried out. It was found that it produced only delicate pink homogenous staining of the outer membranes, without revealing any special structures. Analysis of micrographs of cells stained with ruthenium red showed that a thin layer of surface coat occurs over the whole surface of the cell membrane, while electron dense material was observed to occur below the outer membranes of pellicular alveoli (Figs. 12, 13). Electron microscope photographs also permitted of observing the existence of structures on the dorsal side, similar in appearance to spadices, with an opening in the inside in which undefined, electron dense material was observed (Fig. 10). Their location corresponds to the small structures of unknown function, described by earlier authors (SOLTYNSKA 1971), presenting a silvered appearance and scattered indiscriminately between alveoli (Fig. ll). The characteristic capacity of L. Light RNAse 4 X cryst. to deprive cells of the surface coat was used to establish the part played by the surface coat in reactions connected with conjugation. For this purpose experiments were carried out intended to provide a reply to the question as to whether cells of opposite mating types, deprived of their surface coat, are capable of conjugation and formation of lasting pairs. The plan and result of this experiment are shown in Fig. 16. The experiment showed that cells treated in vivo with L. Light RNAse 4X cryst. preparation are not capable of forming pairs within a time shorter than 7-8 h after mixing. This period corresponds to the time necessary for regeneration of the surface coat rejected as the result of the action of the preparation.
Discussion The positive results of staining with alcian blue specific to acid mucopolysaccharides (CHAYEN 1975) and control digestion with hyaluronidase (specifically digesting hyaluronic acid, which is an acid mucopolysaccharide) show that hyaluronic acid is included in the composition of the stained surface structure (Figs. 1 and 2). This acid would appear to be one of the components of the surface coat in both the Chilodonella species examined. Although digestion with trypsin and pronase does not cause changes in intensity of alcian blue staining, it causes deformation of the pattern, which may indicate that in this case proteins play the part of a "framework" in which mucopolysaccharides are placed. Analysis of electronograms of cells stained with ruthenium red showed that only a thin layer stains over the whole cell surface. It is known (LuFT 1971; BLANQUET 1976) thr.t ruthenium red forms, with the glycocalyx of cells fixed in osmium tetrox-
3'78
M.
GOLEMBIEWSKA
a nd ST.
RADZIKOWSKI
ide, an electron dense clearly visible layer. According to LUFT (1966) this material has to a great degree the character of acid mucopolysaccharides. It may therefore be assumed, on the strength of these results also, that compounds of this particular character are included in the composition of the surface coat in the ciliates examined. The PAS reaction carried out and digestion with iX-amylase did not give an unequivocal answer as to the nature of the stained substances, but showed that the delicate pink colour may derive from glycoproteids and different lipid components of the membrane (PEARSE 1957). Reactions from the effect of different substances on living ciliates show that only Con-A and RNAse 4 X cryst., produced by L. Light, caused characteristic reactions other than reversion. Concanavalina A caused agglutination reactions. It has been shown in earlier papers (WANG 1971, NICOLSON 1971) that this lectin combines specifically with iXD-glucose and iX-D-mannose. It would therefore appear that the agglutination reaction observed is due to the specific combining of these sugars with the outer surface of the cells. The action of the L. Light RNAse 4 X cryst. preparation causes the cells to reject the Eurface coat and form pseudocysts. It would appear that this is not a physiological process, since under normal conditions formation of cysts was never observed in either of these species. Furthermore, simultaneously with partial rejection of the surface coat, the outer membranes of the pellicular alveoli are also damaged. This reaction was also obtained when this preparation was applied after incubation at high temperatures of 90 and 100 °C, which indicates that this reaction is not connected with the effect of RNAse as an enzyme, but more with the presence of some substance polluting the preparation. Information given in the Koch-Light Laboratories Ltd. 1973 catalogue states that the enzyme RNAse is purified by using, inter alia, sulphate of ammonia and ethyl alcohol. Examination was made in vivo of the effect of these compounds on ciliates, but the cells reacted only by reversion, without forming pseudocysts (Table 2). The experiments made with RNAse preparations produced by other firms, which when applied cause only reversion reaction in ciliates, also provide grounds for considering that the reaction is caused only by the L. Light RNAse 4 X cryst. preparation, without having anything in common with enzymatic reaction. Cells fixed and RNAse digested do not exhibit changes in the pattern stained with alcian blue (Table 1). This indicates that this enzyme does not act on the structure itself of the surface coat of these ciliates. It is known that the cell surface plays an important part in specific interactions between cells (WINZLER 1970), particularly in mating reactions (WIESE 1974). The fact that cells deprived of their surface coat do not exhibit the reaction of conjugation and that this reaction cannot take place until 7-8 h after removal of the preparation, i.e. after the period required for the coat completely to re-form, shows that the receptors responsible for agglutination of cells are situated in the outer layer of the surface coat. This is confirmed by numerous earlier data in literature relating
Ultrastructure and Some Properties of the Surface Coat
379
to other Protozoa (COHEN and SIEGEL 1963; METZ 1954; MIYAKE 1969; KITAMURA et al. 1978). Both the results of the present studies and data in literature concerning other cells suggest that the surface coat in Chilodonella consists of a large number of components such asglycoproteids, polysaccharides, glucolipids and that acid mucopolysaccharides stained with alcian blue and ruthenium red are only ono of the parts of a more complex whole. To conclude this discussion reference must also be made to the spadiciform structures occurring in both species on the dorsal side of the cell. They may be compared with the mucocysts occurring in other ciliates, but in the majority of cases resting mucocysts have a paracrystalline structure (HAUSMANN 1972a, 1972b, 1973; SATIR et al. 1973) and this structure was not observed in Chilodonella, in which only the presence of electron dense material was found. In addition rejection of this material outside the cell, and formation of the reticulum characteristic of mucocysts on the surface, was never observed (HAUSMANN 1973). It would seem that the spadiciform organellae in Chilodonella may be connected with production of substances of a protein character, but it is difficult at the present stage to define their functions in the cell.
Acknowledgements The authors are inhebted to dr R. SKOCZYLAS for his advice and critical reading of the manuscript and to Miss J. HULAS for excellent technical assistance.
Literature BENNET, H. S.: Morphological aspect of extracellular polisaccharides. J. Histochem. Cytochem. II (1963): 14-23. - The cell surface: Components and configurations in LIMA DE FARIA, A. (ed.): Handbook of molecular cytology 1261-1293. Amsterdam 1969. BLAXQUET, R. P.: Ul trahistochemical study on the ruthenium red surface staining. Histochemistry 47 (1976): 175-189. BURGER, 1\1. M.: A difference in the architecture of surface membrane of normal and virally transformed cells. Proc. Nat. Acad. Sci. USA. 62 (1969): 994. CHATTOX, E., et LWOFF, A.: La constitution primitive de la strie ciliaire des infusoires. La desmodexie. C. R. Soc. BioI. 118 (1935): 1068-1072. CHAYEN, J., BITENSKY, L. , and BUTCHER, G. R.: Histochemie. Weinheim 1975. COHEN, L. W., and SIEGEL, R. W.: The mating-type substances of Paramecium bursaria. Genet. Res. 4 (1963): 143-150. CORLISS, J. 0.: Silver impregnation of ciliated Protozoa by CHATTON-LwOFF technic. Stain Technol. 28 (1953): 97-100. DIPPEL, R. V.: The site of silver impregnation in Paramecium aurelia. J. Protozool. 9, suppl. 24 (1962). DOBRZANSKA-KACZANOWSKA, J.: Studies of morphology of Ohilodonella cucullulu8 O. F. M. 1786. In: Progress of Protozoology, Internat. Congr. Ser. 91, (1965). DUZGUNES, N.: The Concanavalin A agglutinating system of cell membranes. Biosystems 6 (1975): 209-216.
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EHRET, C. F.; and POWERS, E. L.: The cell surface of Paramecium. Intern. R ev. Cyto!. 8 (1959): 97-133. FAURE·FREMIET, E.: Mecanismes d e la morphogenese chez quelques cilies Gymnostomes Hypo. stomiens. Arch. Anat. Microsc. et Morpho!. Exptl. 39 (1950): 1 - 14. GASIe, G., and GASrc, T.: Removal and regenera tion of the cell coating in tumour cells. Nature 169 (1962): 170. HAUSlI
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RAMBOURG, A.: Morphological and histochemical aspects of glycoproteins at the surface of animal cells. Int. Rev. Cyt. 29 (1971): 47-114. SATIR, B., SCHOOI,EY, C., and SATIR, P.: Membrane fusion in a model system. J. Cell. BioI. 56 (1973): 153-176. SCOTT, J. E., and DORLING, J.: Differential staining of acid glycosaminoglycans (mucopoly. saccharides) by alcyan blue in salt solutions. Histochemie 5 (1965): 221-233. SOLTYNSKA, M.: Morphology and fine structure of Chilodonella cucullulu8 (0. F. M.). Cortex and cytopharyengeal apparatus. Acta Protozool. 9 (1971): 49-82. SZUBINSKA, B., and LUFT, J. H.: Ruthenium red and violet. III. fine structure of Paramecium aurelia. J. Exp. Zool. 171 (1971): 417-441. WANG, J. L., CUNNIGHAM, A. B., and EDELMAN, M. G.: Unusual fragments in the subunit structure of Concanavalin A. Proc. Nat. Acad. Sci. USA 68 (1971): 1130. WIESE, G. E.: Ultrastructural localization of surface Concanavalin A binding sites in an unicellular organism, Amoeba proteu8. Cell Tiss. Res. 150 (1974): 161-166. WINZLER, R. J.: Carbohydrates in cell surfaces. Int. Rev. Cyt. 29 (1970): 77 -125. WOLFE, J.: Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena. Exptl. Cell Res. 87 (1974): 39-46. WYROBA, E., and PRZELlj;CKA, A.: Studies on the surface coat of Paramecium aurelia. I. Ruthenium red staining and enzyme treatment. Z. Zellforsch. 143 (1973): 343 - 353. Authors' address: MALGORZATA GOLEMBIEWSKA and STEFAN RADZIKOWSKI, Department of Cytology, Zoological Institute, University of Warsaw, Krakowskie Przedmie~cie 26/28, Pl· 00· 927/1 Warsaw, Poland.
Zoological Institute, Dept. of Cytology, University of 'Warsaw
Ultrastructure and Some Properties of the Surface Coat of Ohilodonella steini and Ohilodonella cucullulus By MALGORZATA GOLEMBIEWSKA and STEFAN RADZIKOWSKI With 16 Figures
Summary Using specific staining and digestion by enzymes it has been shown that hyaluronic acid and other acid mucopolysaccharides are included in the composition of the surface coat in Chilodonella steini and CMlodonella cucullulus. In both species abrupt rejection of the surface coat from the whole cell surface and formation of pseudocysts take place as a result of the action of L. Light ribonuclease 4 X cryst. preparation. Partial destruction of the outer membranes of the pellicular alveoli was observed in electronograms of cells subjected to the action of this preparation. This reversible reaction is not connected with the action of RNAse itself as an enzyme, but probably by admixtures in it of a different character. This property was utilized to examine the role of the surface coat in the conjugation process. The absence of agglutination reaction in cells of opposite mating types deprived of their surface coat indicates that the receptors responsible for this process are present in this particular layer of the cell.
Introduction In animal cells the cell membrane is covered by a surface coat defined as a, ,glycocalyx" and composed chiefly of substances of a carbohydrate character (BENNETT 1963; GASIC and GASIC 1962 ; MARTINEZ-PALOMO 1970; RAMBOURG 1971). Its chemical composition and the functions it carries out differentiates the cells (BENNETT 1969; SZUBINSKA and LUFT 1971; PINTO da SILVA et al. 1975; WYROBA and PRZELECKA 1973; POLLOCK 1978). A considerable amount of data are to be found in literature relating to the general occurrence of the surface coat in representatives of all groups of Protozoa. It is known that in different ciliates it plays an important part in intercellular interactions preceding the process of conjugation, and also in the process of cell union (MIYAKE 1974; HONDA 1976; WOLFE 1974). In dorso-ventrally flattened representatives of the genus Okilodonella, Ok. steini and Ok. cucullulus, an argentophil reticulum was found to occur on both surfaces of the cell (RADZIKOWSKI 1966; DOBRZANSKA-KACZANOWSKA 1965; RADZIKOWSKI and GOLEMBIEWSKA 1977), and observations in vivo pointed to the presence in the cortex of numerous alveole with fluid contents (KAczANOWSKA-KoWALSKA 1969; RADZIKOWSKI and GOLEMBIEWSKA 1977). 24
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M. GOl.EMBIEWSKA and ST. RADZIKOWSKI
Studies III an eleotron mioroscope showed that the argentophil reticulum visible in vivo and on silvered slides, termed by CHATTON and LWOFF 1935 "argyrome", constitutes the sculpture of the surface resulting from the pellicular membrane system. The borders of pellicular alveoli become silvered (PUYTORAO 1959; EHRET and PoWERS 1959; PITELKA 1961, 1965; DIPPEL 1962). There are, however, no data on the surface coat of these ciliates in the abundant literature on the morphology and morphogenetic processes of these two species (RADZIKOWSKI 1966; KAOZANOWSKA and KOWALSKA 1968; KOWALSKA and KAOZANOWSKA 1969; SOLTYNSKA 1971; RADZIKOWSKI and GOLEMBIEWSKA 1977). An attempt has therefore been made at defining the chemical composition, ultrastructure and part played by the surface coat in the conjugation process in these two species.
Material and Methods The studies were made on two species of the genus Okilodonella,' Ok. steini and Ok. cucullulus. The methods used for their culture have been described in the papers by RADZIKOWSKI (1966) for Ok. cucullulus and RADZIKOWSKI and GOl.EMBIEWSKA (1977) for Ok. steini. The following staining methods were used to define the structure and chemical composition of the surface coat: 1. Staining with alcian blue for acid mucopolysaccharides The material was fixed in a mixture of 96 % ethyl alcohol and formalin in parts of 9: 1. Staining with alcian blue 8G E. Gurr Ltd. London SW 14, was carried out after SCOTT and DORLING (1965). 2. Staining with Ruthenium red for acid mucopolysaccharides Standard Ruthenium red stain produced by Fluka AG, Buchs S. G. was used for staining carried out in accordance with the following procedure, used in electron microscopy (LUFT 1971): A - 5 mg Ruthenium red solution was dissolved in 1 ml of distilled water heated to 60 °e, then cooled B - 3.6 % glutaraldehyde solution in 0.2 cacodylic buffer, pH 7.3 e - 3 mg Ruthenium red solution dissolved in 1 ml of distilled water, heated to 60 °e, then cooled, adding 1 ml of osmium tetroxide and 1 ml pure cacodylic buffer. Solutions A and B were mixed immediately before use. Fixing lasted 1 h at a temperature of
o °e, followed by rinsing in three changes of cacodylic buffer, after which the material was trans· ferred to solution C for three hours. 3. PAS reaction to polysaccharides Reactions with periodic acid and Schiff reagent were made after PEARS 1957. Enzymatic reactions were made using the following enzymes:
1. Proteolytic enzymes Tripsin inhibitor ovomucoid salt·free, Koch.Light Laboratories Ltd. eolnbrook, Bucks, England, at a concentration of 0.1 mg/ml in 0.05 M phosphate buffer with pH 6; Pronase ex Streptomyces gri88u8 Koch-Light Laboratories Ltd. Colnbrook, Bucks, England no. 66269, at a concentration of 0.1 mg/ml in 0.05 M phosphate buffer with pH 6. 2. Carbohydrate-specific for hydrolases lX-amylase ex bacteria, produced by Fluke A. G., concentration 1 %. Diastase from malt, produced by BDH, concentration 1 % Hialuronidase, produced by WSiS, Warsaw, strength 3,200 i. u.
mtrastructure and Some Properties of the Surface Coat
369
3. Nucleases Ribonuclease salt and protease free 40 k Unit vlmgm, 4 X cryst. ex bovine pancreas, produced by L. Light and Co. Ltd., Colnbrook, England. This preparation was used in three variants: not heated. after incubation at 90°C for 5 min, after incubation at 100°C for 30 min. The preparation was heated to 90°C in order to free it of any possible pollution by other enzymes, while incubation at 100°C caused denaturation of the enzyme it~elf. Ribonuclease ex bovine pancreas salt·free, 4 X cryst., and ribonuclease ex bovine pancreas salt.free, 5 X cryst., produced by Koch-Light Laboratories Ltd., Colnbrook, Bucks, England. Ribonuclease Type IA ex bovine pancreas, 5 X cryst., produced by Serva. RNA-se solutions were used at concentrations from 0.5 to 1 % in a veronal buffer and in a culture watar solution with pH 7.7 and 7.4 respectively. The following methods were used for examination of the structure of the cortex and surface coat: 1. Impregnation with salts of silver a) with nitrate of silver after CHATToN-LwoFF, as modified by CORLISS 1953; b) with protargol, produced by Establissement Rogues, Paris for silvering the surface by Bodian method. 2. Acting on living cells using the above enzymes and also organic and inorganic substances a) the following organic compounds were nsed: Concanavalin A in glucose solution, produced by Calbiochem, concentration 500-100 g/ml; Concanavalin A was used on account of its specific combining with (X-D-glucose and (X-Dmannose. (Concanavalin A administered to Stylonychia mytilu8 causes rejection of the surface coat - personal communication by AMMERMANN.) Formalin in concentrations from 0.01 to 0.1 %; Ethyl alcohol in concentrations from 0.1 to 4 %; Both these compounds are included in the composition of the fixing preparation. b) Inorganic compounds used: sulphate of ammonia, which enters into RNA-se production; calcium, potassium, barium and magnesium chloride in concentrations of 10, 20, 30 mM respectively; acetic, phosphe.te and veronal buffers with pH of 6.6 and 7.7. The techniques used in electron microscopy were employed to define the ultrastructure of the surface coat and cortex. The following fixing preparations were used: 2 % glutaraldehyde solution in a phosphate buffer with pH 7.2 and 1 % osmium tetroxide solution in the same buffer. The fixed ciliates were first embedded in 1 % agar-agar, then standard dehydration was carried out, after which the material was embedded in Epon 812 mixture. The material was sectioned on an LKB III ultramicrotome, and contrasted with uranyl acetate and lead citrate. The section were examined under a Tesla BS 500 and JEM-7A (Joelco) electron microscope. The staining method with Ruthenium red has been described earlier in this paper.
Results By staining ciliates with 1 % alcian blue solution it proved possible to discover the characteristic arrangement of small polygons on both the cell surfaces (Figs. 1 and 2), the position of which coincides with the position of pellicular alveoli. It would seem that alcian blue stains only the outer parts of the surface coat (Fig. 3). 24*
370
M.
GOLEMBIEWSKA
and ST.
RADZIKOWSKI
,
. .,
Fig. 1. Part of the ventral surface of Chilodonella steini stailled with alcian blue .
X
1I';,.'1
• . ·-0j!0, ........
,.;.• . r {
:,~.J;
1,500.
Fig. 2. Part of the dorsal surface of Chilodonella steini. Sta ining - a lcian blue. X 1,500. Fig. 3. Part of a Chilodonella cucullulu8 cell after 1 mho act ion of L. Light RNAse 4 X cryst Arrow points to material trailing b ehind cell. Alcian blue. X 1,500.
Ultrastructure and Some Properties of the Surface Coat
371
In order to define the kind or kinds of substances included in the composition of the material stained with alcian blue, the fixed ciliates were digested by enzymes, then stained. The results of these experiments are set out in Table 1. Tabl e I. R esults of staining eells of Chilodonella steini and Chilodonella cucullulus with alcian blue after digestion by enzymes Enzymes
Result of staining
Trypsin Pronase (X· a mylase Diasta se Hyalw'onidase Ribonucleases (va rious)
stains reanily, deformation of pattern stains readily, deformation of pattern no e ffect no e ffect faint st a ining, losses in the staining p attern no e ffect
Analysis of the results given in Table 1 shows that proteolytic enzymes (trypsin and pronase) cause only spatial deformation of the pattern stained with alcian blue, without however affecting intensity of staining, which indicates that they do not digest t.he structures, which are actually stained. Hyaluronidase, on t.he other hand, causes mainly a decrease in intensity of staining and considerable losses in the pattern. In would appear that digestion by the remaining enzymes does not affect the capacity for staining and regularity of the staining pattern. Examination was also made in vivo of the effect on the behaviour of the cells treated by enzymes, organic and inorganic substances, and of the condition of the surface coat of these cells stained with alcian blue. The results of these experiments are given in Table 2. Reactions in v i v 0 due to the effect of factors from column A are given in column B. The state of the surface coat, stained with alcian blue, of cells previously subjected to the action of substances detailed in column A, are shown in column C. + indicates absence of significant differences in the pattern stained with alcian blue in comparison with control cells. - complete absence of any tendency to stain.
Fig. 4. Chilodonella cucullulus cell in the pseudocyst form as the l'esult of the action of RNAse prepara tion. Phase contrast. X 1,500. Fig. 5. Part of Chilodonella cucullulus cell aft er I min action of RNAse preparation. Alcian blue. X 1,500. Fig. 6. Part of Chilodonella cucullulus cell surface after 10 min action of RNAse. Absence of stain. ing re action with alcian blue. X 1,500.
372
M. GOLEMBIEWSKA and ST. RADZIKOWSKI
Table 2. Reactions of cells in vivo, and the state of the surface coat in these cells after treatment by different exogenic factors Kind of substance
Cell reaction
Result of staining
A
B
C
Trypsin Pronase (X·amylase Diastase Hyaluronidase
reversion reversion reversion reversion reversion
+ + + + +
RNAse L. Light 4 X cryst. a) in water and buffer b) after incubation at 90°C c) after incubat.ion at 100°C
reversion and formation of pseudocyst reversion and formation of pseudocyst reversion and formation of pseudocyst
RNAse Koch-Light a) 4 X cryst. in water and buffer b) 5 X cryst. in water and buffer
reversion reversion
+ +
reversion
+
approx. 40 % of cells adhere
+ +
RNAse A Serva a) in water and buffer Organic compounds Concanavalin A Formalin at different concentrations Ethanol at a concentrations of 1- 4 %
reversion with low concentrations reversion and retardation
+
Inorganic compounds Ammonium sulphate Chlorides Buffers
reversion receding reversion
+ +
reversion
+
In ciliates su bj ected in v i v 0 to the effect of 4 X cryst. ribonuclease produced by L. Light we observed, formation of a pseudocyst in which the cell is contained for a certain time (Fig. 5). Incubation of the L. Light ribonuclease preparation at temperatures of 90 and 100 °0 does not cause the preparation to lose its capacity for inducement of pseudocyst formation. Staining of cells with alcian blue after formation of the pseudocyst (Fig. 6) showed that in comparison with the control (Figs. 1 and 2), they are deprived of the characteristic pattern. This pattern occurs in a slightly deformed shape on the walls of the pseudocyst (Fig. 4).
Fig. 7. Part of ciliate surface one hour from time of transfer from RNAse solution to normal culture m edium. Alcian blue. X 1,500. Fig. 8. Part of ciliate surface three hours after transfer tl) cultUl'e medium. Alcia n blue. X 1,500. Fig. 9. Cell sUl'face 7 h from time of transfer to culture medium. Alcian blue. X1,500. Fig. 10. Chilodonclla cuculluZ.us. Electronogram of dorsal surfa ce with spadiciform structure. x 12,000. Fig. 11. Dorsal surface of ciliate silvcred by CHATTON-LwOFF m ethod. Arrows indicate distribution of spadiciform structures.
374
',.
M. GOl'.EMBIEWSKA and ST. RADZIK OW SKI
'w
'"
..
Figs. 12 and 13. Photograph under electron microscope part of cell cross section. Ventral side (12). Ruthenium red. X 12,000. Dorsal side (13). Ruthenium red. X 12,000.
Ultrastructure and Some Properties of thc Surface Coat
375
Figs. 14, 15.and 16. Electronogram of cells after action of RNAse prep a ration. Ruthenium red. x 14,000.
376
M. GOLEMBIEWSKA and ST. RADZIKOWSKI
Fig. 16. Plan of the experiment on the effect of L. Light RNAse 4 X cryst. preparation on conjugation reactions of Ohilodonella steini cells. Control procedure
Experimental procedure
ciliates placed in culture water
ciliates incubated for 10 minutes in 1 % RNAse water solution
8 8 8 8 r
I
n\nj
~
i n
culture
O-fb
Agglutination reaction and formation of first pairs 30 minutes after mixing cells
m e diu m
\/
~ O-fb
Aggulation reaction and formation of first pairs 7 - 8 hours after mixing cells
When cells treated for 10 min in vivo with the L. Light RNAse preparation (Fig. 7) are rinsed in culture fluid in order to free them of RNAse residues and then cultured in a normal medium, formation of the characteristic pattern of small polygons takes place gradually, starting from one hour after rinsing. Considerable parts of a new pattern can be seen in Fig. 8, presenting part of the cell after 3 h regeneration, and its completely re-forms after 7 h. Electronograms of cells stained with ruthenium red, and subjected to the action of the L. Light RNAse preparation, reveal the occurrence of a structure-less pseudocyst round the cells, with fragments of membranes probably originating from damage alveolar membrane (Figs. 14 and 15). In addition, in such cells, immediately below the cortex there are large numbers of vesicles, on the outer surface of which there are numerous ribosomes (Fig. 15), unlike control cells in which structures of this kind were not observed (Figs. 12 and 13). When, however, living ciliates were subjected to the effect of 4 and 5 cryst. RNAse preparations produced by Koch-Light and Serva (Table 2), only intensive the longlasting reversive movement was observed, but without formation of pseudo cysts. The action of Concanavalina A on living cells of Chiloilonella 8teini produced agglutination reactions. About 40 % of the ciliates agglutinate with any given part of the cell surface, forming a compact pellet on the bottom of the dish. The reaction of rejection of surface coat, which takes place in Stylonychia mytilu8 (AMMERMANN
Ultrastructure and Some Properties of the Surface Coat
377
1979 - personal communication) was not, however, observed neither was this lectin observed to effect the result of staining the surface coat with alcian blue. Other enzymes and organic and inorganic substances used in vivo caused only the reversion reaction, without exerting any visible effect on the result of staining with alcian blue (Table 2). In order to obtain additional information as to the character of the surface coat PAS reaction was also carried out. It was found that it produced only delicate pink homogenous staining of the outer membranes, without revealing any special structures. Analysis of micrographs of cells stained with ruthenium red showed that a thin layer of surface coat occurs over the whole surface of the cell membrane, while electron dense material was observed to occur below the outer membranes of pellicular alveoli (Figs. 12, 13). Electron microscope photographs also permitted of observing the existence of structures on the dorsal side, similar in appearance to spadices, with an opening in the inside in which undefined, electron dense material was observed (Fig. 10). Their location corresponds to the small structures of unknown function, described by earlier authors (SOLTYNSKA 1971), presenting a silvered appearance and scattered indiscriminately between alveoli (Fig. ll). The characteristic capacity of L. Light RNAse 4 X cryst. to deprive cells of the surface coat was used to establish the part played by the surface coat in reactions connected with conjugation. For this purpose experiments were carried out intended to provide a reply to the question as to whether cells of opposite mating types, deprived of their surface coat, are capable of conjugation and formation of lasting pairs. The plan and result of this experiment are shown in Fig. 16. The experiment showed that cells treated in vivo with L. Light RNAse 4X cryst. preparation are not capable of forming pairs within a time shorter than 7-8 h after mixing. This period corresponds to the time necessary for regeneration of the surface coat rejected as the result of the action of the preparation.
Discussion The positive results of staining with alcian blue specific to acid mucopolysaccharides (CHAYEN 1975) and control digestion with hyaluronidase (specifically digesting hyaluronic acid, which is an acid mucopolysaccharide) show that hyaluronic acid is included in the composition of the stained surface structure (Figs. 1 and 2). This acid would appear to be one of the components of the surface coat in both the Chilodonella species examined. Although digestion with trypsin and pronase does not cause changes in intensity of alcian blue staining, it causes deformation of the pattern, which may indicate that in this case proteins play the part of a "framework" in which mucopolysaccharides are placed. Analysis of electronograms of cells stained with ruthenium red showed that only a thin layer stains over the whole cell surface. It is known (LuFT 1971; BLANQUET 1976) thr.t ruthenium red forms, with the glycocalyx of cells fixed in osmium tetrox-
3'78
M.
GOLEMBIEWSKA
a nd ST.
RADZIKOWSKI
ide, an electron dense clearly visible layer. According to LUFT (1966) this material has to a great degree the character of acid mucopolysaccharides. It may therefore be assumed, on the strength of these results also, that compounds of this particular character are included in the composition of the surface coat in the ciliates examined. The PAS reaction carried out and digestion with iX-amylase did not give an unequivocal answer as to the nature of the stained substances, but showed that the delicate pink colour may derive from glycoproteids and different lipid components of the membrane (PEARSE 1957). Reactions from the effect of different substances on living ciliates show that only Con-A and RNAse 4 X cryst., produced by L. Light, caused characteristic reactions other than reversion. Concanavalina A caused agglutination reactions. It has been shown in earlier papers (WANG 1971, NICOLSON 1971) that this lectin combines specifically with iXD-glucose and iX-D-mannose. It would therefore appear that the agglutination reaction observed is due to the specific combining of these sugars with the outer surface of the cells. The action of the L. Light RNAse 4 X cryst. preparation causes the cells to reject the Eurface coat and form pseudocysts. It would appear that this is not a physiological process, since under normal conditions formation of cysts was never observed in either of these species. Furthermore, simultaneously with partial rejection of the surface coat, the outer membranes of the pellicular alveoli are also damaged. This reaction was also obtained when this preparation was applied after incubation at high temperatures of 90 and 100 °C, which indicates that this reaction is not connected with the effect of RNAse as an enzyme, but more with the presence of some substance polluting the preparation. Information given in the Koch-Light Laboratories Ltd. 1973 catalogue states that the enzyme RNAse is purified by using, inter alia, sulphate of ammonia and ethyl alcohol. Examination was made in vivo of the effect of these compounds on ciliates, but the cells reacted only by reversion, without forming pseudocysts (Table 2). The experiments made with RNAse preparations produced by other firms, which when applied cause only reversion reaction in ciliates, also provide grounds for considering that the reaction is caused only by the L. Light RNAse 4 X cryst. preparation, without having anything in common with enzymatic reaction. Cells fixed and RNAse digested do not exhibit changes in the pattern stained with alcian blue (Table 1). This indicates that this enzyme does not act on the structure itself of the surface coat of these ciliates. It is known that the cell surface plays an important part in specific interactions between cells (WINZLER 1970), particularly in mating reactions (WIESE 1974). The fact that cells deprived of their surface coat do not exhibit the reaction of conjugation and that this reaction cannot take place until 7-8 h after removal of the preparation, i.e. after the period required for the coat completely to re-form, shows that the receptors responsible for agglutination of cells are situated in the outer layer of the surface coat. This is confirmed by numerous earlier data in literature relating
Ultrastructure and Some Properties of the Surface Coat
379
to other Protozoa (COHEN and SIEGEL 1963; METZ 1954; MIYAKE 1969; KITAMURA et al. 1978). Both the results of the present studies and data in literature concerning other cells suggest that the surface coat in Chilodonella consists of a large number of components such asglycoproteids, polysaccharides, glucolipids and that acid mucopolysaccharides stained with alcian blue and ruthenium red are only ono of the parts of a more complex whole. To conclude this discussion reference must also be made to the spadiciform structures occurring in both species on the dorsal side of the cell. They may be compared with the mucocysts occurring in other ciliates, but in the majority of cases resting mucocysts have a paracrystalline structure (HAUSMANN 1972a, 1972b, 1973; SATIR et al. 1973) and this structure was not observed in Chilodonella, in which only the presence of electron dense material was found. In addition rejection of this material outside the cell, and formation of the reticulum characteristic of mucocysts on the surface, was never observed (HAUSMANN 1973). It would seem that the spadiciform organellae in Chilodonella may be connected with production of substances of a protein character, but it is difficult at the present stage to define their functions in the cell.
Acknowledgements The authors are inhebted to dr R. SKOCZYLAS for his advice and critical reading of the manuscript and to Miss J. HULAS for excellent technical assistance.
Literature BENNET, H. S.: Morphological aspect of extracellular polisaccharides. J. Histochem. Cytochem. II (1963): 14-23. - The cell surface: Components and configurations in LIMA DE FARIA, A. (ed.): Handbook of molecular cytology 1261-1293. Amsterdam 1969. BLAXQUET, R. P.: Ul trahistochemical study on the ruthenium red surface staining. Histochemistry 47 (1976): 175-189. BURGER, 1\1. M.: A difference in the architecture of surface membrane of normal and virally transformed cells. Proc. Nat. Acad. Sci. USA. 62 (1969): 994. CHATTOX, E., et LWOFF, A.: La constitution primitive de la strie ciliaire des infusoires. La desmodexie. C. R. Soc. BioI. 118 (1935): 1068-1072. CHAYEN, J., BITENSKY, L. , and BUTCHER, G. R.: Histochemie. Weinheim 1975. COHEN, L. W., and SIEGEL, R. W.: The mating-type substances of Paramecium bursaria. Genet. Res. 4 (1963): 143-150. CORLISS, J. 0.: Silver impregnation of ciliated Protozoa by CHATTON-LwOFF technic. Stain Technol. 28 (1953): 97-100. DIPPEL, R. V.: The site of silver impregnation in Paramecium aurelia. J. Protozool. 9, suppl. 24 (1962). DOBRZANSKA-KACZANOWSKA, J.: Studies of morphology of Ohilodonella cucullulu8 O. F. M. 1786. In: Progress of Protozoology, Internat. Congr. Ser. 91, (1965). DUZGUNES, N.: The Concanavalin A agglutinating system of cell membranes. Biosystems 6 (1975): 209-216.
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EHRET, C. F.; and POWERS, E. L.: The cell surface of Paramecium. Intern. R ev. Cyto!. 8 (1959): 97-133. FAURE·FREMIET, E.: Mecanismes d e la morphogenese chez quelques cilies Gymnostomes Hypo. stomiens. Arch. Anat. Microsc. et Morpho!. Exptl. 39 (1950): 1 - 14. GASIe, G., and GASrc, T.: Removal and regenera tion of the cell coating in tumour cells. Nature 169 (1962): 170. HAUSlI
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RAMBOURG, A.: Morphological and histochemical aspects of glycoproteins at the surface of animal cells. Int. Rev. Cyt. 29 (1971): 47-114. SATIR, B., SCHOOI,EY, C., and SATIR, P.: Membrane fusion in a model system. J. Cell. BioI. 56 (1973): 153-176. SCOTT, J. E., and DORLING, J.: Differential staining of acid glycosaminoglycans (mucopoly. saccharides) by alcyan blue in salt solutions. Histochemie 5 (1965): 221-233. SOLTYNSKA, M.: Morphology and fine structure of Chilodonella cucullulu8 (0. F. M.). Cortex and cytopharyengeal apparatus. Acta Protozool. 9 (1971): 49-82. SZUBINSKA, B., and LUFT, J. H.: Ruthenium red and violet. III. fine structure of Paramecium aurelia. J. Exp. Zool. 171 (1971): 417-441. WANG, J. L., CUNNIGHAM, A. B., and EDELMAN, M. G.: Unusual fragments in the subunit structure of Concanavalin A. Proc. Nat. Acad. Sci. USA 68 (1971): 1130. WIESE, G. E.: Ultrastructural localization of surface Concanavalin A binding sites in an unicellular organism, Amoeba proteu8. Cell Tiss. Res. 150 (1974): 161-166. WINZLER, R. J.: Carbohydrates in cell surfaces. Int. Rev. Cyt. 29 (1970): 77 -125. WOLFE, J.: Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena. Exptl. Cell Res. 87 (1974): 39-46. WYROBA, E., and PRZELlj;CKA, A.: Studies on the surface coat of Paramecium aurelia. I. Ruthenium red staining and enzyme treatment. Z. Zellforsch. 143 (1973): 343 - 353. Authors' address: MALGORZATA GOLEMBIEWSKA and STEFAN RADZIKOWSKI, Department of Cytology, Zoological Institute, University of Warsaw, Krakowskie Przedmie~cie 26/28, Pl· 00· 927/1 Warsaw, Poland.