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Nucleologenesis in chick erythrocyte nuclei reactivated by cell fusion
J O U R N A L OF U L T R A S T R U C T U R E R E S E A R C H
69, 164-179 (1979)
Nucleologenesis in Chick Erythrocyte Nuclei Reactivated by Cell Fusion DANII~LE HERNANDEZ-VERDUN AND MICHEL BOUTEILLE Laboratoire de Pathologie Cellulaire, Institut Biomddical des Cordeliers, Universitd Pierre et Marie Curie, Paris VI, France Received February 15, 1979 "Dormant" chick erythrocyte nuclei have been fused by Send~ff virus with h u m a n TG cells. A quantitative analysis and a three-dimensional approach using staggered sections of flat embedded heterokaryons were used to analyze the various aspects of nucleolar structures differentiated during chick erythrocyte reactivation 12, 24, and 48 hr after fusion. Three types of nucleolar structures are described: (1) developed nucleoli, (2) prenucleoli, and (3) nucleolar organizer region. Reactivation of RNA synthesis was followed by electron microscope autoradiography after 10, 30, and 180 min [~H]uridine pulse in the three types of nucleolar structures described. Developed nucleoli and prenucleoli were involved in RNA synthesis; the fibrillar component was f£rst labeled in the periphery of the fibrillar center. To conclude, we propose a dynamic model of nucleologenesis which correlates ultrastructural features and RNA synthesis.
In view of recent developments in understanding of correlations between structure and function within the cell nucleus during the cell cycle, the definition of the nucleolus has become ambiguous. At the ultrastructural level, the nucleolus, as described by Bernhard and Granboulan (1968), is a nuclear organelle consisting of fibrils, granules, condensed associated chromatin as well as the more recently demonstrated (Goessens, 1976a; Goessens and Lepoint, 1974; Recher et al., 1969) fibrillar centers. This description represents an "ideal" fully developed nucleolus. The relationships between these various components and the synthesis of R N A have been well established, especially by means of cytochemistry and autoradiography [see Bouteille et al., 1974, and Fakan, 1976, for review and Fakan and Nobis, 1978; Fakan et al., 1976]. However, considerable morphological variations of nucleoli have been observed according to the cell type, the period of the cell cycle, and the physiological state of the cell, as extensively studied by Smetana and Busch (1974). Another concept implies that the term nucleolus should be restricted to structures which are the morphological substrate of
RNA transcription, processing, and storage. This is essentially the biochemical definition. The limits of defining the nucleolus purely in terms of transcription is well illustrated by the residual proteinaceous structures which can still be identified morphologically as nucleolar after 99.9% DNA and 98% RNA extraction (Berezney and Coffey, 1977). A third way of viewing the nucleolus is to consider as its most essential component the DNA, which carries the information necessary for ribosomal RNA transcription, as visualized in the transcription unit by Miller and Beatty (1969). The exact relationship between this ribosomal DNA and the nucleolar organizer region (NOR) is not yet completely elucidated (Phillips and Phillips, 1969; Semeshin et al., 1975). Nevertheless, the NOR is certainly the most significant part of the nucleolus from the cytogenetical point of view, as a particular region of the chromosomes around which the newly formed nucleoli are reconstituted at the end of mitosis. It was necessary, in order to clarify the morphologic definition of the nucleolus, to look for an experimental system in which (1) the reactivation of a resting nucleolus is experimentally initiated from zero time and 164
0022-5320/79/110164-16502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
NUCLEOLAR REACTIVATION then followed by means of electron microscopy; (2) t h e e m e r g e n c e o f n e w l y f o r m e d n u c l e o l i c a n b e v i s u a l i z e d ; (3) t h e s t a g e s o f t h e e m e r g e n c e o f t h e s e n u c l e o l i a r e cont r o l l e d ; a n d (4) t h e d i f f e r e n c e b e t w e e n t h e s t r u c t u r e s i d e n t i f i e d m o r p h o l o g i c a l l y as n u cleolar which are actually sites of transcript i o n a n d t h o s e w h i c h a r e not, c a n b e r e a d i l y established. Such an experimental situation can be f o u n d in c h i c k e r y t h r o c y t e n u c l e i r e a c t i v a t e d b y m e a n s o f cell f u s i o n in h e t e r o k a r yons. M a t u r e e r y t h r o c y t e n u c l e i a r e c o n s i d e r e d as " d o r m a n t " in t e r m s o f s y n t h e s i s ( R i n g e r t z a n d B o l u n d , 1974). N o r i b o s o m a l RNA synthesis can be detected (Zentgraf et al., 1975). C o r r e l a t i v e l y , n o n u c l e o l a r structure can be identified although a small number of resting structures called micron u c l e o l i ( S m e t a n a et al., 1975; S m e t a n a a n d L i k o v s k y , 1971) h a v e b e e n d e s c r i b e d ( S m a l l a n d D a v i e s , 1972). I t is w e l l k n o w n ( H a r r i s , 1965, 1967; R i n g e r t z a n d S a v a g e , 1976; S i d e b o t t o m , 1974, for r e v i e w ) t h a t t h e s e n u c l e i c a n b e i n t r o d u c e d b y cell f u s i o n i n t o t h e c y t o p l a s m o f a c t i v e h o s t cells. T h e y a r e then reactivated by an active phenomenon w h e r e a n e s s e n t i a l r o l e is p l a y e d b y t h e migration of host proteins into the chick n u c l e i ( & p p e l s et al., 1974a, 1974b, 1975; R i n g e r t z et al., 1971). T h e e r y t h r o c y t e n u clei r e s u m e r i b o s o m a l R N A s y n t h e s i s ( C a r l s s o n et al., 1973; H a r r i s et al., 1969) a s nucleolar structures become morphologic a l l y i d e n t i f i e d ( D e a k et al., 1972; D u p u y C o i n et al., 1976; S i d e b o t t o m a n d H a r r i s , 1969), a n d c h i c k r i b o s o m a l R N A c a n b e d e t e c t e d in t h e c y t o p l a s m o f h e t e r o k a r y o n s ( B r a m w e l l , 1978). T h e a i m o f t h e p r e s e n t p a p e r w a s to e m p l o y t h e n u c l e a r r e a c t i v a t i o n m o d e l to d e t e r m i n e (1) if a m o r p h o m e t r i c c y c l e c a n b e d e s c r i b e d for t h e n u c l e o l u s , (2) w h a t n u c l e o l a r c o m p o n e n t s a r e i n v o l v e d in t h e n u c l e o l a r r e c o n s t i t u t i o n , a n d (3) w h e t h e r t h e a p p e a r a n c e of specific n u c l e o l a r s t r u c tures could be correlated with resumed R N A s y n t h e s i s . T h e first a n d t h e s e c o n d
165
points were investigated by means of a quantitative analysis of the various aspects of the nucleolar structures as the course of reactivation progressed. This was combined with a three-dimensional approach using staggered--successive but not fully serial-sections. The third point was analyzed by high-resolution autoradiography after triti a t e d u r i d i n e i n c o r p o r a t i o n . T h e d a t a allowed us to propose a model representing the steps of what can be called a cycle of nucleogenesis. MATERIALS AND METHODS
Cells for Fusion E x p e r i m e n t s The established TG line of human oviduct was used as a source of host cells. The cells were cultured on 75 cm2 plastic flasks (Falcon} in 15 cm3 of Eagle's minimum essential medium (MEM) supplemented with 15% heat-inactivated fetal calf serum (30 min at 56°C). Twenty-four hours before fusion, TG cells were detached from the plastic by a 0.25% trypsin solution for 10 min at 37°C and seeded into 25 cm2 plastic flasks at a concentration of 1.5 × 10~ cells per flask in 10 cm~ of culture medium. Fusion of chick erythrocytes to TG cells was done on a relatively large scale using TG cells at near confluency and in a rapid growth phase. Chick erythrocytes were obtained from the blood of 13-day chick embryos by cutting the allantoic vessels and allowing the blood to accumulate in the allantoic fluid. The allantoic fluid was collected and centrifuged for 5 min at 200g at 4°C. The pellet of chick erythrocytes was washed twice by centrifugation in Earle's salt solution, then suspended in 10 cm3 of fusion medium (see below) and stored until fusion at 4°C. The erythrocyte population contained less than 1% contaminant cells (Appels et al., 1974b).
Fusion Procedure The fusion medium was an Earle's salt solution adjusted to pH 8 with sodium bicarbonate, this pH having been reported to produce optimum fusion yield (Goto and Ringertz, 1974). Cell fusion was induced by means of ultraviolet-inactivatedSendal virus. Samples of 1 cm~ of the allantoic suspension of Sendai virus were irradiated for 4 min at a rate of 25 erg/mm 2 (dosimetre Latarjet), in 35-mm plastic petri dishes. The fusion procedure (Dupuy-Coin et al., 1976) included the following steps: Culture medium was removed; the cells were washed twice with fusion medium and were then cooled at 4°C for 15 rain in 10 ml of fusion medium; a suspension (0.5 to 0.8 cm3) of inactivated Sendai virus was added and the cells were stored again at 4°C for 15 min in order to promote
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HERNANDEZ-VERDUN AND BOUTEILLE
adsorption of viral particles to cell membranes; 50 × 106 chick erythrocytes suspended in fusion medium were then added; the cell mixture was left at 4°C for 15 rain before transfer to 37°C for an additional 45 min; the fusion medium was removed and the cells were washed with Earle's salt solution in order to eliminate unfused elements; finally, the cells were resuspended in 10 ml of culture medium. This time point was considered as zero time after fusion.
[5-3H] Uridine Labeling At different times after fusion (0, 12, 24, and 48 hr) cells were exposed to tritiated uridine, specific activity 28 Ci/mmole (Amersham, United Kingdom), at a final concentration of 100 gCi/cm 3 in 3 cm 3 of culture medium. After incubation at 37°C for 10-30 or 180 min the medium with the radioactive precursor was removed and the cells were washed three times in PBS containing nonradioactive uridine at a final concentration of 1 × 104 mM. A pellet of chick erythrocytes, used as control, was incubated for 30 min in medium containing tritiated uridine under the same conditions.
Electron Microscopy At each time point after fusion, the cells were prepared for electron microscopy whether previously incubated with tritiated uridine or not. The medium was poured off and replaced with 1.6% glutaraldehyde in S6rensen buffer, pH 7.2, for 60 min at 4°C. Then, the cells were rinsed four times with the buffer and postfixed with 2% osmium tetroxide in the same buffer for 60 min. Flat embedding, face-on staggered sections. In order to identify the origin of the nuclei by their difference in size within the heterokaryons, all cells were flat-embedded in situ in epoxy resin Epon and then sectioned face-on (Picard and Tixier-Vidal, 1974). The zones to be analyzed and removed for electron microscopic processing were selected under the light microscope according to the number of heterokaryons. They were outlined with a small circular marking apparatus utilizing a diamond point, then cut out and stuck horizontally on to a block of Epon. Serial sections of the cell monolayers were cut on ultramicrotome (Porter-Blum Sorvall MT2) and collected on six to seven grids. These grids were then processed for high-resolution autoradiography. It was not possible to envisage a complete analysis on contiguous serial sections throughout entire monolayers. However because of the relatively small average size of nucleoli compared to the section thickness, it was possible to obtain a precise data in the three-dimensional arrangement of the nucleoli in chick nuclei in an appreciable number of cases.
Ultrastructural A utoradiography The procedure for nltrastructural autoradiography
has been reported and extensively described elsewhere (Bouteille, 1976). Ilford L4 emulsion was applied onto single grids using the gold interference colored zone of the emulsion film in a platinum loop. After 6 months of exposure in dark boxes at 4°C, the autoradiograms were treated at 18°C by 5-min gold latensification followed by 2-min development in a phenidon-containing developer and then fixed for 5 min. The grids were rinsed three times in distilled water and the sections were poststained with uranyl acetate (20 min) and lead citrate (2 min), the grids being air dried at 37°C for 30 min between the two contrasting agents. As pointed out and documented elsewhere (Bouteille et al., 1976), the combination of gold latensification with phenidon development provides silver grains of size and shape particularly suitable for analysis of the labeling of very small organelles, such as the nucleoli investigated in the present study, with a very high efficiency. The respective size of silver grains and nucleoli allowed precise localization and therefore a quantitative appraisal of the data.
Quantitation A total number of 110 fragments of the flat-embedded cultures were flat sectioned in staggered sections which were collected on 800 grids; grids were then processed for ultrastructural autoradiography. A total number of 199 heterokaryons, that is, cells containing at least two nuclei, one of each origin--host and chick--were photographed at various magnification in staggered sections in order to obtain simultaneously general views at low-magnification and high-resolution analysis. A total of 2800 micrographs were analyzed. These 199 heterokaryons contained 326 chick nuclei, among which 91 were found to be provided with nucleolar structures, 79 with one nucleolus, and 6 with two nucleoli. Due to the bars of the grids and to the fact that not all nuclei were sectioned throughout their entire volume, the actual ration of nucleoli per nucleus was slightly higher. The labeling of the structure investigated was analyzed in terms of grain density, that is, the number of grains per unit area. The background noise was counted at random in each section investigated in parts of the section devoid of cells and was found to be less than three grains per 100 ~m 2. For a definite structure, a minimum density was accepted as criterion of labeling. Thus a nucleus was considered as labeled, when the density of the labeling over its surface was one grain per #m 2, and a nucleofus was considered as labeled when the density of the labeling over its surface was one grain per 0.1 gm 2. The latter was selected because of the smaller size of the nucleolar structures. Nucleoli with less grains than this were counted as unlabeled. The mean of the cumulative density of all nucleoli examined was also calculated and compared to that of the nuclei where they were observed.
NUCLEOLAR REACTIVATION TABLE
167
1
NUCLEOLAR STRUCTURES OBSERVED IN REACTIVATED CHICK ERYTHROCYTES
Time of reactivation (hr)
Nucleolar structures" Number of reactiNumber of nucleolar vated chick erythrostructures cytes (1) (2) (3) 12 122 26 5 20 1 24 189 55 12 38 5 48 15 10 6 4 0 a Three types (see text for details) of nucleolar structures: (1) developed nucleoli, (2) prenucleoli, (3) fibrillar centers or NOR. RESULTS
Criteria of Host and Chick Nuclei Identification One of the difficulties of the present sort of investigation is the identification of chick and host nuclei in h e t e r o k a r y o n s at the ultrastructural level. Nuclei were considered to be of chick origin when three different criteria were met. T h e first two were ultrastructural, i.e., the degree of chromatin condensation which is always higher in chick t h a n in host nuclei and the shape of the chick nuclei which is usually regular, roughly round, and devoid of nuclear pockets. T h e third criterion is the size of the nuclei which is known from previous light microscopical studies (Appels et al., 1974a; Harris, 1967) to be m u c h smaller t h a n the size of the host nuclei even after reactivation. T h e two latter criteria which are valueless in embedded pellets because of the occurrence of tangential sections were easily determined in the present fiat-embedded material (Figs. 6, 13, 17). T h e use of serial face-on sections was especially useful in this respect. T h u s it was possible to gauge with some precision on micrographs at a final magnification of 27 000, the average diameter of reactivating nuclei which was, respectively, 3.3, 4.0, and 4 . 9 / t m for 12-24 and 48 hr after fusion as compared with 2.3 #m for control chick nuclei.
Nucleolar Structures T h e structures identified as nucleolar can be described u n d e r three characteristic aspects: First of all, a definite but small n u m b e r
of structures could be identified as developed nucleoli (Fig. 4). T h e y were seen essentially in the latter stages of reactivation and consisted of a single electron-lucent zone of fibrillar appearance partly surrounded by condensed chromatin. This fibrillar center was contiguous to a dense fibrillar component, opposite to condensed chromatin, In the fibrillar component, a gradient of small granules were observed together with typical vacuoles. In no case was the granular c o m p o n e n t well developed as, for instance, in the host cell. In most cases these four components were found to be more or less disposed along a line with one extremity in the condensed c h r o m a t i n and the other exhibiting the higher density of granules. T h e area of these structures was usually 0.5 #m 2 and the average area of the fibrillar centers in these structures was 0.09/~m 2. In contrast to these well-developed nucleoli, 62% (Table 1) of the nucleolar structures had areas ranging from 0.1 to 0.3 #m 2 and were devoid of granules although the other three components were clearly evident (Figs. 1-3), and in the larger of t h e m small vacuoles were observed. Structures of this type will be referred to as prenucleoli later on in this paper, for reasons given in tbe Discussion. Such nucleoli were found on three occasions in serial sections, occurring, as pairs, situated opposite to a clump of condensed chromatin (Fig. 5). Another series of nucleolar structures were found to consist solely of a fibrillar center adjacent to condensed chromatin but exhibiting no fibrillar nor granular components. It was only possible to verify the
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FIGs. 1-4. Nucleolar structures observed in reactivated chick erythrocytes, 24 hr after fusion: FIGs. 1-3. Prenucleoli at different stages of development. They are composed of a single light fibrillae zone, the fibrillar center (arrow) partly surrounded by condensed chromatin (CC), and the dense fibrillar component. FIG. 4. Developed nucleolus composed of a fibrillar center (arrow) partly surrounded by condensed chromatin (CC) and nucleolar fibrillar component. The fibrogranular component of this nucleolus exhibits typical vacuoles. Figs. 1-4, x 45 000. FIG. 5. Prenucleoli as pairs in an opposite situation observed in a chick erythrocyte nucleus reactivated by fusion in a human cell. Each prenucleolus exhibits one fibriUar center (arrow). X 70 000.
absence of granular and fibrillar components in six cases where, in the sections examined, the entire thickness of the fibrillar center was included in three to four serial sections. This was possible to confirm in six cases (Table 1). However, this situation was probably actually more frequent. The average area of the fibrillar center was 0.05 #m 2. In the rest of the paper for reasons expressed in the Discussion, the fibrillar center will be referred to as NOR. In the late stages of reactivation, 8% of the reactivated erythrocyte nuclei developed granules approximately 40 nm in diameter. These granules were observed in
close vicinity to the fibrillar component (Fig. 20) but not especially associated with condensed chromatin. Uridine Incorporation in Nucleolar Structures The labeling of host nuclei and nucleoli was used as an internal control. In this respect, it was of interest to observe that, despite the presence of one or more foreign nuclei, the kinetics of uridine incorporation was in agreement with current concepts on nuclear and nucleolar RNA synthesis as revealed by ultrastructural autoradiography (Figs. 6-8). In particular, the labeling
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was found essentially over the fibrillar component of the nucleoli at 10 min of incorporation. As usual, very few grains were found over the fibrillar centers. Within 30 min, the labeling of the granular component increased significantly, as did that of the nucleoplasm. The total number of grains was found to increase to a large extent in fibrillar and granular components within 3 hr of incorporation (Fig. 13). By comparison, the labeling of the chick nuclei, present in fusion experiments but not introduced into heterokaryons, as well as those of control samples only consisting of chick erythrocytes, was similar to background level. The labeling of fused chick nuclei in heterokaryons followed roughly the kinetics of incorporation of host nuclei and nucleoli. Chick nuclei in heterokaryons whose host nuclei were highly labeled also tended to exhibit a large number of grains. However, wide variations were observed in the labeling of chick nuclei in the same heterokaryon. This was obviously related to the degree of reactivation. Most attention was paid to the presence or absence of incorporation of tritiated uridine in relation to the stages of morphological development of the nucleolar structures defined above. All developed nuclei observed were labeled after 30 min of tritiated uridine incorporation. The cumulative values of grain count and unit surface area of 11 such nucleoli were 127 grains and 5.36 ~m 2, giving a mean labeling density of 23 grains/#m 2 and a mean nucleolar surface area of 0.48 /~m2. Silver grains were mostly located over the fibrillar component of this developed
• TABLE 2 URIDINE INCORPORATIONIN NUCLEOLAR STRUCTURES
No. of nucleo- Mean labeling Mean labeling lar structures density in nu- density in nuobserved cleolar struc- cleoplasm of tures reactivated erythrocytes (1)" (2) (3)
11 24 2
23 44 0
5 8 4
a Three types (see text for details) of nucleolar structures in 83 reactivated chick erythrocytes after a 30-min pulse: (1) developed nucleoli, (2) prenucleoli, (3) fibrillar centers or NOR. Labeling density: number of grains/~m 2.
nucleoli after a 30-min pulse. The labeling density of the nucleoplasm of the reactivated chick erythrocyte nuclei which contained nucleolar structures was less than that of the nucleolar labeling (Table 2). Most of prenucleoli were covered with a significant number of grains (Figs. 7, 10), i.e., 44 grains//~m2 after a 30-min pulse (Table 2). The mean value of the surface of these structures was 0.17 ~m 2. Some of these nucleoli display a high labeling of 108 grains//~m~ at most. This value must be compared to the value of the background noise estimated to be less than 3 grains/100 /zm2. The labeling was found to be located on the fibrillar component especially around the fibrillar centers of these prenucleoli. About 15% of the prenucleoli were unlabeled (Figs. 17-19). That at least some prenucleoli were actually unlabeled was suggested by the fact that six nucleoli were found to be unlabeled in chick nuclei that were themselves cov-
FIGS. 6-8. Representative electron micrographs of the labeling distribution in a chick-human heterokaryon after a 30-min [~H]uridine pulse. FIG. 6. Two different nuclei, the large one is a h u m a n nucleus and the little one a reactivated chick erythrocyte nucleus. Silver grains are mainly located over the erythrocyte (arrow) and h u m a n (two arrows) nucleoli as compared to the labeling density in the nucleoplasms. × 10 000. FIGS. 7 AND 8. Higher magnification of the same nucleoli (one arrow: chick nucleolus; two arrows: h u m a n nucleolus) shown in Fig. 6. The silver grains are located over the fibrillar component (Fig. 7) around the fibrfllar center (arrow} in this erythrocyte prenucleolus. The labeling is clearly on the fibrillar component (Fig. 8) of the human well-developed nucleolus as well. Fig. 7, × 30 000; Fig. 8, × 20 000.
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HERNANDEZ-VERDUN AND BOUTEILLE
ered by a significant number of grains (Figs. 17-19). Finally, the rare nucleolar structures which consisted only of a fibrillar center, as demonstrated by examination of serial sections, were found to be all unlabeled (Fig. 9). Because of the small size of most nucleolar structures and especially prenucleoli, there was some doubt about the possibility that the silver grains covering them could result from radioactive emissions originating from sources located in their immediate vicinity in the nucleoplasm. In fact, in most cases, the density of grains over them was higher than that of the surrounding nucleoplasm; this point was verified by comparing autoradiographs obtained from serial sections. In all six cases in which this was possible, each of the serial sections examined showed a higher density over the nucleolar structures as illustrated (Figs. 1316). This rules out the possibility of a fortuitous accumulation of grains over these structures. Autoradiographs from serial sections through the prenucleoli which had shown no labeling on examination of the initial sections revealed no grains over these structures in any of the adjacent sections (Figs. 17-19).
TABLE 3 URIDINE INCORPORATION IN NUCLEOLAR STRUCTURESa10 min 180 min
(1) (2) (3) (1) (2) (3) No. of nucleolar structures observed Mean labeling density in nucleolar structures Mean labeling density in nucleoplasm of reactivated erythrocyt{~s
1
10
1
2
5
1
22
32
0
21
58
0
9
8
3
5
15
8
a Nucleolar structures observed in 91 reactivated chick erythrocytes after a 10- and 180-min pulse. (1) Developed nucleoli; (2) prenucleoli; (3) fibriUar center or NOR. Labeling density: number of grains/~m 2.
Kinetic data were also obtained by comparison of autoradiographs of material incubated for 10 and 180 min. Among the 91 chick-reactivated erythrocytes examined, 20 of them contained nucleolar structures (Figs. 11, 12). The most developed nucleoli were all labeled even after 10 min of pulse. In contrast, some prenucleoli were found to be unlabeled after short periods of pulse and, as for the fibrillar centers, none of them was observed to be labeled whatever the time of incorporation (10, 30, and 180 min), (Table 3).
FIGS. 9-12. Uridine incorporation on the nucleolar structures differentiated during the chick erythrocyte reactivation. Figs. 9 and 10: 30-min pulse; Figs. 11 and 12: 180-min pulse. Fro. 9. A fibrillar center (arrow) or NOR, located in the condensed chromatin associated with the nuclear envelope. The fibrillar center (arrow) is free of silver grain. × 52 (DO. FIGS. 10-12. The difference of the labeling density over the prenucleoli after a 30-min (Fig. 10) and a 180min uridine pulse. Silver grains are located in the fibrillar component close to the fibrillar center {arrow). × 52 000. FIG. 13. General view of the labeling distribution in a typical heterokaryon after a 180-min [3H]uridine pulse. In this flat embedded heterokaryon the size of the nucleus is used to identify the chick (small with condensed chromatin) and h u m a n nuclei (large nucleus). Each nucleus contains a nucleolus. Silver grains are distributed on the chick and human nuclei with a similar density. They are also found in the cytoplasm at this time which is different from a 30-min pulse as in Fig. 6. × 10 000. FIGS. 14-16. Staggered sections of the erythrocyte nucleolus of the preceding figure (13). The dense fibrillar component is labeled, x 32 000. Fie. 17. General view of a chick-human heterokaryon containing two erythrocyte nuclei (small ones with condensed chromatin) and one human nucleus after a 30-min [~H]uridine pulse. Note that in the upper chick nucleus the nucleolar fibriUar component is devoid of silver grain, despite a labeled nucleoplasm (arrow). x 6000. FIGS. 18 AND 19. Other sections of the chick nucleus of the preceding figure (Fig. 17) containing an unlabeled prenucleolus. In this case the absence of silver grain over the prenucleolus is confirmed on serial sections. × 20 000.
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HERNANDEZ-VERDUN AND BOUTEILLE DISCUSSION
The present data provide evidence that in heterokaryons, newly formed structures, which can be readily identified as essentially nucleolar, using the widely accepted criteria provided by electron microscopical examination, and in which development is induced by cell fusion (Dupuy-Coin et al., 1976), are actually involved in RNA synthesis. This was especially interesting inasmuch as it provided a convenient experimental model of nucleolar differentiation starting from zero time since chick erythrocytes are known to be devoid of developed nucleoli (Small and Davies, 1972). It has even been shown that these nuclei are not involved in ribosomal RNA synthesis (Zentgraf et al., 1975). The present data are consistent with reports on the resumed ribosomal RNA synthesis in chick erythrocytes reactivated by cell fusion (Carlsson et al., 1973) and with the demonstration that in such nuclei ribosomal RNA processing and transport do take place (Bramwell, 1978). Therefore, the RNA which in the present study was shown by EM autoradiography to be synthesized in reactivated nucleolar structures was presumably ribosomal although this particular technique obviously cannot supply evidence for this. The data were especially informative in the case of the structures we proposed to call prenucleoli, since, due to their size, their identification and the description of their internal structures are not possible by LM examination. This term seems to be justified for several reasons. First of all, comparison of these prenucleoli, which amounted to 62% of the total population of nucleolar structures, with that of more developed nucleoli in this material and with the appearance of fully developed nucleoli in other types of cells (Gimenez-Martin et al., 1977; Smetana and Busch, 1974; Bernhard and Granboulan, 1968), shows that the prenucleoli could be considered as nucleoli provided with all nucleolar components but
the granular component. There is a full line of evidence that the granular component originates from the fibrillar one (see Bouteille et al., 1974; Smetana and Busch, 1974, for review) and that the granules represent the later stages of ribosomal R N A processing as reported by Royal and Simard (1975). Furthermore, the present model can be taken to have the advantage of providing a dynamic view of the nucleologenesis inasmuch as the development of nucleolar structures can be followed step-by-step as the course of reactivation progresses. The ratio of prenucleoli in the population of nucleolar structures was shown to decrease with time while more developed nucleoli augmented, indicating that the prenucleoli were their precursors. The autoradiograph supplies evidence that they could actually be engaged in RNA synthesis. This is in contrast with other situations where fibrillar structures were described as resting nucleoli, especially in avian erythrocyte nuclei (Small and Davies, 1972; Smetana and Busch, 1974) or, after pharmacological inhibition of R N A synthesis (Herzog and Farber, 1975; Phillips and Phillips, 1973). After 10- and 30-min pulse, some prenucleoli were unlabeled (Figs. 17-19), but with a 180-min pulse of tritiated uridine all of them were covered with a significant number of grains. This may be a partial explanation for the increase of the mean labeling density with time of pulse. Another interesting stage was represented by structures consisting only of what can be identified morphologically as fibrillar centers associated with chromatin. The fibrillar centers, first described by Recher et al. (1969), are now interpreted in many types of cells as the counterpart, during interphase, of the nucleolar organizer regions of the chromosomes in mitosis (Goessens, 1976b; Hubert, 1975; Knibiehler et al., 1977; Mitre and Stahl, 1976; Pouchelet et al., 1975; Stahl et al., 1974; Vagner-Capodano et al., 1977). In the present study, this identification was further supported by (1)
NUCLEOLAR REACTIVATION
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FIG. 20. Particular granules developed in close vicinity to fibrillar component in reactivated chick erythrocyte 48 hr after fusion. × 70 000.
the absence of labeling when fibrillar centers were found completely devoid of the dense fibrillar component (Table 1-3, Fig. 9) and (2) the location of the silver grains over this fibrillar component when it was present, that is, in prenucleoli (Fig. 10). From this finding and from the literature data, one is led to interpret the fibrillar center as the first identifiable step of nucleologenesis. At the opposite pole of this cycle of nucleologenesis, were the most developed nucleolar structures which could be observed in reactivated chick nuclei. These were among the nucleolar structures characterized by (1) their larger size, 0.5 #m 2 as compared to 0.3 /tm 2 for prenucleoli and 0.05/tm 2 for fibrillar centers, (2) their increasing ratio as the course of reactivation progresses, and (3) their substructures. In addition to their fibrillar centers and their
highly labeled fibrillar components, they also exhibited a sparse granular component and typical nucleolar vacuoles. In fact, their general appearance was close to, though not identical with, that of fully developed nucleoli as described in the literature. Actually they are similar to nucleoli described during cleavage and early gastrulation in Xenopus embryos (Hay and Gurdon, 1967; Karasaki, 1968). The presence of vacuoles was of particular interest since a relationship has been suggested between nucleolar vacuolization and resumed R N A synthesis in animal (Zybina, 1968) and plant cells (De Barsy et al., 1974; Johnson, 1969). It must be pointed out however, that despite an extensive search and the quantitative approach of this study no fully developed granular component has been observed in chick nucleoli as compared to host human nucleoli. The reasons for this are
178
HERNANDEZ-VERDUN AND BOUTEILLE
4
t 3
t
2
CC
1
FIG. 21. Schematic illustration of the nucleologenesis and uridine incorporation during the course of chick erythrocyte reactivation. (1) The fibrillar center (FC), which corresponds to the NOR and appears as the initiation structure for later stages, is associated with the condensed chromatin (CC). (2) In this prenucleolus, the fibrillar component is not labeled, due for instance, to a low level of activity. (3) In such a prenucleolus, the fibrillar component close to the fibrillar center is labeled (black dots). It is the morphological substrate of RNA synthesis as first detected. (4) In this developed nucleolus, the fibrillogranular component is differentiated and nucleolar vacuoles appear. Uridine incorporation (black dots) is found in the fibrillar and fibriUogranular component of the nucleolus. n o t clear b u t t h e p a r t i c u l a r conditions in w h i c h t h e nucleoli d e v e l o p e d in t h e p r e s e n t situation m a y be p a r t of t h e explanation. As for t h e p a r t i c u l a r large g r a n u l e s w h i c h were f o u n d a r o u n d t h e nucleoli (Fig. 20), t h e y c a n n o t be easily explained either, alt h o u g h a possible physiological difference
between nucleolus-associated perichromatin granules a n d t h o s e a s s o c i a t e d w i t h t h e rest of t h e c h r o m a t i n h a s b e e n p r o p o s e d in a n o t h e r m o d e l ( P u v i o n et al., 1977). I n view of t h e s e results a n d t h e literature data, it is possible to p r o p o s e a d y n a m i c m o d e l of nucleologenesis w h i c h is illust r a t e d a n d discussed in Fig. 21. I n this m o d e l t h e fibrillar c e n t e r s are t h e first p a r t of t h e nucleoli w h i c h are o b s e r v e d a n d app e a r as t h e initiation s t r u c t u r e s for later stages, prenucleoli a n d nucleoli. T h i s m o d e l is applicable to t h e p r e s e n t m a t e r i a l a n d m a n y others, p l a n t cells (De B a r s y et al., 1974), o o c y t e s ( H a y a n d G u r d o n , 1967), a n d e m b r y o n i c cells (Karasaki, 1968; N o e l e t al., 1971), b u t t h e r e is no evidence at t h e prese n t t i m e t h a t this is t h e general model. I n fact, it r e m a i n s possible that, a l t h o u g h general, this cycle of nucleologenesis c a n o n l y be s t u d i e d w h e n its d u r a t i o n is e x p a n d e d either physiologically or e x p e r i m e n t a l l y as in t h e p r e s e n t case. We thank Miss F. Laquerri~re and Mrs. M. Pestmal for their excellent technical assistance, and Mr. Wolfelsperger for photographic work. Human TG cells and Sendai virus were obtained through the courtesy of Dr. J. Belerhadek. We especially thank Professor N. R. Ringertz for his support and helpful criticisms of this work and Dr. Macy O'Hegarty for appreciated suggestions for improvement of the manuscript. This work was partly supported by grants of the Centre National de la Recherche Scientifique (E.R. 189 and A.T.P. "Chromatine"), and the Institut National de la Sant6 et de la Recherche M6dicale (U. 183). REFERENCES APPELS,R., BOLUND,L., GOTO,S., ANDRINGERTZ,N. R. (1974a) Exp. Cell Res. 85, 182-190. APPELS,R., BOLUND,L., AND RINGERTZ, N. R. (1974b) J. Mol. Biol. 87, 339-355. APPELS, R., TALLROTH,E., APPELS,D. M., AND RINGERTZ,N. R. (1975) Exp. Cell Res. 92, 70-78. BEREZNEY,R., AND COFFEY, D. S. (1977) J. Cell Biol. 73, 616-637. BERNHARD,W., AND GRANBOULAN, N. (1968) in DALTON, A. J., AND HAGUENAU,F. (Eds.), The Cell Nucleus, pp. 81-149, Academic Press, New York. BOUTEILLE, M. (1976) J. Microsc. Biol. Cell. 27, 121128. BOUTEILLE, M., FAKAN, S., AND BURGLES, M. J. (1976) J. Microsc. Biol. Cell. 27, 171-176.
NUCLEOLAR REACTIVATION BOUTEILLE, M., LAVAL,M., AND DUPUY-COIN, A. M. (1974) in BUSCH, H. (Ed.), The Cell Nucleus, pp. 371, Academic Press, New York. BRAMWELL, M. E. (1978) Exp. Cell Res. 112, 63-71. CARLSSON, S. A., MOORE, G. P. M., AND RINGEBTZ, N. R. (1973) Exp. Cell Res. 76, 234-241. DEAK,I., SIDEBOTTOM,E., ANDHARRIS,H. (1972) J. Cell Sci. 11, 379-391. DE BARSY, TH., DELTOUR, R., AND BRONCHART, R. (1974) J. Cell Sci. 16, 95-112. DUPUY-CoIN, A. M., EGE, T., BOUTEILLE, M., AND RINGERTZ, N. R. (1976) Exp. Cell Res. 101, 355369. FAKAN, S. (1976) J. Microsc. 106, 159-171. FAKAN, S., AND NOBIS, P. (1978) Exp. Cell Res. 113, 327-337. FAKAN, S., PUVION, E., AND SPOHR, G. (1976) Exp. Cell Res. 99, 155-164. GIMENEZ-MARTIN,G., DE LA TORRE, C., LOPEZ-SAEz, J. F., AND ESPONDA, P. (1977) Cytobiologie 14, 421462. GOESSENS, G. (1976a) Cell Tissue Res. 173, 315-324. GOESSENS, G. (1976b) Exp. Cell Res. 100, 88-94. GOESSENS, G., AND LEPOINT, A. (1974) Exp. Cell Res. 87, 63-72. GOTO, S., AND RINGERTZ,N. R. (1974) Exp. Cell Res. 85, 173-181. HARRIS, H. (1965) Nature (London) 206, 583-588. HARRIS, H. (1967) J. Cell Sei. 2, 23-32. HARRIS, H., SIDEBOTTOM, E., GRACE, D. M., AND BRAMWELL, M. E. (1969) J. Cell Sci. 4, 499-525. HAY, E., AND GURDON,J. B. (1967) J. CellSci. 2, 151162. HERZOG, J., AND FARBER, J. L. (1975) Exp. Cell Res. 93, 502-505. HUBERT, J. (1975) C. R. Acad. Sci. Paris 281, 271273. JOHNSON, J. M. (1969) J. Cell Biol. 43, 197-206. KARASAKI,S. (1968) Exp. Cell Res. 52, 13-26. KNIBIEHLER, B., NAVARRO, A., MIRRE, C., AND STAHL,A. (1977) Exp. Cell Res. 110, 153-157. MACGREGOR,H. C. (1967) J. Cell Sci. 2, 145-150. MILLER, O. L., AND BEATTY, B. R. (1969) J. Cell Physiol. 74, 225-232. MIRRE, C., AND STAHL, A. (1976) J. Ultrastruct. Res. 56, 186-201. NOEL, J. S., DEWEY, W. C., ABEL, J. H., AND THOMPSON, R. P. (1971) J. Cell Biol. 49, 830-847.
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PHILLIPS, S. G., AND PHILLIPS, D. M. (1969) J. Cell Biol. 40, 248-268. PHILLIPS, D. M., AND PHILLIPS, S. G. (1973) J. Cell Biol. 58, 54-63. PICARD, R., AND TIXIER-VIDAL, A. (1974) J. Microsc. Biol. Cell. 20, 80a. POUCHELET,M., GANSMULLER,A., ANTEUNIS,A., AND ROBINEAUX, R. (1975) C. R. Acad. Sci. Paris 280, 2461-2463. PUVION, E., VIRON, A., AND BERNHARD, W. (1977) Biol. Cell. 29, 81-88. I~ECHER, L., WHITESCARVER, J., AND BRIGGS, L. {1969) J. Ultrastruct. Res. 29, 1-14. RINGERTZ, N. R., AND BOLUND, L. (1974) in BUSCH, H. (Ed.), The Cell Nucleus, pp. 417-446, Academic Press, New York. RINGERTZ, N. R., CARLSSON, S. A., EGE, T., AND BOLUND, L. (1971) Proc. Nat. Acad. Sci. USA 68, 3228-3232. RINGERTZ, N. R., AND SAVAGE, R. E. (1976) in RINGERTZ, N. R., AND SAVAGE, R. E. (Eds.), Cells Hybrids, pp. 87-118, Academic Press, New York. ROYAL, A., AND SIMARD, R. (1975) J. Cell Biol. 66, 577-585. SEMESHIN, V. R., SHERUDILO,A. I., AND BELYAEVA, E. S, (1975) Exp. Cell Res. 93, 458-467. SIDEBOTTOM, E. (1974) in BUSCH, H. (Ed.), The Cell Nucleus/, pp. 439-469, Academic Press, New York. SIDEBOTTOM, E., AND HARRIS, H. (1969) J. Cell Sci. 5, 351-364. SMALL, J. V., AND DAVIES, H. G. (1972) Tissue Cell 4, 341-378. SMETANA, K., AND BUSCH, H. (1974) in BUSCH, H. (Ed.), The Cell Nucleus /, pp. 73-147, Academic Press, New York. SMETANA,K., GYORKEY,F., GYORKEY,P., ANDBUSCH, H. (1975) Exp. Cell Res. 91, 143-151. SMETANA,K., AND LIKOVSKY,Z. (1971) Exp. CellRes. 69, 65-71. STAHL, A., LUCIANI, J. M., DEVICTOR,M., CAPODANO, A. M., AND HARTUNG,M. (1974) Exp. Cell Res. 91, 365-371. VAGNER-CAPODANO,A. M., PINNA-DELGROSSI,M. H., AND STAHL, A. (1977) Biol. Cell 29, 209-210. ZENTGRAF, H., SCHEER, U., AND FRANKS, W. W. (1975) Exp, Cell Res. 96, 81-95. ZYBINA, E. V. (1968) Tsitologiya 10, 36-42.
69, 164-179 (1979)
Nucleologenesis in Chick Erythrocyte Nuclei Reactivated by Cell Fusion DANII~LE HERNANDEZ-VERDUN AND MICHEL BOUTEILLE Laboratoire de Pathologie Cellulaire, Institut Biomddical des Cordeliers, Universitd Pierre et Marie Curie, Paris VI, France Received February 15, 1979 "Dormant" chick erythrocyte nuclei have been fused by Send~ff virus with h u m a n TG cells. A quantitative analysis and a three-dimensional approach using staggered sections of flat embedded heterokaryons were used to analyze the various aspects of nucleolar structures differentiated during chick erythrocyte reactivation 12, 24, and 48 hr after fusion. Three types of nucleolar structures are described: (1) developed nucleoli, (2) prenucleoli, and (3) nucleolar organizer region. Reactivation of RNA synthesis was followed by electron microscope autoradiography after 10, 30, and 180 min [~H]uridine pulse in the three types of nucleolar structures described. Developed nucleoli and prenucleoli were involved in RNA synthesis; the fibrillar component was f£rst labeled in the periphery of the fibrillar center. To conclude, we propose a dynamic model of nucleologenesis which correlates ultrastructural features and RNA synthesis.
In view of recent developments in understanding of correlations between structure and function within the cell nucleus during the cell cycle, the definition of the nucleolus has become ambiguous. At the ultrastructural level, the nucleolus, as described by Bernhard and Granboulan (1968), is a nuclear organelle consisting of fibrils, granules, condensed associated chromatin as well as the more recently demonstrated (Goessens, 1976a; Goessens and Lepoint, 1974; Recher et al., 1969) fibrillar centers. This description represents an "ideal" fully developed nucleolus. The relationships between these various components and the synthesis of R N A have been well established, especially by means of cytochemistry and autoradiography [see Bouteille et al., 1974, and Fakan, 1976, for review and Fakan and Nobis, 1978; Fakan et al., 1976]. However, considerable morphological variations of nucleoli have been observed according to the cell type, the period of the cell cycle, and the physiological state of the cell, as extensively studied by Smetana and Busch (1974). Another concept implies that the term nucleolus should be restricted to structures which are the morphological substrate of
RNA transcription, processing, and storage. This is essentially the biochemical definition. The limits of defining the nucleolus purely in terms of transcription is well illustrated by the residual proteinaceous structures which can still be identified morphologically as nucleolar after 99.9% DNA and 98% RNA extraction (Berezney and Coffey, 1977). A third way of viewing the nucleolus is to consider as its most essential component the DNA, which carries the information necessary for ribosomal RNA transcription, as visualized in the transcription unit by Miller and Beatty (1969). The exact relationship between this ribosomal DNA and the nucleolar organizer region (NOR) is not yet completely elucidated (Phillips and Phillips, 1969; Semeshin et al., 1975). Nevertheless, the NOR is certainly the most significant part of the nucleolus from the cytogenetical point of view, as a particular region of the chromosomes around which the newly formed nucleoli are reconstituted at the end of mitosis. It was necessary, in order to clarify the morphologic definition of the nucleolus, to look for an experimental system in which (1) the reactivation of a resting nucleolus is experimentally initiated from zero time and 164
0022-5320/79/110164-16502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
NUCLEOLAR REACTIVATION then followed by means of electron microscopy; (2) t h e e m e r g e n c e o f n e w l y f o r m e d n u c l e o l i c a n b e v i s u a l i z e d ; (3) t h e s t a g e s o f t h e e m e r g e n c e o f t h e s e n u c l e o l i a r e cont r o l l e d ; a n d (4) t h e d i f f e r e n c e b e t w e e n t h e s t r u c t u r e s i d e n t i f i e d m o r p h o l o g i c a l l y as n u cleolar which are actually sites of transcript i o n a n d t h o s e w h i c h a r e not, c a n b e r e a d i l y established. Such an experimental situation can be f o u n d in c h i c k e r y t h r o c y t e n u c l e i r e a c t i v a t e d b y m e a n s o f cell f u s i o n in h e t e r o k a r yons. M a t u r e e r y t h r o c y t e n u c l e i a r e c o n s i d e r e d as " d o r m a n t " in t e r m s o f s y n t h e s i s ( R i n g e r t z a n d B o l u n d , 1974). N o r i b o s o m a l RNA synthesis can be detected (Zentgraf et al., 1975). C o r r e l a t i v e l y , n o n u c l e o l a r structure can be identified although a small number of resting structures called micron u c l e o l i ( S m e t a n a et al., 1975; S m e t a n a a n d L i k o v s k y , 1971) h a v e b e e n d e s c r i b e d ( S m a l l a n d D a v i e s , 1972). I t is w e l l k n o w n ( H a r r i s , 1965, 1967; R i n g e r t z a n d S a v a g e , 1976; S i d e b o t t o m , 1974, for r e v i e w ) t h a t t h e s e n u c l e i c a n b e i n t r o d u c e d b y cell f u s i o n i n t o t h e c y t o p l a s m o f a c t i v e h o s t cells. T h e y a r e then reactivated by an active phenomenon w h e r e a n e s s e n t i a l r o l e is p l a y e d b y t h e migration of host proteins into the chick n u c l e i ( & p p e l s et al., 1974a, 1974b, 1975; R i n g e r t z et al., 1971). T h e e r y t h r o c y t e n u clei r e s u m e r i b o s o m a l R N A s y n t h e s i s ( C a r l s s o n et al., 1973; H a r r i s et al., 1969) a s nucleolar structures become morphologic a l l y i d e n t i f i e d ( D e a k et al., 1972; D u p u y C o i n et al., 1976; S i d e b o t t o m a n d H a r r i s , 1969), a n d c h i c k r i b o s o m a l R N A c a n b e d e t e c t e d in t h e c y t o p l a s m o f h e t e r o k a r y o n s ( B r a m w e l l , 1978). T h e a i m o f t h e p r e s e n t p a p e r w a s to e m p l o y t h e n u c l e a r r e a c t i v a t i o n m o d e l to d e t e r m i n e (1) if a m o r p h o m e t r i c c y c l e c a n b e d e s c r i b e d for t h e n u c l e o l u s , (2) w h a t n u c l e o l a r c o m p o n e n t s a r e i n v o l v e d in t h e n u c l e o l a r r e c o n s t i t u t i o n , a n d (3) w h e t h e r t h e a p p e a r a n c e of specific n u c l e o l a r s t r u c tures could be correlated with resumed R N A s y n t h e s i s . T h e first a n d t h e s e c o n d
165
points were investigated by means of a quantitative analysis of the various aspects of the nucleolar structures as the course of reactivation progressed. This was combined with a three-dimensional approach using staggered--successive but not fully serial-sections. The third point was analyzed by high-resolution autoradiography after triti a t e d u r i d i n e i n c o r p o r a t i o n . T h e d a t a allowed us to propose a model representing the steps of what can be called a cycle of nucleogenesis. MATERIALS AND METHODS
Cells for Fusion E x p e r i m e n t s The established TG line of human oviduct was used as a source of host cells. The cells were cultured on 75 cm2 plastic flasks (Falcon} in 15 cm3 of Eagle's minimum essential medium (MEM) supplemented with 15% heat-inactivated fetal calf serum (30 min at 56°C). Twenty-four hours before fusion, TG cells were detached from the plastic by a 0.25% trypsin solution for 10 min at 37°C and seeded into 25 cm2 plastic flasks at a concentration of 1.5 × 10~ cells per flask in 10 cm~ of culture medium. Fusion of chick erythrocytes to TG cells was done on a relatively large scale using TG cells at near confluency and in a rapid growth phase. Chick erythrocytes were obtained from the blood of 13-day chick embryos by cutting the allantoic vessels and allowing the blood to accumulate in the allantoic fluid. The allantoic fluid was collected and centrifuged for 5 min at 200g at 4°C. The pellet of chick erythrocytes was washed twice by centrifugation in Earle's salt solution, then suspended in 10 cm3 of fusion medium (see below) and stored until fusion at 4°C. The erythrocyte population contained less than 1% contaminant cells (Appels et al., 1974b).
Fusion Procedure The fusion medium was an Earle's salt solution adjusted to pH 8 with sodium bicarbonate, this pH having been reported to produce optimum fusion yield (Goto and Ringertz, 1974). Cell fusion was induced by means of ultraviolet-inactivatedSendal virus. Samples of 1 cm~ of the allantoic suspension of Sendai virus were irradiated for 4 min at a rate of 25 erg/mm 2 (dosimetre Latarjet), in 35-mm plastic petri dishes. The fusion procedure (Dupuy-Coin et al., 1976) included the following steps: Culture medium was removed; the cells were washed twice with fusion medium and were then cooled at 4°C for 15 rain in 10 ml of fusion medium; a suspension (0.5 to 0.8 cm3) of inactivated Sendai virus was added and the cells were stored again at 4°C for 15 min in order to promote
166
HERNANDEZ-VERDUN AND BOUTEILLE
adsorption of viral particles to cell membranes; 50 × 106 chick erythrocytes suspended in fusion medium were then added; the cell mixture was left at 4°C for 15 rain before transfer to 37°C for an additional 45 min; the fusion medium was removed and the cells were washed with Earle's salt solution in order to eliminate unfused elements; finally, the cells were resuspended in 10 ml of culture medium. This time point was considered as zero time after fusion.
[5-3H] Uridine Labeling At different times after fusion (0, 12, 24, and 48 hr) cells were exposed to tritiated uridine, specific activity 28 Ci/mmole (Amersham, United Kingdom), at a final concentration of 100 gCi/cm 3 in 3 cm 3 of culture medium. After incubation at 37°C for 10-30 or 180 min the medium with the radioactive precursor was removed and the cells were washed three times in PBS containing nonradioactive uridine at a final concentration of 1 × 104 mM. A pellet of chick erythrocytes, used as control, was incubated for 30 min in medium containing tritiated uridine under the same conditions.
Electron Microscopy At each time point after fusion, the cells were prepared for electron microscopy whether previously incubated with tritiated uridine or not. The medium was poured off and replaced with 1.6% glutaraldehyde in S6rensen buffer, pH 7.2, for 60 min at 4°C. Then, the cells were rinsed four times with the buffer and postfixed with 2% osmium tetroxide in the same buffer for 60 min. Flat embedding, face-on staggered sections. In order to identify the origin of the nuclei by their difference in size within the heterokaryons, all cells were flat-embedded in situ in epoxy resin Epon and then sectioned face-on (Picard and Tixier-Vidal, 1974). The zones to be analyzed and removed for electron microscopic processing were selected under the light microscope according to the number of heterokaryons. They were outlined with a small circular marking apparatus utilizing a diamond point, then cut out and stuck horizontally on to a block of Epon. Serial sections of the cell monolayers were cut on ultramicrotome (Porter-Blum Sorvall MT2) and collected on six to seven grids. These grids were then processed for high-resolution autoradiography. It was not possible to envisage a complete analysis on contiguous serial sections throughout entire monolayers. However because of the relatively small average size of nucleoli compared to the section thickness, it was possible to obtain a precise data in the three-dimensional arrangement of the nucleoli in chick nuclei in an appreciable number of cases.
Ultrastructural A utoradiography The procedure for nltrastructural autoradiography
has been reported and extensively described elsewhere (Bouteille, 1976). Ilford L4 emulsion was applied onto single grids using the gold interference colored zone of the emulsion film in a platinum loop. After 6 months of exposure in dark boxes at 4°C, the autoradiograms were treated at 18°C by 5-min gold latensification followed by 2-min development in a phenidon-containing developer and then fixed for 5 min. The grids were rinsed three times in distilled water and the sections were poststained with uranyl acetate (20 min) and lead citrate (2 min), the grids being air dried at 37°C for 30 min between the two contrasting agents. As pointed out and documented elsewhere (Bouteille et al., 1976), the combination of gold latensification with phenidon development provides silver grains of size and shape particularly suitable for analysis of the labeling of very small organelles, such as the nucleoli investigated in the present study, with a very high efficiency. The respective size of silver grains and nucleoli allowed precise localization and therefore a quantitative appraisal of the data.
Quantitation A total number of 110 fragments of the flat-embedded cultures were flat sectioned in staggered sections which were collected on 800 grids; grids were then processed for ultrastructural autoradiography. A total number of 199 heterokaryons, that is, cells containing at least two nuclei, one of each origin--host and chick--were photographed at various magnification in staggered sections in order to obtain simultaneously general views at low-magnification and high-resolution analysis. A total of 2800 micrographs were analyzed. These 199 heterokaryons contained 326 chick nuclei, among which 91 were found to be provided with nucleolar structures, 79 with one nucleolus, and 6 with two nucleoli. Due to the bars of the grids and to the fact that not all nuclei were sectioned throughout their entire volume, the actual ration of nucleoli per nucleus was slightly higher. The labeling of the structure investigated was analyzed in terms of grain density, that is, the number of grains per unit area. The background noise was counted at random in each section investigated in parts of the section devoid of cells and was found to be less than three grains per 100 ~m 2. For a definite structure, a minimum density was accepted as criterion of labeling. Thus a nucleus was considered as labeled, when the density of the labeling over its surface was one grain per #m 2, and a nucleofus was considered as labeled when the density of the labeling over its surface was one grain per 0.1 gm 2. The latter was selected because of the smaller size of the nucleolar structures. Nucleoli with less grains than this were counted as unlabeled. The mean of the cumulative density of all nucleoli examined was also calculated and compared to that of the nuclei where they were observed.
NUCLEOLAR REACTIVATION TABLE
167
1
NUCLEOLAR STRUCTURES OBSERVED IN REACTIVATED CHICK ERYTHROCYTES
Time of reactivation (hr)
Nucleolar structures" Number of reactiNumber of nucleolar vated chick erythrostructures cytes (1) (2) (3) 12 122 26 5 20 1 24 189 55 12 38 5 48 15 10 6 4 0 a Three types (see text for details) of nucleolar structures: (1) developed nucleoli, (2) prenucleoli, (3) fibrillar centers or NOR. RESULTS
Criteria of Host and Chick Nuclei Identification One of the difficulties of the present sort of investigation is the identification of chick and host nuclei in h e t e r o k a r y o n s at the ultrastructural level. Nuclei were considered to be of chick origin when three different criteria were met. T h e first two were ultrastructural, i.e., the degree of chromatin condensation which is always higher in chick t h a n in host nuclei and the shape of the chick nuclei which is usually regular, roughly round, and devoid of nuclear pockets. T h e third criterion is the size of the nuclei which is known from previous light microscopical studies (Appels et al., 1974a; Harris, 1967) to be m u c h smaller t h a n the size of the host nuclei even after reactivation. T h e two latter criteria which are valueless in embedded pellets because of the occurrence of tangential sections were easily determined in the present fiat-embedded material (Figs. 6, 13, 17). T h e use of serial face-on sections was especially useful in this respect. T h u s it was possible to gauge with some precision on micrographs at a final magnification of 27 000, the average diameter of reactivating nuclei which was, respectively, 3.3, 4.0, and 4 . 9 / t m for 12-24 and 48 hr after fusion as compared with 2.3 #m for control chick nuclei.
Nucleolar Structures T h e structures identified as nucleolar can be described u n d e r three characteristic aspects: First of all, a definite but small n u m b e r
of structures could be identified as developed nucleoli (Fig. 4). T h e y were seen essentially in the latter stages of reactivation and consisted of a single electron-lucent zone of fibrillar appearance partly surrounded by condensed chromatin. This fibrillar center was contiguous to a dense fibrillar component, opposite to condensed chromatin, In the fibrillar component, a gradient of small granules were observed together with typical vacuoles. In no case was the granular c o m p o n e n t well developed as, for instance, in the host cell. In most cases these four components were found to be more or less disposed along a line with one extremity in the condensed c h r o m a t i n and the other exhibiting the higher density of granules. T h e area of these structures was usually 0.5 #m 2 and the average area of the fibrillar centers in these structures was 0.09/~m 2. In contrast to these well-developed nucleoli, 62% (Table 1) of the nucleolar structures had areas ranging from 0.1 to 0.3 #m 2 and were devoid of granules although the other three components were clearly evident (Figs. 1-3), and in the larger of t h e m small vacuoles were observed. Structures of this type will be referred to as prenucleoli later on in this paper, for reasons given in tbe Discussion. Such nucleoli were found on three occasions in serial sections, occurring, as pairs, situated opposite to a clump of condensed chromatin (Fig. 5). Another series of nucleolar structures were found to consist solely of a fibrillar center adjacent to condensed chromatin but exhibiting no fibrillar nor granular components. It was only possible to verify the
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QO
NUCLEOLAR REACTIVATION
169
FIGs. 1-4. Nucleolar structures observed in reactivated chick erythrocytes, 24 hr after fusion: FIGs. 1-3. Prenucleoli at different stages of development. They are composed of a single light fibrillae zone, the fibrillar center (arrow) partly surrounded by condensed chromatin (CC), and the dense fibrillar component. FIG. 4. Developed nucleolus composed of a fibrillar center (arrow) partly surrounded by condensed chromatin (CC) and nucleolar fibrillar component. The fibrogranular component of this nucleolus exhibits typical vacuoles. Figs. 1-4, x 45 000. FIG. 5. Prenucleoli as pairs in an opposite situation observed in a chick erythrocyte nucleus reactivated by fusion in a human cell. Each prenucleolus exhibits one fibriUar center (arrow). X 70 000.
absence of granular and fibrillar components in six cases where, in the sections examined, the entire thickness of the fibrillar center was included in three to four serial sections. This was possible to confirm in six cases (Table 1). However, this situation was probably actually more frequent. The average area of the fibrillar center was 0.05 #m 2. In the rest of the paper for reasons expressed in the Discussion, the fibrillar center will be referred to as NOR. In the late stages of reactivation, 8% of the reactivated erythrocyte nuclei developed granules approximately 40 nm in diameter. These granules were observed in
close vicinity to the fibrillar component (Fig. 20) but not especially associated with condensed chromatin. Uridine Incorporation in Nucleolar Structures The labeling of host nuclei and nucleoli was used as an internal control. In this respect, it was of interest to observe that, despite the presence of one or more foreign nuclei, the kinetics of uridine incorporation was in agreement with current concepts on nuclear and nucleolar RNA synthesis as revealed by ultrastructural autoradiography (Figs. 6-8). In particular, the labeling
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was found essentially over the fibrillar component of the nucleoli at 10 min of incorporation. As usual, very few grains were found over the fibrillar centers. Within 30 min, the labeling of the granular component increased significantly, as did that of the nucleoplasm. The total number of grains was found to increase to a large extent in fibrillar and granular components within 3 hr of incorporation (Fig. 13). By comparison, the labeling of the chick nuclei, present in fusion experiments but not introduced into heterokaryons, as well as those of control samples only consisting of chick erythrocytes, was similar to background level. The labeling of fused chick nuclei in heterokaryons followed roughly the kinetics of incorporation of host nuclei and nucleoli. Chick nuclei in heterokaryons whose host nuclei were highly labeled also tended to exhibit a large number of grains. However, wide variations were observed in the labeling of chick nuclei in the same heterokaryon. This was obviously related to the degree of reactivation. Most attention was paid to the presence or absence of incorporation of tritiated uridine in relation to the stages of morphological development of the nucleolar structures defined above. All developed nuclei observed were labeled after 30 min of tritiated uridine incorporation. The cumulative values of grain count and unit surface area of 11 such nucleoli were 127 grains and 5.36 ~m 2, giving a mean labeling density of 23 grains/#m 2 and a mean nucleolar surface area of 0.48 /~m2. Silver grains were mostly located over the fibrillar component of this developed
• TABLE 2 URIDINE INCORPORATIONIN NUCLEOLAR STRUCTURES
No. of nucleo- Mean labeling Mean labeling lar structures density in nu- density in nuobserved cleolar struc- cleoplasm of tures reactivated erythrocytes (1)" (2) (3)
11 24 2
23 44 0
5 8 4
a Three types (see text for details) of nucleolar structures in 83 reactivated chick erythrocytes after a 30-min pulse: (1) developed nucleoli, (2) prenucleoli, (3) fibrillar centers or NOR. Labeling density: number of grains/~m 2.
nucleoli after a 30-min pulse. The labeling density of the nucleoplasm of the reactivated chick erythrocyte nuclei which contained nucleolar structures was less than that of the nucleolar labeling (Table 2). Most of prenucleoli were covered with a significant number of grains (Figs. 7, 10), i.e., 44 grains//~m2 after a 30-min pulse (Table 2). The mean value of the surface of these structures was 0.17 ~m 2. Some of these nucleoli display a high labeling of 108 grains//~m~ at most. This value must be compared to the value of the background noise estimated to be less than 3 grains/100 /zm2. The labeling was found to be located on the fibrillar component especially around the fibrillar centers of these prenucleoli. About 15% of the prenucleoli were unlabeled (Figs. 17-19). That at least some prenucleoli were actually unlabeled was suggested by the fact that six nucleoli were found to be unlabeled in chick nuclei that were themselves cov-
FIGS. 6-8. Representative electron micrographs of the labeling distribution in a chick-human heterokaryon after a 30-min [~H]uridine pulse. FIG. 6. Two different nuclei, the large one is a h u m a n nucleus and the little one a reactivated chick erythrocyte nucleus. Silver grains are mainly located over the erythrocyte (arrow) and h u m a n (two arrows) nucleoli as compared to the labeling density in the nucleoplasms. × 10 000. FIGS. 7 AND 8. Higher magnification of the same nucleoli (one arrow: chick nucleolus; two arrows: h u m a n nucleolus) shown in Fig. 6. The silver grains are located over the fibrillar component (Fig. 7) around the fibrfllar center (arrow} in this erythrocyte prenucleolus. The labeling is clearly on the fibrillar component (Fig. 8) of the human well-developed nucleolus as well. Fig. 7, × 30 000; Fig. 8, × 20 000.
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ered by a significant number of grains (Figs. 17-19). Finally, the rare nucleolar structures which consisted only of a fibrillar center, as demonstrated by examination of serial sections, were found to be all unlabeled (Fig. 9). Because of the small size of most nucleolar structures and especially prenucleoli, there was some doubt about the possibility that the silver grains covering them could result from radioactive emissions originating from sources located in their immediate vicinity in the nucleoplasm. In fact, in most cases, the density of grains over them was higher than that of the surrounding nucleoplasm; this point was verified by comparing autoradiographs obtained from serial sections. In all six cases in which this was possible, each of the serial sections examined showed a higher density over the nucleolar structures as illustrated (Figs. 1316). This rules out the possibility of a fortuitous accumulation of grains over these structures. Autoradiographs from serial sections through the prenucleoli which had shown no labeling on examination of the initial sections revealed no grains over these structures in any of the adjacent sections (Figs. 17-19).
TABLE 3 URIDINE INCORPORATION IN NUCLEOLAR STRUCTURESa10 min 180 min
(1) (2) (3) (1) (2) (3) No. of nucleolar structures observed Mean labeling density in nucleolar structures Mean labeling density in nucleoplasm of reactivated erythrocyt{~s
1
10
1
2
5
1
22
32
0
21
58
0
9
8
3
5
15
8
a Nucleolar structures observed in 91 reactivated chick erythrocytes after a 10- and 180-min pulse. (1) Developed nucleoli; (2) prenucleoli; (3) fibriUar center or NOR. Labeling density: number of grains/~m 2.
Kinetic data were also obtained by comparison of autoradiographs of material incubated for 10 and 180 min. Among the 91 chick-reactivated erythrocytes examined, 20 of them contained nucleolar structures (Figs. 11, 12). The most developed nucleoli were all labeled even after 10 min of pulse. In contrast, some prenucleoli were found to be unlabeled after short periods of pulse and, as for the fibrillar centers, none of them was observed to be labeled whatever the time of incorporation (10, 30, and 180 min), (Table 3).
FIGS. 9-12. Uridine incorporation on the nucleolar structures differentiated during the chick erythrocyte reactivation. Figs. 9 and 10: 30-min pulse; Figs. 11 and 12: 180-min pulse. Fro. 9. A fibrillar center (arrow) or NOR, located in the condensed chromatin associated with the nuclear envelope. The fibrillar center (arrow) is free of silver grain. × 52 (DO. FIGS. 10-12. The difference of the labeling density over the prenucleoli after a 30-min (Fig. 10) and a 180min uridine pulse. Silver grains are located in the fibrillar component close to the fibrillar center {arrow). × 52 000. FIG. 13. General view of the labeling distribution in a typical heterokaryon after a 180-min [3H]uridine pulse. In this flat embedded heterokaryon the size of the nucleus is used to identify the chick (small with condensed chromatin) and h u m a n nuclei (large nucleus). Each nucleus contains a nucleolus. Silver grains are distributed on the chick and human nuclei with a similar density. They are also found in the cytoplasm at this time which is different from a 30-min pulse as in Fig. 6. × 10 000. FIGS. 14-16. Staggered sections of the erythrocyte nucleolus of the preceding figure (13). The dense fibrillar component is labeled, x 32 000. Fie. 17. General view of a chick-human heterokaryon containing two erythrocyte nuclei (small ones with condensed chromatin) and one human nucleus after a 30-min [~H]uridine pulse. Note that in the upper chick nucleus the nucleolar fibriUar component is devoid of silver grain, despite a labeled nucleoplasm (arrow). x 6000. FIGS. 18 AND 19. Other sections of the chick nucleus of the preceding figure (Fig. 17) containing an unlabeled prenucleolus. In this case the absence of silver grain over the prenucleolus is confirmed on serial sections. × 20 000.
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HERNANDEZ-VERDUN AND BOUTEILLE DISCUSSION
The present data provide evidence that in heterokaryons, newly formed structures, which can be readily identified as essentially nucleolar, using the widely accepted criteria provided by electron microscopical examination, and in which development is induced by cell fusion (Dupuy-Coin et al., 1976), are actually involved in RNA synthesis. This was especially interesting inasmuch as it provided a convenient experimental model of nucleolar differentiation starting from zero time since chick erythrocytes are known to be devoid of developed nucleoli (Small and Davies, 1972). It has even been shown that these nuclei are not involved in ribosomal RNA synthesis (Zentgraf et al., 1975). The present data are consistent with reports on the resumed ribosomal RNA synthesis in chick erythrocytes reactivated by cell fusion (Carlsson et al., 1973) and with the demonstration that in such nuclei ribosomal RNA processing and transport do take place (Bramwell, 1978). Therefore, the RNA which in the present study was shown by EM autoradiography to be synthesized in reactivated nucleolar structures was presumably ribosomal although this particular technique obviously cannot supply evidence for this. The data were especially informative in the case of the structures we proposed to call prenucleoli, since, due to their size, their identification and the description of their internal structures are not possible by LM examination. This term seems to be justified for several reasons. First of all, comparison of these prenucleoli, which amounted to 62% of the total population of nucleolar structures, with that of more developed nucleoli in this material and with the appearance of fully developed nucleoli in other types of cells (Gimenez-Martin et al., 1977; Smetana and Busch, 1974; Bernhard and Granboulan, 1968), shows that the prenucleoli could be considered as nucleoli provided with all nucleolar components but
the granular component. There is a full line of evidence that the granular component originates from the fibrillar one (see Bouteille et al., 1974; Smetana and Busch, 1974, for review) and that the granules represent the later stages of ribosomal R N A processing as reported by Royal and Simard (1975). Furthermore, the present model can be taken to have the advantage of providing a dynamic view of the nucleologenesis inasmuch as the development of nucleolar structures can be followed step-by-step as the course of reactivation progresses. The ratio of prenucleoli in the population of nucleolar structures was shown to decrease with time while more developed nucleoli augmented, indicating that the prenucleoli were their precursors. The autoradiograph supplies evidence that they could actually be engaged in RNA synthesis. This is in contrast with other situations where fibrillar structures were described as resting nucleoli, especially in avian erythrocyte nuclei (Small and Davies, 1972; Smetana and Busch, 1974) or, after pharmacological inhibition of R N A synthesis (Herzog and Farber, 1975; Phillips and Phillips, 1973). After 10- and 30-min pulse, some prenucleoli were unlabeled (Figs. 17-19), but with a 180-min pulse of tritiated uridine all of them were covered with a significant number of grains. This may be a partial explanation for the increase of the mean labeling density with time of pulse. Another interesting stage was represented by structures consisting only of what can be identified morphologically as fibrillar centers associated with chromatin. The fibrillar centers, first described by Recher et al. (1969), are now interpreted in many types of cells as the counterpart, during interphase, of the nucleolar organizer regions of the chromosomes in mitosis (Goessens, 1976b; Hubert, 1975; Knibiehler et al., 1977; Mitre and Stahl, 1976; Pouchelet et al., 1975; Stahl et al., 1974; Vagner-Capodano et al., 1977). In the present study, this identification was further supported by (1)
NUCLEOLAR REACTIVATION
177
FIG. 20. Particular granules developed in close vicinity to fibrillar component in reactivated chick erythrocyte 48 hr after fusion. × 70 000.
the absence of labeling when fibrillar centers were found completely devoid of the dense fibrillar component (Table 1-3, Fig. 9) and (2) the location of the silver grains over this fibrillar component when it was present, that is, in prenucleoli (Fig. 10). From this finding and from the literature data, one is led to interpret the fibrillar center as the first identifiable step of nucleologenesis. At the opposite pole of this cycle of nucleologenesis, were the most developed nucleolar structures which could be observed in reactivated chick nuclei. These were among the nucleolar structures characterized by (1) their larger size, 0.5 #m 2 as compared to 0.3 /tm 2 for prenucleoli and 0.05/tm 2 for fibrillar centers, (2) their increasing ratio as the course of reactivation progresses, and (3) their substructures. In addition to their fibrillar centers and their
highly labeled fibrillar components, they also exhibited a sparse granular component and typical nucleolar vacuoles. In fact, their general appearance was close to, though not identical with, that of fully developed nucleoli as described in the literature. Actually they are similar to nucleoli described during cleavage and early gastrulation in Xenopus embryos (Hay and Gurdon, 1967; Karasaki, 1968). The presence of vacuoles was of particular interest since a relationship has been suggested between nucleolar vacuolization and resumed R N A synthesis in animal (Zybina, 1968) and plant cells (De Barsy et al., 1974; Johnson, 1969). It must be pointed out however, that despite an extensive search and the quantitative approach of this study no fully developed granular component has been observed in chick nucleoli as compared to host human nucleoli. The reasons for this are
178
HERNANDEZ-VERDUN AND BOUTEILLE
4
t 3
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2
CC
1
FIG. 21. Schematic illustration of the nucleologenesis and uridine incorporation during the course of chick erythrocyte reactivation. (1) The fibrillar center (FC), which corresponds to the NOR and appears as the initiation structure for later stages, is associated with the condensed chromatin (CC). (2) In this prenucleolus, the fibrillar component is not labeled, due for instance, to a low level of activity. (3) In such a prenucleolus, the fibrillar component close to the fibrillar center is labeled (black dots). It is the morphological substrate of RNA synthesis as first detected. (4) In this developed nucleolus, the fibrillogranular component is differentiated and nucleolar vacuoles appear. Uridine incorporation (black dots) is found in the fibrillar and fibriUogranular component of the nucleolus. n o t clear b u t t h e p a r t i c u l a r conditions in w h i c h t h e nucleoli d e v e l o p e d in t h e p r e s e n t situation m a y be p a r t of t h e explanation. As for t h e p a r t i c u l a r large g r a n u l e s w h i c h were f o u n d a r o u n d t h e nucleoli (Fig. 20), t h e y c a n n o t be easily explained either, alt h o u g h a possible physiological difference
between nucleolus-associated perichromatin granules a n d t h o s e a s s o c i a t e d w i t h t h e rest of t h e c h r o m a t i n h a s b e e n p r o p o s e d in a n o t h e r m o d e l ( P u v i o n et al., 1977). I n view of t h e s e results a n d t h e literature data, it is possible to p r o p o s e a d y n a m i c m o d e l of nucleologenesis w h i c h is illust r a t e d a n d discussed in Fig. 21. I n this m o d e l t h e fibrillar c e n t e r s are t h e first p a r t of t h e nucleoli w h i c h are o b s e r v e d a n d app e a r as t h e initiation s t r u c t u r e s for later stages, prenucleoli a n d nucleoli. T h i s m o d e l is applicable to t h e p r e s e n t m a t e r i a l a n d m a n y others, p l a n t cells (De B a r s y et al., 1974), o o c y t e s ( H a y a n d G u r d o n , 1967), a n d e m b r y o n i c cells (Karasaki, 1968; N o e l e t al., 1971), b u t t h e r e is no evidence at t h e prese n t t i m e t h a t this is t h e general model. I n fact, it r e m a i n s possible that, a l t h o u g h general, this cycle of nucleologenesis c a n o n l y be s t u d i e d w h e n its d u r a t i o n is e x p a n d e d either physiologically or e x p e r i m e n t a l l y as in t h e p r e s e n t case. We thank Miss F. Laquerri~re and Mrs. M. Pestmal for their excellent technical assistance, and Mr. Wolfelsperger for photographic work. Human TG cells and Sendai virus were obtained through the courtesy of Dr. J. Belerhadek. We especially thank Professor N. R. Ringertz for his support and helpful criticisms of this work and Dr. Macy O'Hegarty for appreciated suggestions for improvement of the manuscript. This work was partly supported by grants of the Centre National de la Recherche Scientifique (E.R. 189 and A.T.P. "Chromatine"), and the Institut National de la Sant6 et de la Recherche M6dicale (U. 183). REFERENCES APPELS,R., BOLUND,L., GOTO,S., ANDRINGERTZ,N. R. (1974a) Exp. Cell Res. 85, 182-190. APPELS,R., BOLUND,L., AND RINGERTZ, N. R. (1974b) J. Mol. Biol. 87, 339-355. APPELS, R., TALLROTH,E., APPELS,D. M., AND RINGERTZ,N. R. (1975) Exp. Cell Res. 92, 70-78. BEREZNEY,R., AND COFFEY, D. S. (1977) J. Cell Biol. 73, 616-637. BERNHARD,W., AND GRANBOULAN, N. (1968) in DALTON, A. J., AND HAGUENAU,F. (Eds.), The Cell Nucleus, pp. 81-149, Academic Press, New York. BOUTEILLE, M. (1976) J. Microsc. Biol. Cell. 27, 121128. BOUTEILLE, M., FAKAN, S., AND BURGLES, M. J. (1976) J. Microsc. Biol. Cell. 27, 171-176.
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PHILLIPS, S. G., AND PHILLIPS, D. M. (1969) J. Cell Biol. 40, 248-268. PHILLIPS, D. M., AND PHILLIPS, S. G. (1973) J. Cell Biol. 58, 54-63. PICARD, R., AND TIXIER-VIDAL, A. (1974) J. Microsc. Biol. Cell. 20, 80a. POUCHELET,M., GANSMULLER,A., ANTEUNIS,A., AND ROBINEAUX, R. (1975) C. R. Acad. Sci. Paris 280, 2461-2463. PUVION, E., VIRON, A., AND BERNHARD, W. (1977) Biol. Cell. 29, 81-88. I~ECHER, L., WHITESCARVER, J., AND BRIGGS, L. {1969) J. Ultrastruct. Res. 29, 1-14. RINGERTZ, N. R., AND BOLUND, L. (1974) in BUSCH, H. (Ed.), The Cell Nucleus, pp. 417-446, Academic Press, New York. RINGERTZ, N. R., CARLSSON, S. A., EGE, T., AND BOLUND, L. (1971) Proc. Nat. Acad. Sci. USA 68, 3228-3232. RINGERTZ, N. R., AND SAVAGE, R. E. (1976) in RINGERTZ, N. R., AND SAVAGE, R. E. (Eds.), Cells Hybrids, pp. 87-118, Academic Press, New York. ROYAL, A., AND SIMARD, R. (1975) J. Cell Biol. 66, 577-585. SEMESHIN, V. R., SHERUDILO,A. I., AND BELYAEVA, E. S, (1975) Exp. Cell Res. 93, 458-467. SIDEBOTTOM, E. (1974) in BUSCH, H. (Ed.), The Cell Nucleus/, pp. 439-469, Academic Press, New York. SIDEBOTTOM, E., AND HARRIS, H. (1969) J. Cell Sci. 5, 351-364. SMALL, J. V., AND DAVIES, H. G. (1972) Tissue Cell 4, 341-378. SMETANA, K., AND BUSCH, H. (1974) in BUSCH, H. (Ed.), The Cell Nucleus /, pp. 73-147, Academic Press, New York. SMETANA,K., GYORKEY,F., GYORKEY,P., ANDBUSCH, H. (1975) Exp. Cell Res. 91, 143-151. SMETANA,K., AND LIKOVSKY,Z. (1971) Exp. CellRes. 69, 65-71. STAHL, A., LUCIANI, J. M., DEVICTOR,M., CAPODANO, A. M., AND HARTUNG,M. (1974) Exp. Cell Res. 91, 365-371. VAGNER-CAPODANO,A. M., PINNA-DELGROSSI,M. H., AND STAHL, A. (1977) Biol. Cell 29, 209-210. ZENTGRAF, H., SCHEER, U., AND FRANKS, W. W. (1975) Exp, Cell Res. 96, 81-95. ZYBINA, E. V. (1968) Tsitologiya 10, 36-42.