Myelin-like configurations in ochromonas malhamensis

© 1967 by Academic Press Inc.

J, ULTRASTRUCTURE RESEARCH 20, 127-139 1967

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Myelin-like Configurations in Ochromonas malhamensis ~ O. ROGER ANDERSON and O. A. ROELS Marine Biology Division, Lamont Geological Observatory, Columbia University, Palisades, New York 10964 Received January 9, 1967, and in revised form June 2, 1967 The nuclear region of Ochromonas malhamensis is an active site for lipid organization and production of myelin-like bodies. These bodies resemble myelin forms reported in other microorganisms and mammalian tissue. Such lipid bodies may have important implications for understanding membrane biogenesis and mechanisms whereby cells synthesize complex lipid systems. We have therefore examined naturally occurring myelinics in Ochromonas and produced synthetic myelinics in our laboratory to obtain information about the structure and composition of these lipid bodies. We have found that myelin bodies in Ochromonas maIhamensis resemble myelinics produced by dispersion of egg lecithin in water. Fixation with glutaraldehyde prior to osmium tetroxide treatment protects both natural myelinics in Ochromonas malhamensis and synthetic egg lecithin myelinics; both are degraded when fixed with osmium tetroxide alone. The presence of m y e l i n ' f o r m s or multimembranous bodies has been reported in m a n y different tissues. Various forms of myelinics have been reported in plant proplastids (27), adipose tissue in mice (15), and fetal lung tissue in mammals (28). Recent reports by Rudzinska (21, 22) show the presence of membranous systems in food vacuoles of protozoa. Clearly, there are differences in structure and distribution of myelinics produced in these different cell systems. In our study of the role of lipids in biological membrane structure and function, we have observed myelin bodies being secreted into the central vacuole of Ochromonas malhamensis. The secretion of these myelin bodies consistently originated in the nuclear region. In view of the importance of understanding the organization of lipids in, the cell, and the possible implications of these observations for membrane synthesis, we have attempted to study this phenomenon in greater detail: we have produced artificial myelin bodies and compared their structure to those naturally occurring in Ochromonas malhamensis. In addition we have determined the effects of 1 This investigation was supported by Research Grant GM 13660 from the National Institutes of Health, U.S. Public Health Service. Lamont Geological Observatory contribution No. 1075.

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fixatives o n n a t u r a l a n d artificial myelinics made from egg lecithin a n d f o u n d that prefixing with glutaraldehyde protects these myelin bodies, which are degraded when fixed with o s m i u m tetroxide alone. MATERIALS A N D METHODS

Ochromonas malhamensis The O. malhamensis used in this investigation was maintained in axenic culture using a chemically defined medium (l 1). Fresh medium was inoculated with 0.5 % of its volume of a 5-day-old culture grown in Pyrex flasks and kept at 25 °C with 50 foot-candle illumination from cool-white fluorescent tubes. Under these conditions, the cultures approached the end of the logarithmic growth phase after 5 days. All the samples of Ochromonas malhamensis used for our studies were taken during the logarithmic growth phase at the end of the 5-day period. The medium containing the growing cells was centrifuged at 40 g for 5 minutes. The supernatant was decanted and the pellet was resuspended in 0.15 M phosphate buffer containing 5 % w/v glutaraldehyde (Fisher Scientific, Biological grade), pH -7.4. The fixed cells were resedimented and postfixed in 0.15 M phosphate buffer (pH 7.4) containing 1% w/v osmium tetroxide at room temperature for 1 hour. The fixed cells were collected in warm agar (47°C); after solidification, small blocks were cut, dehydrated in a graded series of ethanols, cleared in propylene oxide, and embedded in Epon 812 resin (13). Thin sections were made with glass knives in a Porter-Blum MT-2 ultramicrotome and observed with a Philips EM 200 at 60 kV, equipped with a 25 /z aperture. Sections were poststained for 20 minutes on the grid using alkaline lead citrate (18).

Artificial myelinics Pure egg lecithin was prepared in our laboratory according to the method of Saunders

(24). It was recrystallized repeatedly from 2-butanone. When chromatographed on Adsorbosil 2 (Applied Science, State College, Pennsylvania) with chloroform : methanol : water ( 8 0 : 3 5 : 5 v/v) as the developing solvent, only one sharp spot (Rf-0.66) could be detected on the thin layer. Artificial myelinics were produced by evaporating an ethanol : n-hexane (20 : 80 v/v) solution of pure egg lecithin in a stream of nitrogen; the dry residue was suspended in glass-distilled water or in an aqueous solution of phosphate buffer containing 0.04 % glutaraldehyde, pH = 7.0, to a final concentration of 1 mg lecithin to 1 ml aqueous solvent. After sonication (4) for 5 minutes with an energy input of approximately 60 watts at 20 kilocycles in a total volume of 10 ml aqueous medium, the aqueous suspension of the lipids was deposited as a drop on carbon-reinforced, collodion-covered grids and allowed to dry in a stream of nitrogen. To determine the effect of osmium tetroxide on the structure of the artificial myelinics, a 1% w/v aqueous solution of osmium tetroxide was added to the dried specimen on the grid and allowed to evaporate. In all cases, as a final step, the specimens were negatively stained by adding a drop of a 2% aqueous solution of phosphotungstic acid pH -6.5 (3). After drying, the specimens were observed with a Philips EM 200 microscope at 60 kV accelerating voltage. FIG. i. The nuclear region of Ochromonas malhamensis contains prominent chloroplasts (C). The cytoplasm contains abundant ground substance, endoplasmic reticulum (ER), free ribosomes (R), and tubular mitochondria (M). × 23,000.

MYELIN F I G U R E S IN O. M A L H A M E N S I S

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o. ROGER ANDERSONAND O. A. ROELS RESULTS

The fine structure of Ochromonas malhamensis as shown in Fig. 1, is similar to that reported for other protozoa (2, 9, 10, 17, 22). The mitochondria are tubular and typically chrysophycean. The cytoplasm contains a system of membranes which resemble endoplasmic reticulum (ER) in mammalian cells (16). Free ribosomes ( R ) a r e abundant in the cytoplasm. Portions of chloroplasts (C) are shown: they consist of three-disk thylakoids. The cytoplasm in the vicinity of the nucleus is rich in endoplasmic reticulum. The dictyosome (D) is consistently located in the nuclear region as shown in Fig. 2 and is associated with numerous vesicles (V) which appear scattered throughout the cytoplasm. Spherical bodies are found in the cytoplasm in the nuclear region (see Fig. 3). They are 0.66 # in diameter and contain laminated concentric layers, 50 A thick, surrounding inclusions (I) approximately 0.1 # in diameter, as shown in Figs. 4 and 7. The cytoplasm in the nuclear region (N) also contains systems of concentric membranes (MS) as shown in Fig. 4. In some cases these myelin forms (MS) are associated with the endoplasmic reticulum (ER) or with mitochondria (M). The myelin bodies in the central vacuole appear near the periphery of the nucleus and the chloroplast and project into the large vacuole. In Figs. 3-6 these myelin bodies can be seen in various stages of secretion into the vacuole. After secretion, the myelin bodies in O. malhamensis are suspended in the vacuole (Fig. 6) and appear to undergo disruption producing granular amorphous masses as shown in Figs. 4 and 6 b (AM). The fine structure of the myelin body shows a series of concentric membranes which fold back upon themselves or appear to fuse together. There is no evidence of Palade particles on their surfaces (Fig. 7). These lamellae have a mean thickness of approximately 100 A; the interlamellar space varies, increasing from 140 A at the center to 220 A near the periphery of the body. The lamellar structure of these myelin bodies is very similar to that produced by phospholipids when hydrated in water dispersion as shown in Fig. 8. The myelin bodies in O. malhamensis do not appear in thin sections of the organism when fixed with osmium tetroxide alone; they are consistently observed in the central vacuole when fixed with glutaraldehyde followed by fixation with osmium tetroxide. Cells fixed in cacodylate-buffered, methanol-free formalin and not postfixed in osmium tetroxide were poorly preserved, even when the concentration of formalin

FIG. 2. The dictyosome (D) consistently appears in the nuclear region and is surrounded by numerous microvesicles (V). x 43,000. FIG. 3. A myelin secretory body (arrow) appears in the last stages of secretion into the central vacuole. × 18,000.

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was reduced to 0.2 % w/v. However, thin sections of the formalin-fixed cells showed the presence of dense bodies both in the vacuole and attached to the vacuolar membrane. Furthermore, no dense bodies were observed in the vacuole of formalin-fixed cells when buffered osmium tetroxide was used as a postfixative, suggesting that the formalin does not preserve these bodies against osmium tetroxide degradation. These data support the premise that the presence of secretory bodies in the vacuole is not a specific effect of glutaraldehyde fixation. Electron micrographs made with formalin-fixed Ochromonas cells should be interpreted very cautiously since the preservation of the fine structure of the cells was very poor. It is conceivable that the observed secretory bodies might be a general response of the organism to foreign organic substances. To determine further whether these bodies are produced by the glutaraldehyde osmium fixation, we have examined living organisms with the phase contrast microscope and noted the presence of refractile bodies in the vacuole undergoing Brownian movement. This suggested that the myelin secretory bodies occurred naturally and were altered or destroyed when osmium tetroxide alone was used as a fixative. Conversely, the glutaraldehyde protected the myelinics against osmium tetroxide degradation. We tested this hypothesis by producing pure egg lecithin myelinics in our laboratory and determining the effects of osmium tetroxide on their structure. Fig. 8 a shows a typical artificial myelinic produced by dispersing egg lecithin in water; it is negatively stained with phosphotungstic acid. Application of osmium tetroxide to such a preparation, followed by drying and negative staining, completely destroyed these structures. However, when glutaraldehyde was added to the aqueous dispersion of egg lecithin, followed by osmium tetroxide treatment and subsequent negative staining with phosphotungstic acid, the myelinics were preserved, as shown in Fig. 8 b. Egg lecithin myelinics are therefore destroyed when fixed with osmium tetroxide alone, but are preserved when glutaraldehyde treatment precedes the addition of osmium tetroxide. We, therefore, believe that myelin secretory bodies in cells fixed with glutaraldehyde and postfixed with osmium tetroide are preserved by the protective action of the glutaraldehyde. In contrast, cells fixed with osmium tetroxide alone may be structurally altered due to the disorganization of lipids.

FIG. 4. An enlarged segment of the nuclear region shows a myelin body (arrow) secreted near a chloroplast. Several concentric membrane systems (MS) are located in the cytoplasm near mitochondria (M). The nucleus (N) lies near the vacuole which contains amorphous bodies (AM), which may be secretory bodies undergoing disruption. × 51,000.

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DISCUSSION The presence of myelin bodies has been observed in mammalian (8, 28), protozoan (21, 22) and plant cells (27). Fawcett and Ito (8) reported extensive production of myelin bodies when rat testis germinal tissue was excised and allowed to stand in salt solution. The presence of dense granules on the membranes of these myelin bodies and their continuity with endoplasmic reticulum suggested that these inclusions were formed by reorganization of the endoplasmic reticulum. Rudzinska (21) has identified concentric membranous bodies in Tetrahymena pyriformis which were located near the macronucleus in an indentation of the nuclear membrane or at the periphery of the cytoplasm. These bodies (2-8 # long) contained a dense granular core surrounded by membranes 100 A thick. The occurrence of particles about 125 ~ in diameter on the surface suggested that these might also be part of the endoplasmic reticulum. No particulars of fixing and staining techniques were described. Rudzinska et al. (22) later identified "myelin figures" within food vacuoles of Colpoda maupasi, particularly in later stages before egestion of the vacuole. Osmium tetroxide fixation alone was used in this study; however, these bodies look quite different from those we have observed in O. rnalhamensis. Dense bodies in food vacuoles of many ciliates have been reported near the periphery of the digestive vacuole (14) and Elliott (7) has confirmed an unpublished observation by M/filler that these dense granules do not stain for acid phosphatase. Their function remains obscure. Elliott used glutaraldehyde fixation followed by osmium tetroxide treatment. The presence of myelin bodies in fetal lung tissue of mammals has been reported by Sun (28). These myelin bodies appear near secretory tissue within the lung and appear to be produced during degeneration of the cells. The dense layered m e m branes in these lung myelin bodies are 45-70 A thick with an intermembrane space of 150-400 A. Sun suggested that these bodies are phospholipid complexes which serve as surface-active agents in aiding expansion of the developing lung. Sun used Dalton's fixative containing potassium dichromate and osmium tetroxide. His electron micrographs show excellent preservation of the myelin bodies; this may be due to the stabilizing action of chromate ions for lipid structures as reported by Elbers et al. (6). We have observed myelin bodies in the central vacuole of O. malhamensis fixed with glutaraldehyde and postfixed with osmium tetroxide. These myelin bodies looked FIG. 5. Two myelin bodies (arrow) are shown in the nuclear region still attached to the membrane surrounding the vacuole. × 18,000. FIG. 6. Myelin bodies (arrows) appear suspended in the vacuole. In 6 a the secretory bodies are still intact; in 6 b it appears that disruption has begun giving rise to amorphous granular masses (AM). 6a: x 12,000; 6b: x 10,000.

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O.

ROGER ANDERSON AND O. A. ROELS TABLE I

COMPARATIVE DATA ON NATURALLY OCCURRING AND SYNTHETIC MYELINICS Source of Myelinics Parameter

Lamellar width Interspace Size of body

Egg Lecithin Myelinics

Lung Tissue (Sun, 28)

Digestive vacuole (Rudzinska, 21)

47 A 23 A Variable

45- 70 A 150-400/~ --

100 ~ -2-8/~

Ochromonas malharnensis

80-100 140-220 0.66/~

very similar to those described by Sun (28). W h e n we o m i t t e d g l u t a r a l d e h y d e fixation, a n d used only o s m i u m tetroxide fixing a n d staining, these bodies were never seen. I n an a t t e m p t to determine the structure of these myelin bodies in O. malhamensis a n d the effect of g l u t a r a l d e h y d e fixation on them, we have studied a m o d e l syst e m of synthetic myelinics p r e p a r e d f r o m p u r e egg lecithin. The p r o d u c t i o n of synthetic lipid myelinics in a q u e o u s suspension was a c c o m plished b y B a n g h a m (1), Stoeckenius (26), a n d L u c y a n d G l a u e r t (12), w h o all showed that p h o s p h o l i p i d s , when h y d r a t e d , p r o d u c e d systems of concentric lamellae a p p r o x i m a t e l y 50 A thick. T h e y p r o p o s e d that the lamellae were f o r m e d b y a lipid bilayer in which the h y d r o p h o b i c chains o p p o s e d one a n o t h e r a n d the p o l a r groups projected t o w a r d the a q u e o u s interspace. W e have p r o d u c e d myelinics in o u r l a b o r a t o r y (Figs. 8 a, 8 b) a n d n o t e d the similarity between these synthetic bodies a n d the myelinics f o u n d in Ochromonas. However, there are two characteristics of the secretory bodies in O. malhamensis which differ f r o m artificial myelinics: the spacing of the lamellae is m u c h wider at the p e r i p h e r y of the secretory b o d y t h a n in artificial myelinics a n d the lamellae are a b o u t twice as thick, as is shown in Table I. However, the dimensions a n d a p p e a r a n c e of the myelinics in Ochromonas are very similar to those observed in lung tissue (28). Some of the differences in a p p e a r a n c e between our artificial myelinics a n d the O. FIG. 7. An enlarged segment of Fig. 3 shows the fine structure of a secretory body (7 a). An inclusion (I) is surrounded by concentric lamellae which fold back upon themselves and become confluent along their margins. Fig. 7 b shows a myelin figure (enlarged from Fig. 4), in a late stage of secretion, which contains loosely spiraled lamellae containing dense granules scattered in the interspaces. 7a: × 72,000; 7b: ×48,000. FIo. 8. Negatively stained synthetic myelinics, prepared from egg lecithin, contain concentric lamellae and central dense cores resembling naturally occurring myelinics in Ochromonas. Fig. 8 a shows an egg lecithin myelinic before fixation; Fig. 8 b demonstrates the preservation of an egg lecithin myelinic when glutaraldehyde fixation precedes osmium tetroxide treatment. In the absence of glutaraldehyde fixation these myelinics are destroyed by the osmium treatment. 8 a: x 355,000; 8 b : × 466,000.

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malhamensis secretory body can be attributed to differences in staining technique. The artificial myelinics were negatively stained with potassium phosphotungstate which adsorbs on the polar groups in the aqueous phase leaving the hydrophobic region unstained. The O. malhamensis secretory bodies were stained with osmium tetroxide which reacts with the double bonds in the unsaturated fatty acids and deposits in the hydrophobic phase as well as at the polar groups (5, 20). We have further observed that the appearance in the electron microscope of our myelinics prepared by dispersing pure egg lecithin in an aqueous phase can be influenced by the fixation process: the myelinics were degraded by osmium tetroxide in the absence of glutaraldehyde, but were stabilized when glutaraldehyde fixation preceded the osmium treatment. Sabatini et al. (23) have shown that glutaraldehyde fixation, followed by osmium tetroxide treatment, preserved the mitochondrial matrix and the endoplasmic reticulum of many mammalian tissues better than osmium tetroxide fixation alone. The mechanism whereby glutaraldehyde stabilizes amphipathic lipid systems is not known. However, a tenable explanation is that glutaraldehyde cross-links the polar groups of the lipid molecule, thereby stabilizing the lipid layer. When lecithin layers are formed in a hydrated system, the polar groups project into the aqueous phase. The distance between the positively charged quaternary ammonium groups of two adjacent egg lecithin molecules in monolayers at a surface pressure of 30 dynes per centimeter is about 7.5 A (25). We believe it reasonable to assume a comparable packing distance in lipid multilayers such as synthetic and natural myelinics since the collapse pressure of the pure egg lecithin monolayer is 35-37 dynes per centimeter (Shah and Schulman, 25). This distance will exactly accommodate one glutaraldehyde molecule since the distance between its polar groups is approximately 7.5 A. The formation of ionic linkages between the positive polar groups of the lecithin and the partial negative charge (6-) on the carbonyl groups of the glutaraldehyde would stabilize this system. Riemersma (20) and Criegee (5) have shown that the addition of osmium tetroxide to unsaturated lipids results in the formation of a cyclic osmic ester and subsequently a diol at the original site of the carbon-carbon double bonds. The production of hydroxyl groups in the hydrophobic region of the lipid layer would result in disorganization of the myelin structure unless additional stabilizing factors were present. The addition of glutaraldehyde to such a lipid system apparently provides additional stabilization, perhaps by cross-linking the quaternary ammonium groups and thus preventing disorganization of the lipid. These observations suggest that part of the stabilizing influence of glutaraldehyde in tissue preservation can be attributed to stabilization of amphipathic lipid systems. The observation of myelinics in Ochromonas malhamensis when prefixed with glutaraldehyde could be due to an extra stabilizing influence of the glutaraldehyde fixative.

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We express our sincere appreciation to Dr. S. H. Hutner of the Haskins Laboratories, New York, New York, who supplied the initial culture of Ochromonas malhamensis used in this study. Thanks are also due to Drs. P. J. Halicki and D. O. Shah of Columbia University for reading the manuscript and offering helpful criticisms.

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