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The nucleus of Euglena
JOURNAL OF ULTRASTRUCTURE RESEARCH 62, 251-269 (1978)
The Nucleus of Euglena II. Ultrastructural Modifications of the Nucleus of B12-Deprived Euglena gracilis Z O D I L E B E R T A U X , G I L L E S M O Y N E , * C H R I S T I A N E L A F A R G E - F R A Y S S I N E T , * AND R I C H A R D VALENCIA
Laboratoire de Nutrition et Cin~tique Cellulaires, Facult~ de Pharmacie de l'Universit~ de Paris-Sud, F92290, Ch&tenay-Malabry, France, and *Institut de Recherches Scientifique sur le Cancer, B.P. 8, F-94800, Villejuif, France Received August 17, 1977 The nuclear ultrastructure of Euglena gracilis Z was studied in synchronized cells cultivated in a vitamin B12-deprived medium. Specific and preferential nucleoprotein stains were applied to Epon and frozen ultrathin sections as well as conventional techniques. The evolution of the chromatin structure and of the nucleolar organization was observed as the deficiency increased, while mitosis was synchronously blocked. In normal Euglena, the chromosomes were permanently condensed with a variable degree of packing; under conditions of B12 deficiency, the chromosomal condensation progressively disappeared. Staining of chromatin became ultimately impossible even using specific methods. Only the nucleolar DNA remained normally condensed. However, it was shown that these cells still possess a DNA content equivalent to that found at the end of the S phase. The nucleolus gradually split itself in many lobules. In some cases, nucleolar vacuoles and segregation of the granular and fibrillar components were observed.
Vitamin B~2 is an essential growing factor for some prokaryote microorganisms such as Escherichia coli (Davis and Mingioli, 1950) and LactobacUlus leichmanii (Wright et al., 1948). Apart from the animals, it is also essential for some eukaryote microorganisms such as the flagellates Ochromonas, (Hutner et al., 1949; Ford, 1953) Poteriochromonas (Hutner et al., 1949) and E u g l e n a gracilis (Hutner and Provasoli, 1955; Epstein et al., 1962). Without vitamin B12, L. leichmanii grows into giant elongated cells that have been claimed to represent a model of the pernicious anemia megaloblast (Beck et al., 1962, 1965). Similarly, when E. gracilis cells are deprived of vitamin B12 during their exponential growing phase, cell division stops and the cellular volume increases up to 40 times the normal volume (Bertaux, 1976). This hypertrophy results from the synthesis of RNA, proteins, and glucidic substances accumulating in these cells. Regarding DNA, during mitosis inhibition, it remains at a level equivalent to that found at the end of
the S phase (Bertaux and Valencia, 1975; Bertaux, 1976). In this work, we have studied the nuclear ultrastructure of Euglena cells deprived of vitamin B12. This avitaminosis leads to unbalances in the R N A / D N A and protein/DNA ratios that can reach up to 10 times their normal values. This abnormal metabolism cannot fail to induce alterations of the nuclear activity and ultrastructure. We have studied the ultrastructure of the nucleus of B12-deprived cells as compared with that of the normal nucleus, observed either in interphase and investigated with the same techniques (Moyne et al., 1975), or during a synchronized cell cycle (Frayssinet et al., 1975; Bertaux, 1976; Bertaux et al., 1976). We have also distinguished between two kinds of B12-deprivation procedures and their ultrastructural consequences: Avitaminosis beginning during the exponential growing phase results in a severe deficiency while during slow growth the consequences
251 0022-5320/78/0623-0251502.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
BERTAUX ET AL.
252
o f B12 d e p r i v a t i o n a r e less d r a s t i c ( B e r t a u x a n d V a l e n c i a , 1973). MATERIALS AND METHODS
I. Cell Culture Autotroph cultures of Euglena gracilis Z (Cambridge No. 1224-5 D) were cultivated in Cramer and Myers' inorganic salt medium (1952). This medium is considered as autotrophic with regard to the carbon source. Vitamins B1 and B1~ provide negligible amounts of carbon for energetic needs, while citrate, at pH 6.8, is exclusively present as a chelator (Hutner et al., 1966}. Erlenmeyer flasks (750-ml volume) conraining 250 ml of culture medium were mechanically shaken in a 25°C stove to ensure aeration of cultures; when larger quantities of cells were required, culture was carried out in 2000-ml toxin flasks containing 1500 ml of culture medium, bubbled with a mixture of air-CO2 (95:5). Under these conditions, at the end of the exponential phase, the cell concentration was 10fold higher than in the Erlenmeyer cultures (Bertaux, 1976). The cells were synchronized by successive photoperiods of light (14 hr) and dark (10 hr), according to Edmunds (1965). Deficiencies were initiated by transferring the ceils into a medium deprived of vitamin B12. To obtain a deficiency during the exponential growth phase, the cell concentration at the onset of mitosis inhibition had to near 104 cells/ml. On the contrary, a concentration of 10~ cells/ml was required when the deprivation was carried out in stationary phase. In toxin flasks bubbled with the air-CO2 mixture, the cell concentration could be increased, respectively, up to i0 ~ and 106 cells/ml (Bertaux, 1976). Cell concentrations and the cellular mean volumes were monitored with a Coulter counter fitted with a mean cell volume device.
II. B i o c h e m i c a l Determinations The nucleic acids were extracted according to Schmidt and Thannh~iuser (1945), as modified by Smillie and Krotkov (1960). RNA was determined by Mejbaum's technique (1939) and DNA by Giles' and Myers' (1965). Protein determination was carried out according to Lowry et al. (1951).
III. Microscopy (a) Light microcopy. Whole cells fixed on microscope slides were stained by Feulgen's technique. (b) Electron microscopy. All the techniques used in this work were previously described in detail (Moyne et al., 1975). Three types of staining methods were used: conventional uranyl and lead staining (Reynolds, 1963), the EDTA regressive method preferential for ribonucleoproteins (Bernhard, 1969), and DNAspecific stains such as the Schiff-thallium technique
(Moyne, 1973), and the osmium ammine method (Cogliati and Gautier, 1973). RESULTS I. OBTAINING B12-DEPRIVED CELLS
(a) E x p o n e n t i a l Growing P h a s e B12-deprived E u g l e n a cells w e r e o b t a i n e d b y t r a n s f e r r i n g n o r m a l s y n c h r o n i z e d cells i n t o B12-deprived m e d i u m . T h e m a x i m u m cell c o n c e n t r a t i o n , b e t w e e n t w o c h a n g e s of c u l t u r e m e d i u m , c o u l d n o t e x c e e d 105 c e l l s / m l in o r d e r to r e m a i n in e x p o n e n t i a l growing phase. In the same way, mitosis i n h i b i t i o n h a d to o c c u r w h e n t h e cell conc e n t r a t i o n w a s n e a r 104 c e l l s / m l ( C h a r t 1). U n d e r t h e s e f a v o r a b l e c o n d i t i o n s , t h e cell u l a r m e t a b o l i s m w a s a c t i v e , m a k i n g feasible an easy study of the effects of vitamin B12 d e f i c i e n c y . C e l l s t r a n s f e r r e d in B i 2 - d e p r i v e d m e d i u m w e r e still a b l e t o d i v i d e five o r six t i m e s { C h a r t 1). T h e i r m e a n v o l u m e v a r i e d i n a . cyclic w a y d u r i n g t h e d i f f e r e n t cell cycles. F r o m 4000 # m 3 a t t h e e n d o f t h e l i g h t photoperiod, at the very beginning of the e x p o n e n t i a l g r o w t h p h a s e (103 c e l l s / m l ) , it d e c r e a s e d d o w n t o 1000 ~ m ~ a f t e r t h e cell division, a t t h e e n d o f t h e e x p o n e n t i a l p h a s e (105 c e l l s / m l ) . A f t e r t h e b e g i n n i n g of m i t o sis i n h i b i t i o n (7 d a y s ' d e f i c i e n c y ) , t h e m e a n v o l u m e i n c r e a s e d r e g u l a r l y so t h a t s e v e r a l d a y s o f b l o c k l e d to a l a r g e c e l l u l a r h y p e r trophy. I n t h e e x a m p l e s h o w n , t h e m e a n cell v o l u m e w a s 22000 # m ~ a f t e r 9 d a y s of m i tosis block.
(b) S t a t i o n a r y Growing P h a s e This type of deficiency was obtained w h e n m i t o s i s i n h i b i t i o n b e g a n w i t h a cell c o n c e n t r a t i o n h i g h e r t h a n 10 ~ c e l l s / m l . G r o w i n g c o n d i t i o n s w e r e less f a v o r a b l e , a n d cell m e t a b o l i s m w a s slowed. T h u s , h y pertrophy was less marked (maximum m e a n cell v o l u m e = 2200 # m ~) a n d t h e e x t e r n a l cell m o r p h o l o g y d i s p l a y e d s o m e d e f o r m a t i o n s ( B e r t a u x a n d V a l e n c i a , 1973; B e r t a u x , 1976). However, our main goal was to induce a
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CHART 1. (A) Synchronous growth of Euglena between the first and the sixth day of deprivation, starting from the first transfer in medium free of vitamin B12. After six generations of apparently normal cells, cell division was blocked after the second transfer between the sixth and the fifteenth day of deficiency. (a) After a block of 2 days: Beginning of nucleolar fragmentation (Fig. 4); the chromosomes appear as residual chromatin. (b) 8.day block: The nucleoli are entirely fragmented in lobules; chromatin is no longer stainable; extended clear zones mark the location of former chromosomes (Fig. 5). (B) Mean cell volume of deprived Euglena. Decreasing cyclic evolution of the volume during the exponential phase between the first and the sixth day of deficiency, before mitosis block. The volume increases during the 14-hr light period; it decreases during the dark period when the cells divide. Linear increase of the mean cell volume after beginning of the divisions block between the sixth and the fifteenth day of deficiency (maximum mean cell volume = 22 000 ttm:~). 253
254
BERTAUX ET AL.
synchronized block of the cell divisions in order to determine at which stage of the cell cycle it occurred. We have therefore mostly studied B12-deprived cells in the exponential growing phase.
also a significant correlation of the amounts of protein and RNA with the cellular vol. ume (P < 0.001; Chart 3). Regarding DNA, the regression line slope is slightly positive, with no significant correlation. The progressive protein and RNA inII. BIOCHEMICAL EFFECTS creases in the deprived cells explain their Data from the literature revealed that enormous hypertrophy. On the contrary, vitamin B12 deficiency induces marked in- the mean amount of DNA did not vary creases in the amounts of RNA and pro- during all the cell division block. The teins in rapidly growing cells or tissues of amount of DNA was equivalent to the highvarious types: human (White et al., 1953; est level observed in normal cells at the end Glazer et al., 1954) or unicellular (Beck et of the S phase. al., 1965; Carell et al., 1970). In contradistinction, results on the (b) Stationary Growing Phase amounts of DNA were more ambiguous. The results obtained in the stationary For some authors they were lowered growing phase are summarized in Table 1. (Soldo, 1965; Pogo et al., 1966), whereas for Deprived cells in stationary phase disothers they were not modified (Marinone, played only a slight hypertrophy since their 1954) or even increased (Davidson et al., maximal volume was 2200 gm 3. This implies 1948; Perugini et al., 1961; Carell et al., a 25% increase as compared with control 1970). It was thus necessary to determine cells cultivated under the same conditions the variations in the amounts of macromol- (stationary phase). However, the volume of ecules in Euglena cells during the normal the deprived cells more than doubled in synchronized cycle in order to appreciate comparison with control cells in G1 phase the meaning of the data, including the ul- in spite of a similar macromolecular comtrastructural effects, obtained in BI2-de- position (Table 1). Indeed, the amounts of prived cells. DNA, RNA, and proteins found in the deprived cells in the stationary phase are at (a) Exponential Growing Phase a minimum level, equal to that of the G1 The evolution of the amounts of DNA, phase for exponentially growing control RNA, and protein in normal Euglena cells ceils. Therefore, the volume increase is not is shown on Chart 2 as a function of the attributable to RNA and protein synthesis cellular volume. The same values are shown as in the preceding case but more probably again in the left part of Chart 3 (between to an accumulation of glucidic material 1000 and 3000 #m 3) for the comparison with such as paramylon. the corresponding amounts in Bi2-deprived III. ULTRASTRUCTURAL EFFECTS cells of much larger volume (up to 20,000 ~m3). The ultrastructure of the normal EuThus, the normal values are grouped glena nucleus was previously described in within three well-delimited zones. The de- detail (Moyne et al., 1975; Bertaux et al., terminations were obtained from 117 sam- 1976). The chromosomes persist in conples collected at different stages of the cell densed form throughout the cell cycle. The cycle. The regression lines (Chart 2) dem- nucleolus does not disappear during mitosis onstrate a significant correlation (P < and divides after stretching itself. During 0.001) of the different amounts with the the cell cycle, several degrees of chromocellular volume. some condensation were observed: Packing The values for the deprived Euglena was more intense just after mitosis, dewere obtained from 82 samples. They show creased during G1 (Fig. 1), and was at a
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
255
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CHART 2. Regression lines demonstrating the correlations of the DNA, RNA, and protein contents in normal synchronous Euglena with the mean cell volume. Correlation coefficients: r (DNA) = 0.79; r (RNA) = 0.89; r (proteins) = 0.72. All these correlations are significant with a probability smaller than 10-~. m i n i m u m in G2 a n d d u r i n g m i t o s i s . I n t h e nucleolus, granular and fibrillar parts were o b s e r v e d . T h e l a t t e r w e r e o r g a n i z e d in conc e n t r i c s t r u c t u r e s " ( F r a y s s i n e t et al., 1975) o f a d i a m e t e r o f a b o u t 0.5 ttm (Fig. 1). W e could demonstrate the localization of nu-
cleolar DNA as rings within these particul a r s t r u c t u r e s ( M o y n e etal., 1975).
(a) Nuclear Hypertrophy D u r i n g v i t a m i n B12 d e f i c i e n c y i n t h e exponential growing phase, the cellular hy-
256
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CHART3. Regression lines demonstrating the correlations of the DNA, RNA, and protein contents in B12deprived Euglena with the mean cell volume. The short regression lines in the left part of the chart are a reduced representation of Chart 2 which permits comparison at the same scale of the macromolecular contents in control and deprived Euglena. Correlation coefficients: r (DNA) = 0.13; there is no correlation of the DNA concentration per ceil with the volume of the deprived cells; r (RNA) = 0.6?; r (proteins) = 0.72; these correlations are significant with a probability smaller than 10-3. p e r t r o p h y also involved t h e nucleus. W e carried o u t an a p p r o x i m a t e c o m p u t a t i o n of the nuclear volumes from measurements of the n u c l e a r d i a m e t e r s on e l e c t r o n micrographs. I t s h o w e d t h a t t h e n u c l e a r v o l u m e i n c r e a s e d a b o u t p r o p o r t i o n a t e l y to t h e w h o l e cell volume. T h e v o l u m e o f n o r m a l nuclei (diameter, 5 t o 7.5 ~tm) v a r i e d d u r i n g t h e cell cycle b e t w e e n 65 a n d 220 # m 3. A f t e r t h e two d a y s of mitosis inhibition, t h e nuclear v o l u m e i n c r e a s e d to 400/~m 3 (diame-
ter, 9 #m), a n d after 8 d a y s it was larger t h a n 2000 # m s {diameter, 16 #m). T h e nucleolus e n l a r g e d p r o p o r t i o n a t e l y . D u r i n g t h e s t a t i o n a r y phase, t h e n u c l e u s did n o t enlarge. Its v o l u m e varied within t h e size limits of t h e n o r m a l nucleus.
(b) Ultrastructural Modifications of the Nucleus W e will essentially describe t h e ultras t r u c t u r e o f t h e n u c l e u s o f d e p r i v e d Eu-
Euglena N U C L E U S A N D V I T A M I N B12 D E F I C I E N C Y TABLE 1
257
deprived cells, chromatin seemed to be largely dispersed throughout the whole nucleoplasm since only small clear zones conControls B ,2taining no granules marked the probable Deprived location of the residual condensed chroExponential Station- _ _ phase (117) ary matin. phase When adapted to ultrathin frozen secBeginmid(28)" Stationning Sb ary tions, the same technique revealed a differof phase GL~ (42)" ent nucleoplasmic structure: We observed Volume (#m 3) 1000 2000 1800 2200 U-shaped fibrils (Fig. 10) identical to those D N A (#g/10 ~ 3.2 5.3 3.9 3.1 visualized in the normal cell (Moyne et al., cells) 1975). RNA (#g/106 28 48 30 32 2. Chromosomes. Conventional staining. cells) In normal Euglena cells, chromosomes Proteins 260 350 360 231 (~g/106 cells) were always densely stained by conventional techniques (Fig. 1) during the entire a M e a n values; n u m b e r of s a m p l e s given in parentheses. cell cycle (Bertaux et al., 1976). When BlZ b Values obtained from t h e regression lines. deficiency occurred in the exponential growing phase, the chromosomes progresglena cells in the exponential growing sively disappeared as dense chromatin. phase since it was this type of deficiency Two or three days after inhibition of cell that induced the most important nuclear division, a few small clumps of dense chromodifications. matin persisted (Fig. 3). They often bor1. Nucleoplasm. Conventional staining. dered large electron-lucent zones (Figs. 3 After the conventional procedure, i.e., dou- and 4) sometimes in contact with the nuble fixation, Epon embedding, and uranyl cleolus (Fig. 7). Ultimately, even this residlead staining, we did not observe any dif- ual dense chromatin disappeared (Figs. 5 ference regarding the nucleoplasm of the and 6), leaving only electron-lucent zones deprived (Figs. 2-4) and control cells (Fig. on the sites formerly occupied by dense 1). However, in deprived cells with mitosis chromatin. These electron-lucent zones blocked for several days, we often saw clus- consisted of fibrils about 100 A thick (Fig. ters of 10 to 30 large granules (Figs. 6 and 7). 8). Their diameter varied from 650 to 2300 When cells were deprived of vitamin B12 A, and they were sometimes in contact with so that the block of cell division occurred the nucleolus (Fig. 8). in stationary phase, the structure of chroIn normal Euglena, we have described matin was not modified. The chromosomes isolated dense granules (Moyne et al., 1975) were long and ramified (Fig. 2), a typical but they were much rarer than those ob- aspect of normal cells in G1 phase (Bertaux served in deprived cells. et al., 1976). Preferential R N P staining. The nucleoSpecific DNA staining. The fibrillar plasm appeared normal when observed structure of the residual dense chromatin after staining by the regressive EDTA was revealed on Epon sections using the method. It was filled by a large number of specific DNA stain osmium ammine (Cogdense granules 300 to 500 /~ in diameter, liati and Gautier, 1973) (Fig. 16). The residregularly dispersed within a finely fibrillar ual chromatin was sometimes dispersed matrix relatively less electron dense (Fig. within the nucleoplasm as fine fibers of 9). However, it was more homogeneous thickness ranging from 20 to 45 A (Fig. 16). than in normal cells where the localization The clumps of residual dense chromatin of the chromosomes was marked by large were about 0.1 to 0.2 ~m in their longest bleached zones (Moyne et al., 1975). In B12- dimension (Fig. 4). They were therefore 10 MACROMOLECULE CONTENTS OF CONTROL AND B12DEPRIVED CELLS
258
B E R T A U X E T AL.
FIGS. 1-3.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
times smaller than the electron-lucent zones (Fig. 6), themselves twice larger than t h e mean size of normal sectioned chromosomes in G1 phase. Thus, even a specific staining technique was not able to reveal the large amount of DNA contained in these nuclei (7 #g/106 cells). The light microscopic Feulgen's reaction "was also carried out on whole normal cells. Clumped chromatin intensely stained was thus revealed. The same reaction was applied to deprived cells in exponential growing phase. After 8 days of mitosis inhibition, the reaction was still visible but entirely : diffuse. 3. Nucleolus. Conventional staining. Immediately after the beginning of mitosis inhibition, 7 days after the initiation of B12 deficiency, one or several lobules parted from the nucleolar periphery (Figs. 3 and 4). This phenomenon increased as the deprivation carried on. After 8 days of cell division block, the nucleoli were largely and sometimes entirely fragmented (Figs. 5 and 10). We observed a similar sequence in cells deprived of vitamin B12 during the stationary growing phase (Fig. 2). However, the parted lobules never numbered more than three or four. Their size displayed some variability: 0.5 to 1.5 #m in diameter for lobules with one annular structure while in normal cells they were never seen to be larger than 0.5 #m (Fig. 1). In a few cases, the nucleolus was only little fragmented, but we observed a segregation of the granular and fibrillar components (Figs. 6 and 7) with a nucleolar "vacuolization." Preferential RNP staining. The regres-
259
sive EDTA stain bleaches preferentially DNA-containing structures. In partly fragmented nucleoli, this technique revealed on Epon sections that each nucleolar lobule contains a notable quantity of DNP (Fig. 9). Thus, during B12 defiency, the nucleolar DNA keeps its staining properties unlike the nucleoplasmic DNA. On ultrathin frozen sections, clear circular zones were also seen in each lobule of a totally fragmented nucleolus (Fig. 10). Specific staining of the nucleolar DNA. We could clearly demonstrate the presence of DNA in the nucleoli of B12-deprived cells using either the Schiff-thallium (Figs. 11 and 12) or the osmium ammine technique (Figs. 13-15) on Epon or frozen ultrathin sections. The DNA was generally contained in annular structures (Figs. 13 and 14), 0.4 to 0.8 gm in diameter, in the center of the lobules. The DNA-containing structures sometimes consisted of a double ring (Fig. 15) similar to the cockade structures visible in the nucleoli of normal cells (Fig. 1). Thus, in B12-deprived cells the localization and structure of the nucleolar DNA are similar to that of normal cells. In cells deprived of vitamin B12 in the exponential growing phase, nucleolar DNA was more abundant than in normal cells : We could count up to 15 DNA rings per section of nucleolus (Fig. 14), while in normal cells, with the same technique, we found two or three. However, after conventional staining, up to six rings could be counted in normal cells (Fig. 1). The number of DNA rings in cells where B12 deprivation had been initiated in the stationary growing phase was equivalent to that of the normal cell (Figs. 11 and 12).
FIG. 1. Nucleus of a control cell at the end of G1 phase (cell volume = 1800 gm~). Uranyl lead staining. The chromosomes (chr) persist in the condensed state. In the nucleolus (nu), DNA-containing annular structures (A) are surrounded by nucleolar granules. × 30 000. FIG. 2. Nucleus of a cell in stationary phase after 8 days of vitamin B,2 deficiency (cell volume = 2000 ~m~). Uranyl lead staining. The chromosomes (chr) are still condensed and larger than in Fig. 1. Nucleolus (nu) slightly fragmented. × 22 000. FIG. 3. Nucleus of a cell in exponential growing phase after 7 days of vitamin B,2 deficiency and 1 day of mitosis inhibition (cell volume = 5000 gm:~). Uranyl lead staining. Chromatin has largely disappeared and light zones (L) localize the former chromosomes. They are bordered with residual dense chromatin (--~). Fragmen-. tation of the nucleolus (nu) is beginning. Annular structure (A). ×8 500.
260
B E R T A U X E T AL. DISCUSSION
We have studied simultaneously the modifications of the nuclear ultrastructure and of the macromolecular composition of cells in vitamin BI2 deficiency, as compared with normal cells. We aimed at discovering a relationship between structure and function that could explain the observed phenomena. The choice of Euglena gracilis Z has a double justification: (i) It is a good model for the study of vitamin BI2 deficiency. The essential role of
this growing factor in cell division was demonstrated in pernicious anemia and in several microorganisms. Euglena is interesting as a eukaryotic cell which, like animal cells, • makes use of "true" vitamin BI2 unlike the prokaryotic microorganisms able to use cobalamine analogs (Valencia, 1974). Above all, it is one of the rare eukaryotic cells whose experimental BI2 deprivation can be easily realized: This is due to the simplicity of its culture medium, mineral with the exception of vitamins B, and BIe, and to its photosynthetic faculties.
FIGS. 4 AND 5. Nuclei of cells in exponential growing phase in vitamin B,2 deficiency. Uranyl lead staining. FIG. 4. Deficiency, 8 days; mitosis inhibition, 2 days (cell volume = 6500 #m:J). Increased fragmentation of the nucleolus (nu) as compared with Fig. 3. Each nucleolar fragment is centered by an annular structure (A). Light zones (L). Residual chromatin (--*). ×15 500. FIG. 5. Deficiency, 14 days; mitosis inhibition, 8 days (cell volume = 22,000 #m:~). Residual chromatin rarer than in Fig. 4. Nucleolus (nu) entirely fragmented. ×34 000. FIGS. 6-8. Nuclei of cells in the exponential growing phase cultivated without vitamin B,~. Uranyl lead staining. FIG. 6. Deficiency 14 days; mitosis inhibition, 8 days (cell volume = 22,000/~m:~). Electron-lucent light zones (L) seem the only remnants of chromatin. In the nucleolus (nu), granular components (g) are separated from the fibrillar and chromatin components that remain intermingled. Nucleoplasmic dense granules (--~). Nucleolar vacuoles (v). ×12 000. FIG. 7. Deficiency, 11 days; mitosis inhibition, 5 days (cell volume = 14,000 #ma). A fibrillar light zone (L) bordered with dense chromatin (chr) is in contact with the nucleolus. A DNA-containing annular structure (A) is visible. Nucleolar fibrils (f) and granules (g). ×30 000. FIG. 8. Same deficiency as in Fig. 6. Electron-dense granules (-*) are visible in contact with the fragmented nucleolus (nu). Compare with Fig. 6 where identical granules are visible within the nucleoplasm. ×30 000. FIGS. 9 AND 10. Nuclei of cells in exponential phase cultivated without vitamin B~2. FIG. 9. Deficiency, 9 days; mitosis inhibition, 3 days (cell volume = 8000 #m:J). EDTA preferential stain for ribonueleoproteins. Moderately fragmented nucleolns. Bleached regions (--*) assumed to contain DNA are visible in each nucleolar fragment. ×32 000. FIG. 10. Deficiency, 14 days; mitosis inhibition, 10 days (cell volume = 24,000 #m3). Ultrathin frozen section. Ribonucleoprotein preferential staining. The nucleolus (nu) is entirely fragmented. A light zone (L) is visible in the nucleoplasm otherwise filled with RNP fibrils identical with the U-fibrils observed in normal cells (-*). ×33 000
FIGS. 11-14. Cells cultivated without vitamin B,2. DNA-specific stains: Schiff-thailium reaction (Figs. 11 and 12) or osmium ammine (Figs. 13 and 14). FIG. 11. Nucleolar DNA of a cell in stationary phase; 15 days of cultivation. The nucleolar DNA (--)) is revealed as a well-localized circular formation in the nucleolar body (nu). Chromosomes (chr).)<36000. FIG. 12. Same as in Fig. 11. × 33000. FIG. 13. High magnification of an annular structure. Cell in exponential phase. Mitosis blocked for 5 days. Ultrathin frozen section. This method reveals nucleolar DNA as a finely fibrillar structure. ×51000. FIG. 14. Nucleus of a cell in the exponential growing phase. Mitosis inhibition for 3 days. In the nncleoplasm, some residual chromatin (---~} is visible. In the nucleolus, DNA is present in numerous circular structures. x32 000.
FIGS. 15 AND 16. Cells in the exponential growing phase cultivated for 10 days without vitamin B,2. Mitosis inhibition for 4 days. Specific DNA stain with osmium ammine. FIG. 15. Detail of a nucleolus. Each nucleolar fragment contains one or two annular DNA structures (--~). x56 000. FIG. 16. Residual nucleoplasmic chromatin. The fibrillar structure of the residual chromatin is clearly visible. No DNA fibers are visible in the surrounding nucleoplasm. ×100000.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
FIGs. 4-5.
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FIGS. 6-8.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
F~GS. 9-10.
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FrGs. 11-14.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
FIGS. 15-16.
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BERTAUX ET AL.
(ii) Cultures of Euglena can be brought to an excellent degree of synchrony. They allow attainment of large quantities of cells at each phase of the cell cycle, and thus they permit parallel biochemical and ultrastructural observations. The synchronization holds during the deficiency, even when mitosis is blocked since synchronous synthesis of RNA and proteins still occurs during the successive photoperiods (Bertaux and Valencia, 1973; Bertaux, 1976). We chose to study B,2 deficiency mostly during the exponential growing phase when the absence of vitamin B,2 is the only limiting factor for cells with an active metabolism. Under these conditions, the need for vitamin B~2 is very high and makes more drastic the effects of the avitaminosis. I. NUCLEOPLASMANDCHROMATIN The inhibition of cell division during B12 deficiency does not seem to be linked to a quantitative lack of DNA synthesis, since it occurs when the cells contain about twice as much DNA as in G, phase. But the ultrastructural modifications of chromatin do not allow the exclusion of qualitative alterations. The last replication could be incorrect and could induce modifications of the secondary or/and tertiary molecular structure that could explain the morphological changes observed: (i) Dense chromatin disappeared progressively; after 8 days of cell division block, the nucleoplasm contained only electronlucent zones that seemed to correspond to the former localization of chromosomes. (ii) Under the light microscope, Feulgen's reaction showed that DNA is well localized in chromatin clumps in normal cells, while its repartition becomes diffuse in deprived cells. (iii) DNA reactions at the ultrastructural level also revealed a disappearance of chromatin clumps leaving, at last, small masses of residual dense chromatin much reduced in volume as compared with normal chromatin. It was mentioned above that the quantity
of DNA is not reduced during B12 deficiency under our conditions of checked exponential growth. Consequently, the cytochemical results can be explained by a great dispersion of DNA in deprived cells. If we add that the cellular volume increases 20-fold, it is clear that DNA fibers highly dispersed in the nucleoplasm can be no longer demonstrated by cytochemistry. In normal cells, we could visualize no dispersed chromatin with the exception of fibers linking chromosomes together-and to the nucleolus (Moyne et al., 1975). In spite of these structural modifications, the transcriptional abilities of the cell are not noticeably decreased. It is even conceivable that the synthesis of messenger . RNA would be facilitated by the dispersed state of the chromatin. Regarding the morphology, a similar phenomenon was described by Heath {1966) and Menzies et al. (1966) in the megaloblast. They found chromatid breaks and incomplete contraction of the chromosomes. It is also interesting to note that the unbalanced increase of the R N A / D N A ratio is as marked in Euglena as in pernicious anemia blood cells (White et al., 1953; Glazer et al., 1954). These facts tend to complete the parallel between pernicious anemia blood cells and B12-deficient Euglena. In addition, we could never observe in B12-deprived Euglena the microtubules of the division spindle visible in control dividing cells. This absence can be explained either because the cells are blocked in G2 phase, before prophase, or because the formation of the spindle fibers itself is inhibited by the deficiency. II. NUCLEOLUS The main nucleolar alteration consists of a kind of fragmentation that has not been previously described as far as we know. The nucleolar fragments can comprise several annular structures at the beginning of the deficiency {Fig. 4), but with a longer deprivation, the nucleolus seems to be split into
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY as many lobules as annular structures (Fig. 5). This is different from the classical nucleolar segregation related to interruption of RNA synthesis, generally after the action of drugs (Busch and Smetana, 1970) and characterized by a separation of the fibrillar, granular, and chromatin components of the nucleolus. In B12-deprived Euglena we have occasionally observed a nucleolar segregation accompanied by a discrete fragmentation. However, biochemistry demonstrated that the transcriptional activity remained very high in these deprived cells. Whatever the severity of the deficiency, nucleolar DNA kept a normal condensation and stainability, even in nucleoli entirely fragmented (Fig. 15). This DNA was still transcribed after mitosis inhibition, at least during the next four cycles. Ribosomal RNA synthesis continued at a normal level as shown by extraction and electrophoretic separation of the RNAs (Bertaux, 1976). Nucleolar DNA is transcribed, but is is also likely to be replicated several times in spite of the inhibition of mitosis. Indeed, we could observe up to 15 sections of DNA rings per nucleolus. This is far more than the number of DNA rings or even of annular structures visible in sections of control Euglena nucleoli. The likely replication of the nucleolar DNA would show that some DNA synthesis is still possible during B~2 deficiency. This would support the hypothesis explaining the mechanism of the division block by qualitative alterations of the chromosomal DNA molecules rather than by a stop at the end of the S phase. On the other hand, this is consistent with the hypothesis that the block of the division mechanism in B12-deficient Euglena would occur either at the end of the S phase or in phase G2, in any case before the outset of mitosis. Whatever might be the interpretation of the nucleolar fragmentation, it is accompanied by an increasing degranulation, parallel to the deficiency, that could explain the relative decrease in RNA synthesis near the lethal period (after 8 days of mitosis inhibition). Chart 3 shows the
267
regression line of the RNA concentration per cell as a function of the cell volume, during the increasing B~2 deficiency; it is clear that the slope of the deprived cells, 1.23, is smaller than that of the control cells, 2.1. It is also worth noting that for deprived cells of volume smaller than 12,000/tin 3, the increase in RNA concentration per cell is roughly parallel to that of the control cells. When the cells grow over this volume, the accumulation of RNA is much more limited, thus implying a decrease of the level of synthesis. We have also observed that Euglena cells of high volume, at the extreme stage of deficiency, are very unlikely to survive when vitamin B12 is added to the culture medium. On the other hand, the deficiency is rapidly and entirely reversible when the cellular volume is smaller than 12,000/zm 3. We have described B~2 deficiency in Euglena cells during exponential growth under controlled conditions. It leads to drastic consequences: block of cell division with a DNA content equal to that of normal G2 phase, enormous cell hypertrophy, nucleolar fragmentation, and loss of contrast of the chromosomes. Cell death is regularly observed after a fortnight. B~2 deficiency in Euglena approaching the stationary phase did not induce similar effects with the exception of a slight nucleolar fragmentation. The chromosomes remained visible, and the DNA content was equal to that of the normal G~ phase. We cannot exclude the possibility that the high cell concentration and slowing metabolism could induce complex effects compensating each other, and rendering less visible the phenomena of B12 deficiency. Regarding comparison of B12-deprived Euglena with pernicious anemia blood cells, the latter evidently depend on an environment much more complex than our model. They are subject to a variety of factors difficult to analyze. Consequently, their characteristics probably issue from the influence of these factors added to a pattern of B12 deprivation intermediate be-
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tween the two extremes described in our cell system. CONCLUSION
The similar effects of vitamin B12 deficiency in pernicious anemia and in Euglena allow consideration of this phytoflagellate as a model of pernicious anemia blood cells, as far a s B12 metabolism is concerned. In this work, we demonstrated that in the exponential growing phase, B~2 avitaminosis induces the following: A cellular hypertrophy resulting from the accumulation of RNA, protein, and glucidic substances stored in the cells. It leads to a volume increase up to 20-fold the normal one. The nuclear and nucleolar hypertrophies are analogous. A block of the cell division in G2 phase. During all the block the quantity of cellular DNA is maintained at a level equal to that of the end of the S phase. Important modifications of the chromatin structure. After a few days of mitosis inhibition, chromosomes are no longer visible. They appear only as extended unstained regions that we interpreted as resuiting from chromatin decondensation. DNA was no longer visualized after specific staining in spite of the large quantity of DNA contained in each nucleus. On the other hand, we did not observe any structural modification of the RNP in Epon sections as well as in frozen sections where the U fibrils persisted. Regularly observed nucleolar fragmentation. At an early stage, the fragments comprise several annular structures. Later, they are made of single isolated lobules. Fragmentation of the nucleolus is accompanied by a progressive degranulation. However, the nucleolar DNA can be revealed as a ring-shaped structure whatever the severity of the nucleolar alterations. In the stationary growing phase, the following are observed: Apart from some alterations of the shape of the cells, we noticed only a moderate hypertrophy (volume doubled as compared
with control cells in G1 phase). Cellular DNA is at its lowest level, comparable to that of control cells in GI phase. The structure of the chromatin is not modified. The chromosomes appear as dense and very long bodies. The nucleolar fragmentation is limited to two or three pieces. To sum up, B12 avitaminosis is a severe condition for Euglena cells deprived of the vitamin during the exponential growing phase. It is much milder for cells in the stationary growing phase, with a slower metabolism. We offer grateful thanks to Mrs. Evelyne Pichard and Miss Annie Viron for their excellent technical assistance. REFERENCES BECK, W. S., GOULIAN, M., AND HOOK, S. (1962) Bioch#n. Biophys. Acta 55, 470-478. BECK, W. S., GOULIAN,M., AND KASHKET, S. (1965) Trans. Assoc. Amer. Physicians 78, 341-361. BERNHARD, W. (1969) J. Ultrastruet. Res. 27, 250-265. BERTAUX, O. (1976) Etude M~tabolique et Cytologique de Euglena gracilis Z Synchrone. R61e de la Vitamine B12 danE le ContrLle de la Division Cellulaire, Thesis, University of Paris VI. BERTAUX, O., FRAYSSINET, C., AND VALENCIA, R. (1976) C. R. Acad. Sci. Ser. D, 282, 1293-1296. BERTAUX, O., AND VALENCIA, R. (1973) C. R. Acad. Sci. Ser. D, 276, 753-756. BERTAUX, O., AND VALENCIA, R. (1975) LeE Cycles Cellulaires et leur Blocage chez plusieurs Protistes, International colloquium of CNRS, No. 240, pp 331-343. BuscH, H., AND SMETANA,K. (1970) The Nucleolus, Academic Press, New York. CARELL, E. F., JOHNSTON, P. L., AND CHRISTOPHER, A. R. (1970) J. Cell Biol. 47, 525-530. COGLIATI,R., AND GAUTIER, A. (1973) C. R. Acad. SeN. Ser. D, 276, 3041-3044. CRAMER, U., AND MYERS, J. (1952) Arch. Mikrobiol. 17, 384-402. DAVIDSON, J. N., LESLIE, I., AND WHITE, J. C. (1948) J. Pathol. Baeteriol. 60, 1-20. DAVIS, B. D., AND MINGIOLI, E. S. (1950) J. Bacteriol. 50, 17-28. EDMUNDS, L. i . , JR. (1965) J. Cell. Comp. Physiol. 66, 147-182. EPSTEIN, S. S., WEISS, J. B., CAUSELEY, D., AND BUSH, P. (1962) J. Protozool. 9, 336-339. FORD, J. E, (1953) Brit. J. Nutr. 7, 299-306. FRAYSSINET, C., BERTAUX, O., AND VALENCIA, R.
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(1975) LeE Cycles Cellulaires et leur Blocage chez plusieurs Protistes. International colloquium of CNRS, No. 240, pp. 291-296. GILES, K. W., AND MYERS, A. (1965) Nature (London)~ 206, 93-94. GLAZER, n. S,, MUELLER, J. F., JARROLD, W., SAKU~ RAI, K., WILL, J. J., AND VILTER, R. W. (1954) J. Lab. Clin. Med. 43, 905-913. HEATH, C. W., JR. (1966) Blood J. Haematol. 27, 800-815. HUTNER, S. H., AND PROVASOLI, L. (1955) In Biochemistry and Physiology of Protozoa, Vol. 2, p. 18, Academic Press, New York. HUTNER, S. H., PROVASOLI,L., AND FILFUS, J. (1949) Ann. N.Y. Acad. Sci. 55, 852-862. HUTNER, S. H., ZAHALSKY, A. C., AARONSON, S., BAKER, H., AND FRANK, O. (1966) Methods Cell Physiol. 2, 217-228. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANI)ALL, R. J. (1951) J. Biol. Chem. 193, 265-275. MARINONE, G. (1954) Rev. Hematol. 9, 341-343. MEJBAUM, W. (1939) Hoppe Seyler's Z. Physiol.
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Chem. 258, 117-120. MENZIES, R. C., CROSSEN~ P. E., FITZGERALD,P. H., AND GUNZ, F. W. (1966) Blood 28, 581-594. MOYNE, G. (1973) J. Ultrastruct. Res. 45, 102-123. MOYNE, G., BERTAUX, O., AND PUVION, E. (1975) J. Ultrastruct. Res. 52, 362-376. PERUGINI, S., SILINGARDI,V., AND ZAMPORI,O. (1961) Haematologica 46, 565-580. POGO, B. T. G., UBERO, I. R,, AND POGO, A. 0. (1966) Exp. Cell Res. 42, 58-66. REYNOLDS, E. S. (1963) J. Cell Biol. 17,208-212. SCHMIDT, G., ANY) THANNHAUSER, S. J. (1945). J. Biol. Chem. 161, 83-89. SMILLIE, R. M., AND KROTKOV, G. (1960) Canad. J. Bot. 38, 31-49. SOLDO, A. T. (1955) Arch. Biochem. Biophys. 55, 71-76. VALENCIA,R: (1974) J. Physiol. 69, 5A-76A. WHITE, J. C., LESLIE, I., AND DAVIDSON,J. N. (1953) J. Pathol. Bacteriol. 64, 292-306. WRIGHT, L. D,, SKEGGS, H. R., AND HUFF, J. W. (1948) J. Biol. Chem. 175, 475-476.
The Nucleus of Euglena II. Ultrastructural Modifications of the Nucleus of B12-Deprived Euglena gracilis Z O D I L E B E R T A U X , G I L L E S M O Y N E , * C H R I S T I A N E L A F A R G E - F R A Y S S I N E T , * AND R I C H A R D VALENCIA
Laboratoire de Nutrition et Cin~tique Cellulaires, Facult~ de Pharmacie de l'Universit~ de Paris-Sud, F92290, Ch&tenay-Malabry, France, and *Institut de Recherches Scientifique sur le Cancer, B.P. 8, F-94800, Villejuif, France Received August 17, 1977 The nuclear ultrastructure of Euglena gracilis Z was studied in synchronized cells cultivated in a vitamin B12-deprived medium. Specific and preferential nucleoprotein stains were applied to Epon and frozen ultrathin sections as well as conventional techniques. The evolution of the chromatin structure and of the nucleolar organization was observed as the deficiency increased, while mitosis was synchronously blocked. In normal Euglena, the chromosomes were permanently condensed with a variable degree of packing; under conditions of B12 deficiency, the chromosomal condensation progressively disappeared. Staining of chromatin became ultimately impossible even using specific methods. Only the nucleolar DNA remained normally condensed. However, it was shown that these cells still possess a DNA content equivalent to that found at the end of the S phase. The nucleolus gradually split itself in many lobules. In some cases, nucleolar vacuoles and segregation of the granular and fibrillar components were observed.
Vitamin B~2 is an essential growing factor for some prokaryote microorganisms such as Escherichia coli (Davis and Mingioli, 1950) and LactobacUlus leichmanii (Wright et al., 1948). Apart from the animals, it is also essential for some eukaryote microorganisms such as the flagellates Ochromonas, (Hutner et al., 1949; Ford, 1953) Poteriochromonas (Hutner et al., 1949) and E u g l e n a gracilis (Hutner and Provasoli, 1955; Epstein et al., 1962). Without vitamin B12, L. leichmanii grows into giant elongated cells that have been claimed to represent a model of the pernicious anemia megaloblast (Beck et al., 1962, 1965). Similarly, when E. gracilis cells are deprived of vitamin B12 during their exponential growing phase, cell division stops and the cellular volume increases up to 40 times the normal volume (Bertaux, 1976). This hypertrophy results from the synthesis of RNA, proteins, and glucidic substances accumulating in these cells. Regarding DNA, during mitosis inhibition, it remains at a level equivalent to that found at the end of
the S phase (Bertaux and Valencia, 1975; Bertaux, 1976). In this work, we have studied the nuclear ultrastructure of Euglena cells deprived of vitamin B12. This avitaminosis leads to unbalances in the R N A / D N A and protein/DNA ratios that can reach up to 10 times their normal values. This abnormal metabolism cannot fail to induce alterations of the nuclear activity and ultrastructure. We have studied the ultrastructure of the nucleus of B12-deprived cells as compared with that of the normal nucleus, observed either in interphase and investigated with the same techniques (Moyne et al., 1975), or during a synchronized cell cycle (Frayssinet et al., 1975; Bertaux, 1976; Bertaux et al., 1976). We have also distinguished between two kinds of B12-deprivation procedures and their ultrastructural consequences: Avitaminosis beginning during the exponential growing phase results in a severe deficiency while during slow growth the consequences
251 0022-5320/78/0623-0251502.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
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o f B12 d e p r i v a t i o n a r e less d r a s t i c ( B e r t a u x a n d V a l e n c i a , 1973). MATERIALS AND METHODS
I. Cell Culture Autotroph cultures of Euglena gracilis Z (Cambridge No. 1224-5 D) were cultivated in Cramer and Myers' inorganic salt medium (1952). This medium is considered as autotrophic with regard to the carbon source. Vitamins B1 and B1~ provide negligible amounts of carbon for energetic needs, while citrate, at pH 6.8, is exclusively present as a chelator (Hutner et al., 1966}. Erlenmeyer flasks (750-ml volume) conraining 250 ml of culture medium were mechanically shaken in a 25°C stove to ensure aeration of cultures; when larger quantities of cells were required, culture was carried out in 2000-ml toxin flasks containing 1500 ml of culture medium, bubbled with a mixture of air-CO2 (95:5). Under these conditions, at the end of the exponential phase, the cell concentration was 10fold higher than in the Erlenmeyer cultures (Bertaux, 1976). The cells were synchronized by successive photoperiods of light (14 hr) and dark (10 hr), according to Edmunds (1965). Deficiencies were initiated by transferring the ceils into a medium deprived of vitamin B12. To obtain a deficiency during the exponential growth phase, the cell concentration at the onset of mitosis inhibition had to near 104 cells/ml. On the contrary, a concentration of 10~ cells/ml was required when the deprivation was carried out in stationary phase. In toxin flasks bubbled with the air-CO2 mixture, the cell concentration could be increased, respectively, up to i0 ~ and 106 cells/ml (Bertaux, 1976). Cell concentrations and the cellular mean volumes were monitored with a Coulter counter fitted with a mean cell volume device.
II. B i o c h e m i c a l Determinations The nucleic acids were extracted according to Schmidt and Thannh~iuser (1945), as modified by Smillie and Krotkov (1960). RNA was determined by Mejbaum's technique (1939) and DNA by Giles' and Myers' (1965). Protein determination was carried out according to Lowry et al. (1951).
III. Microscopy (a) Light microcopy. Whole cells fixed on microscope slides were stained by Feulgen's technique. (b) Electron microscopy. All the techniques used in this work were previously described in detail (Moyne et al., 1975). Three types of staining methods were used: conventional uranyl and lead staining (Reynolds, 1963), the EDTA regressive method preferential for ribonucleoproteins (Bernhard, 1969), and DNAspecific stains such as the Schiff-thallium technique
(Moyne, 1973), and the osmium ammine method (Cogliati and Gautier, 1973). RESULTS I. OBTAINING B12-DEPRIVED CELLS
(a) E x p o n e n t i a l Growing P h a s e B12-deprived E u g l e n a cells w e r e o b t a i n e d b y t r a n s f e r r i n g n o r m a l s y n c h r o n i z e d cells i n t o B12-deprived m e d i u m . T h e m a x i m u m cell c o n c e n t r a t i o n , b e t w e e n t w o c h a n g e s of c u l t u r e m e d i u m , c o u l d n o t e x c e e d 105 c e l l s / m l in o r d e r to r e m a i n in e x p o n e n t i a l growing phase. In the same way, mitosis i n h i b i t i o n h a d to o c c u r w h e n t h e cell conc e n t r a t i o n w a s n e a r 104 c e l l s / m l ( C h a r t 1). U n d e r t h e s e f a v o r a b l e c o n d i t i o n s , t h e cell u l a r m e t a b o l i s m w a s a c t i v e , m a k i n g feasible an easy study of the effects of vitamin B12 d e f i c i e n c y . C e l l s t r a n s f e r r e d in B i 2 - d e p r i v e d m e d i u m w e r e still a b l e t o d i v i d e five o r six t i m e s { C h a r t 1). T h e i r m e a n v o l u m e v a r i e d i n a . cyclic w a y d u r i n g t h e d i f f e r e n t cell cycles. F r o m 4000 # m 3 a t t h e e n d o f t h e l i g h t photoperiod, at the very beginning of the e x p o n e n t i a l g r o w t h p h a s e (103 c e l l s / m l ) , it d e c r e a s e d d o w n t o 1000 ~ m ~ a f t e r t h e cell division, a t t h e e n d o f t h e e x p o n e n t i a l p h a s e (105 c e l l s / m l ) . A f t e r t h e b e g i n n i n g of m i t o sis i n h i b i t i o n (7 d a y s ' d e f i c i e n c y ) , t h e m e a n v o l u m e i n c r e a s e d r e g u l a r l y so t h a t s e v e r a l d a y s o f b l o c k l e d to a l a r g e c e l l u l a r h y p e r trophy. I n t h e e x a m p l e s h o w n , t h e m e a n cell v o l u m e w a s 22000 # m ~ a f t e r 9 d a y s of m i tosis block.
(b) S t a t i o n a r y Growing P h a s e This type of deficiency was obtained w h e n m i t o s i s i n h i b i t i o n b e g a n w i t h a cell c o n c e n t r a t i o n h i g h e r t h a n 10 ~ c e l l s / m l . G r o w i n g c o n d i t i o n s w e r e less f a v o r a b l e , a n d cell m e t a b o l i s m w a s slowed. T h u s , h y pertrophy was less marked (maximum m e a n cell v o l u m e = 2200 # m ~) a n d t h e e x t e r n a l cell m o r p h o l o g y d i s p l a y e d s o m e d e f o r m a t i o n s ( B e r t a u x a n d V a l e n c i a , 1973; B e r t a u x , 1976). However, our main goal was to induce a
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CHART 1. (A) Synchronous growth of Euglena between the first and the sixth day of deprivation, starting from the first transfer in medium free of vitamin B12. After six generations of apparently normal cells, cell division was blocked after the second transfer between the sixth and the fifteenth day of deficiency. (a) After a block of 2 days: Beginning of nucleolar fragmentation (Fig. 4); the chromosomes appear as residual chromatin. (b) 8.day block: The nucleoli are entirely fragmented in lobules; chromatin is no longer stainable; extended clear zones mark the location of former chromosomes (Fig. 5). (B) Mean cell volume of deprived Euglena. Decreasing cyclic evolution of the volume during the exponential phase between the first and the sixth day of deficiency, before mitosis block. The volume increases during the 14-hr light period; it decreases during the dark period when the cells divide. Linear increase of the mean cell volume after beginning of the divisions block between the sixth and the fifteenth day of deficiency (maximum mean cell volume = 22 000 ttm:~). 253
254
BERTAUX ET AL.
synchronized block of the cell divisions in order to determine at which stage of the cell cycle it occurred. We have therefore mostly studied B12-deprived cells in the exponential growing phase.
also a significant correlation of the amounts of protein and RNA with the cellular vol. ume (P < 0.001; Chart 3). Regarding DNA, the regression line slope is slightly positive, with no significant correlation. The progressive protein and RNA inII. BIOCHEMICAL EFFECTS creases in the deprived cells explain their Data from the literature revealed that enormous hypertrophy. On the contrary, vitamin B12 deficiency induces marked in- the mean amount of DNA did not vary creases in the amounts of RNA and pro- during all the cell division block. The teins in rapidly growing cells or tissues of amount of DNA was equivalent to the highvarious types: human (White et al., 1953; est level observed in normal cells at the end Glazer et al., 1954) or unicellular (Beck et of the S phase. al., 1965; Carell et al., 1970). In contradistinction, results on the (b) Stationary Growing Phase amounts of DNA were more ambiguous. The results obtained in the stationary For some authors they were lowered growing phase are summarized in Table 1. (Soldo, 1965; Pogo et al., 1966), whereas for Deprived cells in stationary phase disothers they were not modified (Marinone, played only a slight hypertrophy since their 1954) or even increased (Davidson et al., maximal volume was 2200 gm 3. This implies 1948; Perugini et al., 1961; Carell et al., a 25% increase as compared with control 1970). It was thus necessary to determine cells cultivated under the same conditions the variations in the amounts of macromol- (stationary phase). However, the volume of ecules in Euglena cells during the normal the deprived cells more than doubled in synchronized cycle in order to appreciate comparison with control cells in G1 phase the meaning of the data, including the ul- in spite of a similar macromolecular comtrastructural effects, obtained in BI2-de- position (Table 1). Indeed, the amounts of prived cells. DNA, RNA, and proteins found in the deprived cells in the stationary phase are at (a) Exponential Growing Phase a minimum level, equal to that of the G1 The evolution of the amounts of DNA, phase for exponentially growing control RNA, and protein in normal Euglena cells ceils. Therefore, the volume increase is not is shown on Chart 2 as a function of the attributable to RNA and protein synthesis cellular volume. The same values are shown as in the preceding case but more probably again in the left part of Chart 3 (between to an accumulation of glucidic material 1000 and 3000 #m 3) for the comparison with such as paramylon. the corresponding amounts in Bi2-deprived III. ULTRASTRUCTURAL EFFECTS cells of much larger volume (up to 20,000 ~m3). The ultrastructure of the normal EuThus, the normal values are grouped glena nucleus was previously described in within three well-delimited zones. The de- detail (Moyne et al., 1975; Bertaux et al., terminations were obtained from 117 sam- 1976). The chromosomes persist in conples collected at different stages of the cell densed form throughout the cell cycle. The cycle. The regression lines (Chart 2) dem- nucleolus does not disappear during mitosis onstrate a significant correlation (P < and divides after stretching itself. During 0.001) of the different amounts with the the cell cycle, several degrees of chromocellular volume. some condensation were observed: Packing The values for the deprived Euglena was more intense just after mitosis, dewere obtained from 82 samples. They show creased during G1 (Fig. 1), and was at a
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
255
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CHART 2. Regression lines demonstrating the correlations of the DNA, RNA, and protein contents in normal synchronous Euglena with the mean cell volume. Correlation coefficients: r (DNA) = 0.79; r (RNA) = 0.89; r (proteins) = 0.72. All these correlations are significant with a probability smaller than 10-~. m i n i m u m in G2 a n d d u r i n g m i t o s i s . I n t h e nucleolus, granular and fibrillar parts were o b s e r v e d . T h e l a t t e r w e r e o r g a n i z e d in conc e n t r i c s t r u c t u r e s " ( F r a y s s i n e t et al., 1975) o f a d i a m e t e r o f a b o u t 0.5 ttm (Fig. 1). W e could demonstrate the localization of nu-
cleolar DNA as rings within these particul a r s t r u c t u r e s ( M o y n e etal., 1975).
(a) Nuclear Hypertrophy D u r i n g v i t a m i n B12 d e f i c i e n c y i n t h e exponential growing phase, the cellular hy-
256
BERTAUX E T AL.
PROTEINS 3000 JJG/IO6 CELLS
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CHART3. Regression lines demonstrating the correlations of the DNA, RNA, and protein contents in B12deprived Euglena with the mean cell volume. The short regression lines in the left part of the chart are a reduced representation of Chart 2 which permits comparison at the same scale of the macromolecular contents in control and deprived Euglena. Correlation coefficients: r (DNA) = 0.13; there is no correlation of the DNA concentration per ceil with the volume of the deprived cells; r (RNA) = 0.6?; r (proteins) = 0.72; these correlations are significant with a probability smaller than 10-3. p e r t r o p h y also involved t h e nucleus. W e carried o u t an a p p r o x i m a t e c o m p u t a t i o n of the nuclear volumes from measurements of the n u c l e a r d i a m e t e r s on e l e c t r o n micrographs. I t s h o w e d t h a t t h e n u c l e a r v o l u m e i n c r e a s e d a b o u t p r o p o r t i o n a t e l y to t h e w h o l e cell volume. T h e v o l u m e o f n o r m a l nuclei (diameter, 5 t o 7.5 ~tm) v a r i e d d u r i n g t h e cell cycle b e t w e e n 65 a n d 220 # m 3. A f t e r t h e two d a y s of mitosis inhibition, t h e nuclear v o l u m e i n c r e a s e d to 400/~m 3 (diame-
ter, 9 #m), a n d after 8 d a y s it was larger t h a n 2000 # m s {diameter, 16 #m). T h e nucleolus e n l a r g e d p r o p o r t i o n a t e l y . D u r i n g t h e s t a t i o n a r y phase, t h e n u c l e u s did n o t enlarge. Its v o l u m e varied within t h e size limits of t h e n o r m a l nucleus.
(b) Ultrastructural Modifications of the Nucleus W e will essentially describe t h e ultras t r u c t u r e o f t h e n u c l e u s o f d e p r i v e d Eu-
Euglena N U C L E U S A N D V I T A M I N B12 D E F I C I E N C Y TABLE 1
257
deprived cells, chromatin seemed to be largely dispersed throughout the whole nucleoplasm since only small clear zones conControls B ,2taining no granules marked the probable Deprived location of the residual condensed chroExponential Station- _ _ phase (117) ary matin. phase When adapted to ultrathin frozen secBeginmid(28)" Stationning Sb ary tions, the same technique revealed a differof phase GL~ (42)" ent nucleoplasmic structure: We observed Volume (#m 3) 1000 2000 1800 2200 U-shaped fibrils (Fig. 10) identical to those D N A (#g/10 ~ 3.2 5.3 3.9 3.1 visualized in the normal cell (Moyne et al., cells) 1975). RNA (#g/106 28 48 30 32 2. Chromosomes. Conventional staining. cells) In normal Euglena cells, chromosomes Proteins 260 350 360 231 (~g/106 cells) were always densely stained by conventional techniques (Fig. 1) during the entire a M e a n values; n u m b e r of s a m p l e s given in parentheses. cell cycle (Bertaux et al., 1976). When BlZ b Values obtained from t h e regression lines. deficiency occurred in the exponential growing phase, the chromosomes progresglena cells in the exponential growing sively disappeared as dense chromatin. phase since it was this type of deficiency Two or three days after inhibition of cell that induced the most important nuclear division, a few small clumps of dense chromodifications. matin persisted (Fig. 3). They often bor1. Nucleoplasm. Conventional staining. dered large electron-lucent zones (Figs. 3 After the conventional procedure, i.e., dou- and 4) sometimes in contact with the nuble fixation, Epon embedding, and uranyl cleolus (Fig. 7). Ultimately, even this residlead staining, we did not observe any dif- ual dense chromatin disappeared (Figs. 5 ference regarding the nucleoplasm of the and 6), leaving only electron-lucent zones deprived (Figs. 2-4) and control cells (Fig. on the sites formerly occupied by dense 1). However, in deprived cells with mitosis chromatin. These electron-lucent zones blocked for several days, we often saw clus- consisted of fibrils about 100 A thick (Fig. ters of 10 to 30 large granules (Figs. 6 and 7). 8). Their diameter varied from 650 to 2300 When cells were deprived of vitamin B12 A, and they were sometimes in contact with so that the block of cell division occurred the nucleolus (Fig. 8). in stationary phase, the structure of chroIn normal Euglena, we have described matin was not modified. The chromosomes isolated dense granules (Moyne et al., 1975) were long and ramified (Fig. 2), a typical but they were much rarer than those ob- aspect of normal cells in G1 phase (Bertaux served in deprived cells. et al., 1976). Preferential R N P staining. The nucleoSpecific DNA staining. The fibrillar plasm appeared normal when observed structure of the residual dense chromatin after staining by the regressive EDTA was revealed on Epon sections using the method. It was filled by a large number of specific DNA stain osmium ammine (Cogdense granules 300 to 500 /~ in diameter, liati and Gautier, 1973) (Fig. 16). The residregularly dispersed within a finely fibrillar ual chromatin was sometimes dispersed matrix relatively less electron dense (Fig. within the nucleoplasm as fine fibers of 9). However, it was more homogeneous thickness ranging from 20 to 45 A (Fig. 16). than in normal cells where the localization The clumps of residual dense chromatin of the chromosomes was marked by large were about 0.1 to 0.2 ~m in their longest bleached zones (Moyne et al., 1975). In B12- dimension (Fig. 4). They were therefore 10 MACROMOLECULE CONTENTS OF CONTROL AND B12DEPRIVED CELLS
258
B E R T A U X E T AL.
FIGS. 1-3.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
times smaller than the electron-lucent zones (Fig. 6), themselves twice larger than t h e mean size of normal sectioned chromosomes in G1 phase. Thus, even a specific staining technique was not able to reveal the large amount of DNA contained in these nuclei (7 #g/106 cells). The light microscopic Feulgen's reaction "was also carried out on whole normal cells. Clumped chromatin intensely stained was thus revealed. The same reaction was applied to deprived cells in exponential growing phase. After 8 days of mitosis inhibition, the reaction was still visible but entirely : diffuse. 3. Nucleolus. Conventional staining. Immediately after the beginning of mitosis inhibition, 7 days after the initiation of B12 deficiency, one or several lobules parted from the nucleolar periphery (Figs. 3 and 4). This phenomenon increased as the deprivation carried on. After 8 days of cell division block, the nucleoli were largely and sometimes entirely fragmented (Figs. 5 and 10). We observed a similar sequence in cells deprived of vitamin B12 during the stationary growing phase (Fig. 2). However, the parted lobules never numbered more than three or four. Their size displayed some variability: 0.5 to 1.5 #m in diameter for lobules with one annular structure while in normal cells they were never seen to be larger than 0.5 #m (Fig. 1). In a few cases, the nucleolus was only little fragmented, but we observed a segregation of the granular and fibrillar components (Figs. 6 and 7) with a nucleolar "vacuolization." Preferential RNP staining. The regres-
259
sive EDTA stain bleaches preferentially DNA-containing structures. In partly fragmented nucleoli, this technique revealed on Epon sections that each nucleolar lobule contains a notable quantity of DNP (Fig. 9). Thus, during B12 defiency, the nucleolar DNA keeps its staining properties unlike the nucleoplasmic DNA. On ultrathin frozen sections, clear circular zones were also seen in each lobule of a totally fragmented nucleolus (Fig. 10). Specific staining of the nucleolar DNA. We could clearly demonstrate the presence of DNA in the nucleoli of B12-deprived cells using either the Schiff-thallium (Figs. 11 and 12) or the osmium ammine technique (Figs. 13-15) on Epon or frozen ultrathin sections. The DNA was generally contained in annular structures (Figs. 13 and 14), 0.4 to 0.8 gm in diameter, in the center of the lobules. The DNA-containing structures sometimes consisted of a double ring (Fig. 15) similar to the cockade structures visible in the nucleoli of normal cells (Fig. 1). Thus, in B12-deprived cells the localization and structure of the nucleolar DNA are similar to that of normal cells. In cells deprived of vitamin B12 in the exponential growing phase, nucleolar DNA was more abundant than in normal cells : We could count up to 15 DNA rings per section of nucleolus (Fig. 14), while in normal cells, with the same technique, we found two or three. However, after conventional staining, up to six rings could be counted in normal cells (Fig. 1). The number of DNA rings in cells where B12 deprivation had been initiated in the stationary growing phase was equivalent to that of the normal cell (Figs. 11 and 12).
FIG. 1. Nucleus of a control cell at the end of G1 phase (cell volume = 1800 gm~). Uranyl lead staining. The chromosomes (chr) persist in the condensed state. In the nucleolus (nu), DNA-containing annular structures (A) are surrounded by nucleolar granules. × 30 000. FIG. 2. Nucleus of a cell in stationary phase after 8 days of vitamin B,2 deficiency (cell volume = 2000 ~m~). Uranyl lead staining. The chromosomes (chr) are still condensed and larger than in Fig. 1. Nucleolus (nu) slightly fragmented. × 22 000. FIG. 3. Nucleus of a cell in exponential growing phase after 7 days of vitamin B,2 deficiency and 1 day of mitosis inhibition (cell volume = 5000 gm:~). Uranyl lead staining. Chromatin has largely disappeared and light zones (L) localize the former chromosomes. They are bordered with residual dense chromatin (--~). Fragmen-. tation of the nucleolus (nu) is beginning. Annular structure (A). ×8 500.
260
B E R T A U X E T AL. DISCUSSION
We have studied simultaneously the modifications of the nuclear ultrastructure and of the macromolecular composition of cells in vitamin BI2 deficiency, as compared with normal cells. We aimed at discovering a relationship between structure and function that could explain the observed phenomena. The choice of Euglena gracilis Z has a double justification: (i) It is a good model for the study of vitamin BI2 deficiency. The essential role of
this growing factor in cell division was demonstrated in pernicious anemia and in several microorganisms. Euglena is interesting as a eukaryotic cell which, like animal cells, • makes use of "true" vitamin BI2 unlike the prokaryotic microorganisms able to use cobalamine analogs (Valencia, 1974). Above all, it is one of the rare eukaryotic cells whose experimental BI2 deprivation can be easily realized: This is due to the simplicity of its culture medium, mineral with the exception of vitamins B, and BIe, and to its photosynthetic faculties.
FIGS. 4 AND 5. Nuclei of cells in exponential growing phase in vitamin B,2 deficiency. Uranyl lead staining. FIG. 4. Deficiency, 8 days; mitosis inhibition, 2 days (cell volume = 6500 #m:J). Increased fragmentation of the nucleolus (nu) as compared with Fig. 3. Each nucleolar fragment is centered by an annular structure (A). Light zones (L). Residual chromatin (--*). ×15 500. FIG. 5. Deficiency, 14 days; mitosis inhibition, 8 days (cell volume = 22,000 #m:~). Residual chromatin rarer than in Fig. 4. Nucleolus (nu) entirely fragmented. ×34 000. FIGS. 6-8. Nuclei of cells in the exponential growing phase cultivated without vitamin B,~. Uranyl lead staining. FIG. 6. Deficiency 14 days; mitosis inhibition, 8 days (cell volume = 22,000/~m:~). Electron-lucent light zones (L) seem the only remnants of chromatin. In the nucleolus (nu), granular components (g) are separated from the fibrillar and chromatin components that remain intermingled. Nucleoplasmic dense granules (--~). Nucleolar vacuoles (v). ×12 000. FIG. 7. Deficiency, 11 days; mitosis inhibition, 5 days (cell volume = 14,000 #ma). A fibrillar light zone (L) bordered with dense chromatin (chr) is in contact with the nucleolus. A DNA-containing annular structure (A) is visible. Nucleolar fibrils (f) and granules (g). ×30 000. FIG. 8. Same deficiency as in Fig. 6. Electron-dense granules (-*) are visible in contact with the fragmented nucleolus (nu). Compare with Fig. 6 where identical granules are visible within the nucleoplasm. ×30 000. FIGS. 9 AND 10. Nuclei of cells in exponential phase cultivated without vitamin B~2. FIG. 9. Deficiency, 9 days; mitosis inhibition, 3 days (cell volume = 8000 #m:J). EDTA preferential stain for ribonueleoproteins. Moderately fragmented nucleolns. Bleached regions (--*) assumed to contain DNA are visible in each nucleolar fragment. ×32 000. FIG. 10. Deficiency, 14 days; mitosis inhibition, 10 days (cell volume = 24,000 #m3). Ultrathin frozen section. Ribonucleoprotein preferential staining. The nucleolus (nu) is entirely fragmented. A light zone (L) is visible in the nucleoplasm otherwise filled with RNP fibrils identical with the U-fibrils observed in normal cells (-*). ×33 000
FIGS. 11-14. Cells cultivated without vitamin B,2. DNA-specific stains: Schiff-thailium reaction (Figs. 11 and 12) or osmium ammine (Figs. 13 and 14). FIG. 11. Nucleolar DNA of a cell in stationary phase; 15 days of cultivation. The nucleolar DNA (--)) is revealed as a well-localized circular formation in the nucleolar body (nu). Chromosomes (chr).)<36000. FIG. 12. Same as in Fig. 11. × 33000. FIG. 13. High magnification of an annular structure. Cell in exponential phase. Mitosis blocked for 5 days. Ultrathin frozen section. This method reveals nucleolar DNA as a finely fibrillar structure. ×51000. FIG. 14. Nucleus of a cell in the exponential growing phase. Mitosis inhibition for 3 days. In the nncleoplasm, some residual chromatin (---~} is visible. In the nucleolus, DNA is present in numerous circular structures. x32 000.
FIGS. 15 AND 16. Cells in the exponential growing phase cultivated for 10 days without vitamin B,2. Mitosis inhibition for 4 days. Specific DNA stain with osmium ammine. FIG. 15. Detail of a nucleolus. Each nucleolar fragment contains one or two annular DNA structures (--~). x56 000. FIG. 16. Residual nucleoplasmic chromatin. The fibrillar structure of the residual chromatin is clearly visible. No DNA fibers are visible in the surrounding nucleoplasm. ×100000.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
FIGs. 4-5.
261
262
B E R T A U X E T AL.
FIGS. 6-8.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
F~GS. 9-10.
263
264
BERTAUX E T AL.
FrGs. 11-14.
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY
FIGS. 15-16.
265
266
BERTAUX ET AL.
(ii) Cultures of Euglena can be brought to an excellent degree of synchrony. They allow attainment of large quantities of cells at each phase of the cell cycle, and thus they permit parallel biochemical and ultrastructural observations. The synchronization holds during the deficiency, even when mitosis is blocked since synchronous synthesis of RNA and proteins still occurs during the successive photoperiods (Bertaux and Valencia, 1973; Bertaux, 1976). We chose to study B,2 deficiency mostly during the exponential growing phase when the absence of vitamin B,2 is the only limiting factor for cells with an active metabolism. Under these conditions, the need for vitamin B~2 is very high and makes more drastic the effects of the avitaminosis. I. NUCLEOPLASMANDCHROMATIN The inhibition of cell division during B12 deficiency does not seem to be linked to a quantitative lack of DNA synthesis, since it occurs when the cells contain about twice as much DNA as in G, phase. But the ultrastructural modifications of chromatin do not allow the exclusion of qualitative alterations. The last replication could be incorrect and could induce modifications of the secondary or/and tertiary molecular structure that could explain the morphological changes observed: (i) Dense chromatin disappeared progressively; after 8 days of cell division block, the nucleoplasm contained only electronlucent zones that seemed to correspond to the former localization of chromosomes. (ii) Under the light microscope, Feulgen's reaction showed that DNA is well localized in chromatin clumps in normal cells, while its repartition becomes diffuse in deprived cells. (iii) DNA reactions at the ultrastructural level also revealed a disappearance of chromatin clumps leaving, at last, small masses of residual dense chromatin much reduced in volume as compared with normal chromatin. It was mentioned above that the quantity
of DNA is not reduced during B12 deficiency under our conditions of checked exponential growth. Consequently, the cytochemical results can be explained by a great dispersion of DNA in deprived cells. If we add that the cellular volume increases 20-fold, it is clear that DNA fibers highly dispersed in the nucleoplasm can be no longer demonstrated by cytochemistry. In normal cells, we could visualize no dispersed chromatin with the exception of fibers linking chromosomes together-and to the nucleolus (Moyne et al., 1975). In spite of these structural modifications, the transcriptional abilities of the cell are not noticeably decreased. It is even conceivable that the synthesis of messenger . RNA would be facilitated by the dispersed state of the chromatin. Regarding the morphology, a similar phenomenon was described by Heath {1966) and Menzies et al. (1966) in the megaloblast. They found chromatid breaks and incomplete contraction of the chromosomes. It is also interesting to note that the unbalanced increase of the R N A / D N A ratio is as marked in Euglena as in pernicious anemia blood cells (White et al., 1953; Glazer et al., 1954). These facts tend to complete the parallel between pernicious anemia blood cells and B12-deficient Euglena. In addition, we could never observe in B12-deprived Euglena the microtubules of the division spindle visible in control dividing cells. This absence can be explained either because the cells are blocked in G2 phase, before prophase, or because the formation of the spindle fibers itself is inhibited by the deficiency. II. NUCLEOLUS The main nucleolar alteration consists of a kind of fragmentation that has not been previously described as far as we know. The nucleolar fragments can comprise several annular structures at the beginning of the deficiency {Fig. 4), but with a longer deprivation, the nucleolus seems to be split into
Euglena NUCLEUS AND VITAMIN B12 DEFICIENCY as many lobules as annular structures (Fig. 5). This is different from the classical nucleolar segregation related to interruption of RNA synthesis, generally after the action of drugs (Busch and Smetana, 1970) and characterized by a separation of the fibrillar, granular, and chromatin components of the nucleolus. In B12-deprived Euglena we have occasionally observed a nucleolar segregation accompanied by a discrete fragmentation. However, biochemistry demonstrated that the transcriptional activity remained very high in these deprived cells. Whatever the severity of the deficiency, nucleolar DNA kept a normal condensation and stainability, even in nucleoli entirely fragmented (Fig. 15). This DNA was still transcribed after mitosis inhibition, at least during the next four cycles. Ribosomal RNA synthesis continued at a normal level as shown by extraction and electrophoretic separation of the RNAs (Bertaux, 1976). Nucleolar DNA is transcribed, but is is also likely to be replicated several times in spite of the inhibition of mitosis. Indeed, we could observe up to 15 sections of DNA rings per nucleolus. This is far more than the number of DNA rings or even of annular structures visible in sections of control Euglena nucleoli. The likely replication of the nucleolar DNA would show that some DNA synthesis is still possible during B~2 deficiency. This would support the hypothesis explaining the mechanism of the division block by qualitative alterations of the chromosomal DNA molecules rather than by a stop at the end of the S phase. On the other hand, this is consistent with the hypothesis that the block of the division mechanism in B12-deficient Euglena would occur either at the end of the S phase or in phase G2, in any case before the outset of mitosis. Whatever might be the interpretation of the nucleolar fragmentation, it is accompanied by an increasing degranulation, parallel to the deficiency, that could explain the relative decrease in RNA synthesis near the lethal period (after 8 days of mitosis inhibition). Chart 3 shows the
267
regression line of the RNA concentration per cell as a function of the cell volume, during the increasing B~2 deficiency; it is clear that the slope of the deprived cells, 1.23, is smaller than that of the control cells, 2.1. It is also worth noting that for deprived cells of volume smaller than 12,000/tin 3, the increase in RNA concentration per cell is roughly parallel to that of the control cells. When the cells grow over this volume, the accumulation of RNA is much more limited, thus implying a decrease of the level of synthesis. We have also observed that Euglena cells of high volume, at the extreme stage of deficiency, are very unlikely to survive when vitamin B12 is added to the culture medium. On the other hand, the deficiency is rapidly and entirely reversible when the cellular volume is smaller than 12,000/zm 3. We have described B~2 deficiency in Euglena cells during exponential growth under controlled conditions. It leads to drastic consequences: block of cell division with a DNA content equal to that of normal G2 phase, enormous cell hypertrophy, nucleolar fragmentation, and loss of contrast of the chromosomes. Cell death is regularly observed after a fortnight. B~2 deficiency in Euglena approaching the stationary phase did not induce similar effects with the exception of a slight nucleolar fragmentation. The chromosomes remained visible, and the DNA content was equal to that of the normal G~ phase. We cannot exclude the possibility that the high cell concentration and slowing metabolism could induce complex effects compensating each other, and rendering less visible the phenomena of B12 deficiency. Regarding comparison of B12-deprived Euglena with pernicious anemia blood cells, the latter evidently depend on an environment much more complex than our model. They are subject to a variety of factors difficult to analyze. Consequently, their characteristics probably issue from the influence of these factors added to a pattern of B12 deprivation intermediate be-
268
BERTAUX E T AL.
tween the two extremes described in our cell system. CONCLUSION
The similar effects of vitamin B12 deficiency in pernicious anemia and in Euglena allow consideration of this phytoflagellate as a model of pernicious anemia blood cells, as far a s B12 metabolism is concerned. In this work, we demonstrated that in the exponential growing phase, B~2 avitaminosis induces the following: A cellular hypertrophy resulting from the accumulation of RNA, protein, and glucidic substances stored in the cells. It leads to a volume increase up to 20-fold the normal one. The nuclear and nucleolar hypertrophies are analogous. A block of the cell division in G2 phase. During all the block the quantity of cellular DNA is maintained at a level equal to that of the end of the S phase. Important modifications of the chromatin structure. After a few days of mitosis inhibition, chromosomes are no longer visible. They appear only as extended unstained regions that we interpreted as resuiting from chromatin decondensation. DNA was no longer visualized after specific staining in spite of the large quantity of DNA contained in each nucleus. On the other hand, we did not observe any structural modification of the RNP in Epon sections as well as in frozen sections where the U fibrils persisted. Regularly observed nucleolar fragmentation. At an early stage, the fragments comprise several annular structures. Later, they are made of single isolated lobules. Fragmentation of the nucleolus is accompanied by a progressive degranulation. However, the nucleolar DNA can be revealed as a ring-shaped structure whatever the severity of the nucleolar alterations. In the stationary growing phase, the following are observed: Apart from some alterations of the shape of the cells, we noticed only a moderate hypertrophy (volume doubled as compared
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