JOURNAL OF ULTRASTRUCTURE RESEARCH 62, 213-219 (1978)

The Nucleolar Origin of Certain Perichromatin-Like Granules: A Study with o~-Amanitin

MASSIMO DERENZINI ~ AND GILLES MOYNE 2

Institut de Recherches Scientifiques sur le Cancer, B.P. n°8, F-94800 Villejuif, France

Received September 26, 1977

Isolated rat hepatocytes treated with a-amanitin developed nuclear accumulations of ribonu- cleoprotein granules similar to perichromatin granules. Serial sections permitted the demonstration of a structural continuity between these particles and the nucleolus. This morphological evidence, added to that of the suppression of RNP granules by pretreatment with actinomycin D, supports the possibility that at least a part of the perichromatin granules originates in the nucleolus.

T h e or ig in a n d f u n c t i o n of n u c l e a r r ibo- n u c l e o p r o t e i n p a r t i c l e s k n o w n as p e r i c h r o - m a t i n g r a n u l e s a r e n o t y e t c l e a r l y def ined . R e c e n t p a p e r s f rom th i s l a b o r a t o r y h a v e s u g g e s t e d t h a t s o m e of t h e m c o m e f rom t h e nuc l eo lu s (4, 10, 14, 15). D u r i n g a s t u d y of t h e u l t r a s t r u c t u r a l ef fec ts of a - a m a n i t i n , we o b s e r v e d m a n y p e r i c h r o m a t i n - l i k e g r a n u l e s

~that s e e m e d r e l a t e d to t h e nuc leo lus . W e r e p o r t t h e s e o b s e r v a t i o n s t h a t f a v o r t h e h y p o t h e s i s o f a n u c l e o l a r o r ig in for a t l e a s t s o m e o f t h e p e r i c h r o m a t i n g ranu les .

MATERIALS AND METHODS

Material. Hepatocytes were isolated from 3-month- old WAG rats after 18 hr of fasting with water ad libitum. Tbe obtainment and culture of the cells were as previously described (13). The experiments began 2 hr after isolation of the cells.

Drug treatment, a-Amanitin (Boehringer, Mann- helm) was dissolved in the culture medium at concen- trations of 1, 2, 5, 10, 20, and 50 #g/ml. Actinomycin D (Sigma) was used at a concentration of 0.05 ~g/mL The following experiments were carried out: a-aman- itin alone (1 or 2 hr); actinomycin D alone (1 hr); actinomycin D (I hr) followed by a-amanitin (2 hr); a-amanitin (1 or 2 hr) followed by aetinomycin D (1 hr).

Electron microscopy. After each experiment, the cells were fixed in 1.6% glutaraldehyde in S6rensen's phosphate buffer prior to acetone dehydration and Epon-Araldite embedding. Thin sections were stained either with uranyl acetate and lead citrate or with the uranyl-EDTA-lead method preferential for ribonu-

~ Present address: Istituto di Patologia Generale, Via S. Giacomo 141-40126, Bologna, Italy.

2 To whom requests for reprints should be sent.

cleoprotein staining (1). Serial sections were mounted on slot grids coated with a Formvar-carbon film.

RESULTS

Defini t ion o f the E x p e r i m e n t a l Condi t ions

N o u l t r a s t r u c t u r a l a l t e r a t i o n was ob- s e r v e d w h e n t h e cel ls were t r e a t e d w i th 1 t t g / m l of a - a m a n i t i n . W i t h 2 a n d 5 g g / m l , we n o t e d a n u c l e o l a r f r a g m e n t a t i o n a n d a s l igh t i n c r e a s e in t h e n u m b e r of p e r i c h r o -

m a t i n g ranu les . D o s e s f r o m 10 to 50 # g / m l i n d u c e d la rge a c c u m u l a t i o n s o f p e r i c h r o - m a t i n g ranu les . In add i t i on , t h e nuc l eo lu s was f r a g m e n t e d a n d i t s c o m p o n e n t s were s e g r e g a t e d .

A l t h o u g h f r a g m e n t a t i o n a n d s e g r e g a t i o n

o f t h e n u c l e o l u s were o b s e r v e d a f t e r I h r o f a - a m a n i t i n t r e a t m e n t , t h e a c c u m u l a t i o n s of p e r i c h r o m a t i n g r a n u l e s were l a r g e r a f t e r 2 hr . T h e r e f o r e , t h i s p a p e r d e s c r i b e s t h e re- su l t s of e x p e r i m e n t s c a r r i e d o u t w i t h a- a m a n i t i n for 2 h r a t a dose o f 10 t tg /ml , i.e., t h e m i n i m u m dose a n d t i m e ef fec t ive for i n d u c i n g a n a c c u m u l a t i o n of p e r i c h r o m a t i n g ranu les .

Ultras tructural A l t era t ions after ~-Aman- itin Trea tmen t

I. Nucleolus. F r a g m e n t a t i o n of t h e nu- c leo lus was n o t o b s e r v e d in al l t h e nuclei , b u t s e g r e g a t i o n was a l w a y s p re sen t . I n t h e s e g r e g a t e d nucleol i , t h e f ib r i l l a r zone was c l e a r l y s e p a r a t e d f r o m t h e g r a n u l e s (Figs. 2 a n d 3), in c o n t r a s t to c on t ro l ce l ls {Fig.

213 0022-5320/78/0623-0213502.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

214 DERENZINI AND MOYNE

FIGs. 1-3

NUCLEOLAR PERICHROMATIN-LIKE GRANULES 215

1). Two kinds of nucleolar granules could be recognized after ei ther uranyl - lead or u r a n y l - E D T A - l e a d staining (Fig. 3). Gran- tiles of the first type showed an electron dens i ty very similar to tha t of the fibrils. T h e y were in direct contact with the fibril- lar zone. The second type was less electron dense, and it did not touch the fibrillar zone but was constantly in contact with granules of the first type. Quite often, however, only one type of granule was visible; this is very likely to be due to the sectioning incidence.

II. Periehromatin- l ike granules. Marked accumulations of perichromatin- like granules were observed in nuclei t rea ted for 2 hr with a-amanit in (Figs. 2 and 3). The i r contrast af ter E D T A staining was similar to tha t of the per ichromat in granules observed in control cells (Fig. 1), but, as they were more irregular in shape, we called them perichromatin-l ike gran- ules. T h e y were generally clustered and especially numerous close to the nucleolus- associated chromat in (Figs. 2 and 3). In a- amani t in- t rea ted cells, we could not ob- serve particles identical to normal perichro- mat in granules.

Serial sections demonst ra ted tha t the nu- cleolus was in immediate contact with some perichromatin-l ike granules (Figs. 3-5, 6-8). This topographical proximity does not im- ply an actual continuity between the nu- cleolus and perichromatin-l ike granules; however, our pictures strongly suggested such an interpretat ion. Fur thermore , those perichromatin-l ike granules in contact with the nucleolus resembled very much those dispersed in the nucleoplasm. It thus seemed logical to consider the eventual i ty

of their common origin. Finally, serial sec- tioning of nuclear regions filled with peri- chromatin-l ike granules bu t devoid of nu- cleolus has repeatedly demonst ra ted tha t a nucleolar body could be found in the vicin- i ty of the perichromatin-l ike granule accu- mulat ions (Figs. 6-8).

W h e n p e r i c h r o m a t i n - l i k e g ranu le s touched the nucleolar body, it was always through type 2 nucleolar granules (Fig. 3) or through nucleolus-associated chromatin.

Effect of Actinomycin D

Since we supposed tha t some perichro- matin-like granules might contain nucleolar RNA, it was logical to observe how inhibi- t ion of the nucleolar transcription would affect them. Th e inhibition was obtained with act inomycin D at very low doses known to suppress primarily nucleolar tran- scription {11). Actinomycin D alone in- duced the classical nucleolar segregation. With respect to the accumulat ion of peri- chromatin-like granules, the results are summarized in Table I.

Clearly, act inomycin D, used ei ther alone or before a-amanitin, prevented the for- mat ion of perichromatin-l ike granules (Fig. 10). Contrarily, act inomycin D used after a-amani t in did not suppress perichromatin- like granules (Fig. 9).

DISCUSSION

Several investigators have described the action of a-amanit in in ra t and mouse liver cells. T h e y all observed nucleolar fragmen- ta t ion and segregation (3, 7, 8, 12). With respect to the per ichromatin granules, how- ever , they repor ted ei ther an increased

FIG. i. Nucleus of a control hepatocyte. Uranyl-EDTA-lead staining method. In the nucleolus (nu), the granular and fibrillar components are intermingled. Some perichromatin granules (arrowheads) are visible on the border of dense chromatin (chr). × 37 000.

FIG. 2. Nucleus of a hepatocyte treated for 2 hr with a-amanitin. Uranyl-lead stain. Two nucleolar fragments are visible. Segregation of the granular (g) and fibrillar (f) components. Numerous perichromatin-like granules are located near the nucleolus-associated chromatin, x 22 000.

FIG. 3. Nucleus of a hepatocyte treated for 2 hr with a-amanitin. Uranyl-EDTA-lead staining method. In the segregated nucleolus, it is possible to distinguish the fibrils (f) and two kinds of granules: type 1 (gl) and type 2 (g2). Many perichromatimlike granules (arrowheads), more irregular in shape than control perichromatin granules, are visible near dense chromatin (chr) or in contact with type 2 nucleolar granules, x 37 000.

216 DERENZINI

number ( 7, 12) orno apparent variation (8). In isolated rat hepatocytes, we also have

demonstrated the fragmentation of the nu- cleolar body and a segregation of the dis- persed fragments. In addition to this class- ical observation, we could distinguish two kinds of granules within the nucleolus, one electron dense (type 1) and one of lower density (type 2). Only type 2 granules were in contact with the perichromatin-like granules which were very numerous throughout the rest of the nucleus. We also showed that the nucleolus is in close con- tact with perichromatin-like granules, sug- gesting a structural continuity.

The differences between our results and

AND MOYNE

previous observations could be explained" by our use of cultivated cells maintained iw vitro. Under these conditions the concen- tration of a-amanitin remained constan~ during the entire experiment, whereas th~ clearance of the drug was very fast in poi- soned animals (2). For this reason, th~ doses that we used in vitro are difficult tc compare with those injected into animals. However, they can reasonably be consid- ered as high doses that are able not only to, block the extranucleolar RNA polymerase II (6) but also to alter the synthesis o~ nucleolar RNA (16} which is linked to nu- cleolar segregation.

In 1969, Monneron and Bernhard sug~

FIGS. 4 and 5. Serial sections of the nucleus of a hepatocyte treated with a-amanitin. Uranyl-lead stain. Groups of perichromatin-like granules (arrowheads) are in intimate contact with the nucleolus (nu) from which they seem to emanate. Note the abundance of perichromatin-like granules in all the chromatin. × 28 000.

Fins. 6-8. Serial sections of the nucleus of a hepatocyte treated with a-amanitin. Uranyl-lead stain. Perichromatin-like granules seemingly dispersed in chromatin (star) can be shown to be close to a nucleolus (nu). (Fig. 6) × 22 000; (Figs. 7 and 8) × 19 000.

NUCLEOLAR PERICHROMATIN-LIKE GRANULES 217

FIGS, 6-8

218 DERENZINI AND MOYNE

FIGS. 9 and 10. Uranyl-EDTA-lead staining method. Type 1 and 2 nucleolar granules (ga, g:), nucleolar fibrils (f), perichromatin-like granules (arrowheads).

Fro. 9. Nucleus of a hepatocyte treated with a-amanithl prior to actinomycin D. Numerous perichromatin- like granules are in contact with type 2 granules or with chromatin. × 28 000.

FIG. 10. Nucleus of a hepatocyte treated with actinomycin D prior to a-amanitin. Perichromatin-like granules are very scarce. × 26 000.

TABLE I

EFFECT OF TREATMENT ON ACCUMULATION OF PERICHROMATIN-LIKE PARTICLES

Treatment Accumula- Figure tion of per- No. ichromatin-

like gran- ules

Actinomycin D" - - - a-Amanitin b + 3 Actinomycin D/a-amanitin - 10 a-Amanitin/actinomycin D + 9

Actinomycin D: 0.05 #g/ml, 1 hr. aoAmanitin: 10 tLg/ml, 2 hr.

gested that the perichromatin granules might contain messenger RNA coming from the perichromatin fibrils (9). More recently, the same cell system, isolated rat hepatocytes, was submitted to the action of

several other drugs. Treatments with cor- dycepin (14), camptothecin (4), and afla" toxin B1 (G. Moyne et al., unpublished re= sults) all resulted in an accumulation of perichromatin granules in the nucleolar re-, gion, suggesting that some of these granules, might originate in the nucleolus. These three drugs induce a nucleolar segregation. ~

With a-amanitin the accumulation of, particles differing slightly from normal per- ichromatin granules was not strictly peri- nucleolar. Yet, the continuity between the nucleolus and some of these perichromatin- like granules supports the hypothesis of' their nucleolar origin. Finally, the action of actinomycin D prior to treatment with a- amanitin prevented the formation of peri- chromatin-like granules. This is another in-

NUCLEOLAR PERICHROMATIN-LIKE GRANULES 219

dication of their probable origin since tran- ?cription of the nucleolar RNA is the pri- mary target of actinomycin D at low con- centration. ~. On the basis of our morphological obser-

vations and the analogy with the situation ih the normal nucleolus (5), we present as a hypothesis the following sequence for the origin of the perichromatin-like granules in a-amanitin-treated cells: nucleolar fibrils ---) type 1 nucleolar granules --~ type 2 nu- cleolar granules--, perichromatin-like gran- ~les.

CONCLUSIONS

Our observations of rat hepatocytes treated with a-amanitin suggest that peri- chromatin-like granules contain nucleolar material, Here, as in the other known cases of perinucleolar accumulations of perichro- matin granules, a specific pathologic con- ~dition is necessary for their appearance, 0amely, an interruption of nucleolar RNA maturation.

The authors thank Dr. W: Bernhard for the interest he took in their work. They are grateful to Professor E, H. Leduc for correcting the English manuscript. M. D. was recipient of an EMBO fellowship.

REFERENCES

I. BERNHARD, W. (1969) J. Ultrastruct. Res. 27, 250-265.

2. FIUME, L., BUSI, C., CAMPADELLI-FIUME, G., AND FRANCESCHI, C. (1975) Experientia 31, 1233-1234.

3. FIUME, L., AND LASCHI, R. (1965) Sperimentale 115, 288-297.

4. GAJKOWSKA, B., PuvION, E., AND BERNHARD, W. (1977) J. Ultrastruct. Res. 60, 335-347.

5. GRANBOULAN, N,, AND GRANBOULAN, P. (1965) Exp. Cell Res. 38, 604-619.

6. LINDEL, T. J., WEINBERG, F., MORRIS, P. W., ROEDER, R. G., AND RUTTER, W. J. (1970) Sci- ence 170, 447-448.

7. MARINOZZI, V., AND FIUME, L. (1971) Exp. Cell Res. 67, 311-322.

8. MEYER-SCHULTZ, F., AND PORTE, A. (1971) Cy- tobiologie 3, 387-400.

9. MONNERON, A., AND BERNHARD, W. (1969) J. Ultrastruct. Res. 27, 266-288.

10. MOYNE, G., NASH, R. E., AND PUVION, E. (1977) Biol. Cell. 30, 5-16.

11. PERRY, R. P. (1962) Exp. Cell Res. 29, 400-406. 12. PETROV, P,, AND SEKERIS, C. E. (1971) Exp. Cell

Res. 69, 393-401. 13. PUVION, E., GARRIDO, J., AND VIRON, A. (1974) C.

R, Acad. Sci. Ser. D 279, 509-512. 14. PuvIoN, E., MOYNE, G., AND BERNHARD, W.

(1976) J. Microsc. Biol. Cell. 25, 17-32. 15. PUVION, E., VIRON, A., AND BERNHARD, W. (1977)

Biol. Cell. 29, 81-88. 16. TATA, J. R., HAMILTON, M. J., AND SHIELD, D.

(1972) Nature (London) 238, 161-164.