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The fine structure of a nucleolar constituent
© 1969 by Academic Press, Inc. J. ULTRASTRUCTLIRE RESEARCH
29, 1-14 (1969)
T h e Fine S t r u c t u r e of o N u c l e o l o r C o n s t i t u e n t 1 L. RECHER, J. WHITESCARVER,AND L. BRIGGS Southern California Cancer Center, Research Division, California Hospital Medical Center, Los Angeles 90015 Received January 27, 1969, and in revised form April 17, 1969 The fine structure and macromolecular composition of a nucleolar constituent has been studied. For convenience of communication the term "fibrillar center" has been used to designate this constituent. Fibrillar centers are rounded structures that are associated with the fibrillar nucleolonema. They are composed primarily of pepsin digestible proteins and contain fine fibrils of 50 A that resist pepsin and ribonuclease digestion. Individual fibrils of the fibrillar centers and the fibrillar nucleolonema under certain circumstances stain intensely with lead citrate. The substance reacting with lead has not been identified. It is likely hat the lead-positive material of the centers differs from that of the associated fibrillar nucleolonema because of slight differences in resistance of the material of the two nucleolar zones to pepsin and ribonuclease digestion. Electron microscopic analysis of vertebrate cell nucleoli has shown that the nucleolar organization is complex. At least four principal components have been differentiated: the fibrillar and granular components, the nucleolar chromatin, and the amorphous matrix (1). In most nucleoli, the fibrillar and granular components form a filamentous network for which the term "nucleolonema" has been adopted (1, 4, 5). In the meshes of the nucleolonema, inclusions of condensed chromatin can be present that on occasions are seen to coalesce with the nucleolus-associated chromatin located on the nucleolar surface (6). Frequently, a fibrillar substance of loose texture is observed in the meshes of the nucleolonema that invariably is associated with the fibrillar component. Schoefl (15) referred to this substance as an amorphous material and noted that it stained less intensely after pepsin digestion and could no longer be distinguished after digestion with pepsin followed by ribonuclease. Terzakis (21) considered it a fibrillar substance with a moderately dense amorphous matrix, but differentiated it from the fibrillar nucleolonema that is associated with a dense amorphous matrix. In this laboratory, ultrastructural studies that included the use of various enzyme digestion procedures were carried out on human tissue culture cells. Particular atten1 This research was supported in part by the Albert Soiland Cancer Foundation. 1 -- 691837 J. Ultrastrueture.Research
2
RECHER~ WHITESCARVER,AND BRIGGS
tion was paid to this fibrillar substance and its association with the fibrillar nucleolonema. The results are summarized in this report.
MATERIALS A N D METHODS
Tissue culture. The rapidly growing CMP cell line (obtained from Dr. D. Rounds, Pasadena Foundation for Medical Research, Pasadena, California) has been used in most initial studies. The cell line derives from a human adenocarcinoma and has been propagated in our laboratory on Eagle's Minimum Essential Medium (MEM), supplemented with 20 % calf serum or 30 % fetal calf serum. In more recent studies, the cell line No. 180, derived from a metastatic carcinoma of the cervix, was employed. This cell line was established in our laboratory and is being propagated on Eagle's MEM supplemented with 30 % fetal calf serum. All cultures were grown in T-flasks. Electron microscopy. The cells were detached with a rubber policeman from the glass surface of the T-flasks, pelleted at 1000 g, fixed with cacodylate-buffered 2 % glutaraldehyde for 30 min and then washed twice with Millonig's buffer for 10 rain. Some cell pellets were immediately dehydrated, while others were posffixed with 1% osmium tetroxide in Millonig's buffer and then dehydrated in 70 % and 100 % ethyl alcohol. All specimens were embedded in Epon, by the method of Luft (10). For enzyme digestion studies, the cells were fixed with cacodylate buffered 2 % glutaraldehyde for 15-20 rain, washed twice with 0.01 M tris(hydroxymethyl)aminomethane (Tris), then digested for 1 or 2 hours at 37 ° with the following enzymes: ribonuclease (lyophilized, phosphate free, Worthington) or ribonuclease (5 x crystallized, Calbiochem) dissolved in 0.01 M Tris (1 mg/ml); pepsin (2 x crystallized, Worthington) dissolved in 0.1 N HC1 (0.1 mg/ml or 1 mg/ml); trypsin (1-300, Nutritional Biochemicals Corp.) dissolved in phosphatebuffered saline (2.5 mg/ml). After digestions, the cells were washed with 0.01 M Tris, pelleted in Millonig's buffer, postfixed with 1% osmium tetroxide in Millonig's buffer, and routinely processed. In some cases ribonuclease digestion was followed by incubation in 5 % trichloroacetic acid (TCA) for 10 rain at 4 °. Cell samples were incubated in 0.01 M T r i s or 0.1 N HC1 to serve as controls for ribonuclease and pepsin digestion. In a few instances, cells were suspended in the hypotonic buffer RSB (0.01 M NaC1, 0.01 M Tris, 0.0015 M MgCI~, pH 7.4) for 5-10 rain prior to processing for electron microscopy (11). Sections were cut on an LKB Ultrotome with a diamond knife at a thickness of 500-800 ~ . They were collected on Formvar-coated copper screens, stained for 5 rain with a saturated solution of uranyl acetate in 50 % ethanol, washed with distilled water, and then stained with lead citrate for 3 rain (13). If only lead citrate was used, the staining time was 5 rain. The sections were thinly coated with carbon and examined in a Siemens Elmiskop IA. FIG. 1. Nucleolus of No. 180 cell showing multiple fibrillar centers (FC) that are walled off by the dense fibrillar nucleolonema (FN). Fixation: glutaraldehyde. Stain: uranyl acetate and lead citrate. x 40,000. FIG. 2. Nucleolus of No. 180 cells showing one fibrillar center (FC) located at the periphery. Fixation: glutaraldehyde. Stain: uranyl acetate and lead citrate, x 60,000. Fla. 3. Nucleolus of CMP cell showing a vacuolar structure (V) that appears to have developed from a fibrillar center. Fixation: glutaraldehyde. Stain: uranyl acetate and lead citrate, x 40,000.
RECHER, WHITESCARVER,AND BRIGGS
RESULTS
General morphology The term "fibrillar center" has been adopted to designate the loose fibrillar substance associated with the fibrillar component of the nucleolonema (fibrillar nucleolonema). The term was chosen because of the central location of the substance within the fibrillar nucleolonema and because of its fibrillar fine structure. Fibrillar centers are consistent nucleolar components of cells of the CMP and the No. 180 cell lines. They are rounded structures of 300-900 m/z diameter that in all instances are associated with the fibrillar nucleolonema. The centers located within the nucleolar body are usually walled off by the fibrillar nucleolonema (Fig. 1). Those located at the periphery show association with the nucleolonema where they border on nucleolar substance (Fig. 2). Occasionally, fibrillar centers are found that have lost contact with the nucleolonema. Such separation ultimately seems to result in the formation of nucleolar vacuoles (Fig. 3). In general, the fibrillar centers have a low electron density that contrasts with the high electron density of the associated fibrillar nucleolonema. The difference in density of the two nucleolar zones is perhaps less evident in cells fixed with glutaraldehyde (Figs. 1 and 2) than it is in cells fixed with glutaraldehyde and osmic acid (Fig. 4). The fibrils of the centers and the associated nucleolonema may under certain circumstances appear extremely electron dense (Fig. 5). In Fig. 6, it can be seen that lead citrate staining is responsible for this effect. The intense staining of these fibrils is never observed in nucleoli of cells fixed with glutaraldehyde (Figs. 1 and 2). Postfixation with osmic acid is required. It can also be observed in cells fixed with osmic acid alone. The staining intensity by lead citrate varies among individual centers even though they may be part of the same nucleolus. Variations of staining intensity are also observed in the associated fibrillar nucleolonema. In most instances, a strongly lead-positive center is associated with a strongly lead-positive nucleolonema. In a series of experiments, tissue culture cells were briefly exposed to the hypotonic buffer RSB prior to processing for electron microscopy. Treatment of cells with RSB .causes shrinkage of the fibrillar centers and enhances their intense staining by lead citrate (Fig. 7). The centers partially separate from the associated nucleolonema, but few bridges remain that demonstrate the continuity of the two nucleolar zones. The centers often are Y-shaped and appear to be an integral part of the nucleolonema. Fro. 4. Nucleolus of CMP cell showing three fibrillar centers (FC)associated with extremely electron dense fibrillar nucleolonema. Fixation: glutaraldehyde and osmium tetroxide. Stain: uranyl acetate and lead citrate, x 40,000. Fro. 5. Nucleolus of CMP cell with two peripheral fibrillar centers (FC). Note the intense staining of individual fibrils of the centers and the associated fibrillar nucleolonema (FN). Fixation: glutaraldehyde and osmium tetroxide. Stain: uranyl acetate and lead citrate, x 60,000.
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RECHER, WHITESCARVER, AND BRIGGS
The texture of the lead positive material differs in the two nucleolar zones, the centers having a coarser texture than the fibrillar nucleolonema. The fibrillar centers and the fibrillar nucleolonema are not the only structures that show intense staining with lead citrate. A few strongly lead-positive granules are scattered throughout the granular portions of nucleoli. Interchromatin granules stain heavily with lead citrate. Extra nucleolar fibrillar bodies also have the same staining characteristic.
Enzyme digestion studies Ribonuclease. Ribonuclease digestion results in a loss of electron density of the nucleoli (Fig. 8). The fibrillar and granular components can still be differentiated, but lose their distinct appearance. The nucleolar chromatin has a higher electron density than other nucleolar components and is therefore more prominent. Ribonuclease digestion does not visibly change the fibrillar centers. It also does not seem to abolish the lead reaction of the centers. A weak lead reaction is occasionally observed in the associated fibrillar nucleolonema, but this is not a consistent finding. Pepsin. Pepsin appears to remove gradually the proteinaceous matrix of nucleoli, ultimately leaving behind a fibrillar framework. It clearly exposes the basic structure of the fibrillar centers (Fig. 9). The centers are composed of fine fibrils that average 50 ~ in thickness. These fibrils are interlaced and are not limited to the centers, but can often be traced deep into the fibrillar nucleolonema. They appear to be resistant to pepsin digestion even if the digestion time is 2 hours or longer and the concentration of the enzyme 1 mg/ml 0.1 N HC1 (Fig. 10). Prolonged pepsin digestion appears to eliminate the lead stainability of the fibrillar centers and nearly eliminate that of the fibrillar nucleolonema. Ribonuclease and pepsin. The fibrils of the fibrillar centers appear to resist double digestion by ribonuclease and pepsin, although they may be reduced in numbers or appear fragmented (Figs. 11 and 12). No lead-positive material can be detected following double digestion. Trypsin. Trypsin is not as effective as pepsin in removing the proteins of cells, but some proteolytic effect can be noted in the cytoplasm and the nucleus. The nucleoli appear generally pale; that apparently is due to some loss of proteinaceous matrix.
FIG. 6. Nucleolus of CMP cell with two fibrillar centers (FC). Note the intense staining of the fibrils of the centers and the fibrillar nucleolonema (FN) by lead citrate. Fixation: glutaraldehyde and osmium tetroxide. Stain: lead citrate (5 rain), x 80,000. FIG. 7. Nucleolus of CMP cell treated with RSB prior to processing for electron microscopy. The fibrillar centers (FC) appear shrunken and contain a strongly lead-positive coarse material. The associated fibrillar nucleolonema (FN) contains lead-positive material of a fine texture. Fixation: glutaraldehyde and osmium tetroxide. Stain: lead citrate, x 60,000.
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RECHER, WHITESCARVER,AND BRIGGS
The lead stainability of the fibrillar centers a n d the fibrillar n u c l e o l o n e m a is not abolished by trypsin digestion (Fig. 13).
DISCUSSION Fibrillar centers are distinct nucleolar constituents. Considering their fine structure and composition, one feels justified to treat t h e m as a separate entity rather t h a n to include them i n one of the other n u c l e o l a r components. A l t h o u g h m a n y authors
(1, 3, 5, 7, 8, 14, 15, 18, 21, 22, 23)
have referred to struc-
tures resembling fibrillar centers u n d e r a variety of different names, relatively little is k n o w n a b o u t their nature. D u t t a et al. (3) described intranucleolar bodies ("nucleololi") in light a n d electron microscopic preparations of nerve cells of the m o n k e y cerebellar cortex. These nucleololi appear to be identical to the fibrillar centers described in this report. Cytochemical tests indicated that nucleo!oli c o n t a i n proteins a n d b o u n d lipids b u t n o D N A or R N A . The authors (3), therefore, suggested that these structures m a y be concerned with p r o t e i n synthesis. Schoefl (15), o n the basis of enzyme digestion studies, concluded that fibrillar centers are composed largely of proteins, b u t c o n t a i n also R N A . Y a s u z u m i a n d Sugihara (23) observed almost complete extraction of the filamentous elements of fibrillar centers (referred to as "clear zones" by these authors) after ribonuclease digestion. O u r enzyme digestion studies have shown that the fibrillar centers are primarily composed of pepsin digestible proteins. A fine fibrillar framework remains following
FIG. 8. Nucleolus of No. 180 cell that has been digested with ribonuclease (1 mg/ml) for 2 hours at 37°. The lead-positive material is still visible within the fibrillar center (FC). A few specks of leadpositive material are also seen in the associated nucleolonema (arrows). Fixation: glutaraldehyde prior to digestion, osmium tetroxide after digestion. Stain: uranyl acetate and lead citrate, x 60,000. FIG. 9. Nucleolus of No. 180 cell that has been digested with pepsin (0.1 mg/ml) for 1 hour at 37°. The fibrils of the fibrillar centers (FC) evidently resisted digestion. They average 50 ~ in thickness and appear to be continuous with fibrils of the associated nucleolonema. A few specks of lead-positive material are observed (arrows). Fixation: glutaraldehyde prior to digestion, osmium tetroxide after digestion. Stain: uranyl acetate and lead citrate, x 60,000. FIG. 10. Nucleolus of No. 180 cell that has been digested with pepsin (1 mg/ml) for 2 hours at 37°. The fibrils of the fibrillar centers (FC) are still recognized, although they may appear somewhat fragmented. Note the absence of lead-positive material. Fixation: glutaraldehyde prior to digestion, osmium tetroxide following digestion. Stain: uranyl acetate and lead citrate, x 60,000. FIG. 11. Nucleolus of No. 180 cell that has been digested with ribOnuclease (1 mg/ml) and pepsin (1 mg/ml) for 1 hour each at 37°. Although most of the nucleolar substance is removed, a fibrillar center (FC) with fragmented fibrils can be recognized. Fixation: glutaraldehyde prior to digestions, osmium tetroxide after digestions. Stain: uranyl acetate and lead citrate, x 30,000. FIG. 12. Same as Fig. 11 showing the fibrillar center at a higher magnification. The fibrils of the center resisted ribonuclease and pepsin digestion, x 80,000. FIG. 13. Nucleolus of No. 180 cell that has been digested with trypsin (2.5 mg/ml) for 1 hour at 37°. The lead-positive material resisted digestion. Note its distribution over almost the entire fibrillar nucleolonema. Fixation: glutaraldehyde prior to digestion, osmium tetroxide following digestion. Stain: uranyl acetate and lead citrate, x 40,000.
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12
RECHER, WHITESCARVER, AND BRIGGS
pepsin digestion that shows continuity with a fibrillar network of the associated nucleolonema. This fibrillar framework appears to resist not only pepsin digestion, but also ribonuclease digestion. The question therefore is being raised whether these fibrils are composed of DNA. An attempt was made to digest cells with deoxyribonuclease but technical difficulties were encountered. Deoxyribonuclease was found to have no effect on glutaraldehyde-fixed cells, even if deoxyribonuclease digestion was preceded by pepsin digestion. The formalin fixative used by Swift (18)was tried, but the preservation of the nuclei was not good enough for proper evaluation of details. Further attempts are being made to develop a workable deoxyribonuclease digestion method. Incorporation of thymidine-~H with subsequent autoradiography will also be attempted. It is not believed that a negative Feulgen reaction (3) excludes the possibility of D N A being present in fibrillar centers, because the amount is likely to be too small to be detectable in the light microscope. The possibility that the fibrils of fibrillar centers contain RNA has to be considered. The RNA might be hybridized to D N A and thus be inaccessible for ribonuclease. Our findings, however, differ from those of Schoefl (15) and Yasuzumi and Sugihara (23) in that ribonuclease had no visible effect on fibrillar centers. The intense staining by lead citrate appears to be a characteristic feature of the fibritlar centers and the fibrillar nucleolonema. Little is known about the nature of this lead reaction. The lead-positive substance is found primarily along the fibrils of the two nucleolar zones. It is therefore possible that the component which reacts with lead is bound to or forms part of the macromolecules that compose these fibrils. It was hoped that enzyme digestion studies would point to a macromolecular species as being specifically involved in the lead reaction, but the digestion results are not conclusive. Pepsin appears to be most effective in abolishing the lead staining reaction, but whether this is due to the removal of a specific protein that can bind lead is uncertain. It is possible that peptic digestion masks the lead reaction or solubilizes and removes other nonproteinacious substances that may contain the lead positive material. Trypsin apparently has no effect on the lead stainability of either the fibrillar centers or the fibrillar nucleolonema. A weak lead reaction was occasionally observed in the fibrillar nucleolonema following ribonuclease digestion, but the finding was not consistent enough to allow any conclusions. Studinski and Love (16) demonstrated certain nucleolar constituents by lead precipitation in unfixed cultured cells. They referred to these constituents as nucleolini and described them as granules and hollow spherules. These nucleolini, the hollow spherules in particular, appear to have some resemblance to the fibrillar centers described in this report. It is of interest that both these structures can be demonstrated by lead precipitation. Studinski and Love (16) speculated that phosphates might be involved in the reaction. Tandler (19) described a similar lead precipitation reaction
NUCLEOLAR FINE STRUCTURES
13
to demonstrate nucleoli of unfixed cells. He identified the nucleolar precipitate as lead orthophosphate (20). The lead precipitation described by these authors (16, 19) succeeded only when unfixed cells were exposed to lead acetete. Any type of fixation abolished the reaction. The lead reaction described in this report occurs under somewhat different conditions. The cells are fixed with glutaraldehyde and osmic acid, dehydrated with ethyl alcohol, embedded in Epon and then exposed to lead citrate. But this difference may be more apparent than real, in principle the reaction may be the same. Phosphates may also be involved. The lead reaction in order to occur requires fixation with osmic acid. What role osmic acid could possibly play is unknown at the present time. It is likely that other conditions also may determine the outcome of the reaction because the lead precipitation did not always occur when it was expected. Further investigation is needed before the nature and significance of this reaction can be appreciated. Sankaranarayanan and Busch (14) described dense granules in nucleoli of Walker 256 carcinosarcoma cells. The granules were embedded in a fibrillar matrix in the "light regions" or "pars amorpha." It is likely that they are identical to the leadpositive material of the fibrillar centers described in this report because of their location. The authors (14) did use osmium tetroxide as a fixative and stained their sections with lead hydroxide. Nucleolar granules of high electron density have also been observed in plant cells by Lafontaine (9), Porter (12), and Sun (17) and in Amoeba proteus by Cohen (2). It is possible that these granules and the lead-positive material are of identical nature and are a regular component of nucleoli. The close structural relationship of the fibrillar centers and the fibrillar nudeolonema and the high affinity to lead suggests a close functional relationship of the two nucleolar zones. The fibrillar centers may be transitory structures that are being developed for a certain function and then regress. Variations of lead stainability may reflect different states of functional activity. Vacuoles as illustrated in Fig. 3 may be burned-out centers. REFERENCES
1 . BERNHARD,W. and GRANBOULAN,N., in DALTON, A. J. and HAGUENAU,F. (Eds.), Ultrastructure in Biological Systems, Vol. 3: The Nucleus, p. 81. Academic Press, New York, 1968. 2. COHEN,A. J., J. Biophys. Biochem. Cytol. 3, 859 (1957). 3. DUTTA, C. R., SIEGESMUND, K. A. and Fox, C. A., J. Ultrastruct. Res. 8, 542 (1963). 4. ESTABLE,C., SWIFT,H., BERNHARD,W., GALL,J., PERRY,R. and S1RLIN,J., Natl. Cancer Inst. Monograph 23, 573 (1966).
14
RECHER, WHITESCARVER~AND BRIGGS
5. FAWCETT, D., The Cell. An Atlas of Fine Structure, p. 26. Saunders, Philadelphia, Pennsylvania, 1966. 6. GRAN~OULAN,N. and GRANBOULAN,P., Exptl. Cell Res. 34, 71 (1964). 7. HEINE, U., LANGLO~S,A. J. and BEARD, J. W., Cancer Res. 26, 1847 (1966). 8. JEZEQUEL,A. M. and BERNHARD,W., J. Microscopie 3, 279 (1964). 9. LAEONTA~NE,J. G., J. Biophys. Biochem. CytoI. 4, 229 (1958). 10. LUF~C,J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). 11. PENMAN,S., SMITH,I., HOLTZMAN,E. and GREE~ERG, H., Natl. Cancer Inst. Monograph 23, 489 (1966). 12. PORTER,K. R., Proc. 4th Intern. Congr. Electron Mikroscopy, Berlin, 1958 Vol. 2, p. 186. Springer, Berlin, 1960. 13. REYNOLDS,E. W., J. Cell Biol. 17, 208 (1963). 14. SANKARANARAYANAN,K. and BuscH, H., Exptl. Cell Res. 38, 434 (1965). 15. SCHOEFL,G. I., J. Ultrastruct. Res. 10, 224 (1964). 16. STUDINS~:~,G. P. and LovE, R., Stain Technol. 39, 397 (1964). 17. SUN, C. N., ExptL Cell Res. 25, 213 (1961). 18. SwiFt, H., Exptl. Cell Biol., Suppl. 9, 54 (1963). 19. TANDLER,C. J., J. Histochem. Cytochem. 4, 331 (1956). 20. - - - - ibid. 5, 489 (1957). 21. TERZArdS, J. A., J. Cell Biol. 27, 293 (1965). 22. TOKUYASU,K., MADDEN, S. C. and ZELD~S,L. J., J. Cell Biol. 39, 630 (1968). 23. YASUZUMI,G. and SUGIHARA,R., Exptl. Cell Res. 37, 207 (1965).
29, 1-14 (1969)
T h e Fine S t r u c t u r e of o N u c l e o l o r C o n s t i t u e n t 1 L. RECHER, J. WHITESCARVER,AND L. BRIGGS Southern California Cancer Center, Research Division, California Hospital Medical Center, Los Angeles 90015 Received January 27, 1969, and in revised form April 17, 1969 The fine structure and macromolecular composition of a nucleolar constituent has been studied. For convenience of communication the term "fibrillar center" has been used to designate this constituent. Fibrillar centers are rounded structures that are associated with the fibrillar nucleolonema. They are composed primarily of pepsin digestible proteins and contain fine fibrils of 50 A that resist pepsin and ribonuclease digestion. Individual fibrils of the fibrillar centers and the fibrillar nucleolonema under certain circumstances stain intensely with lead citrate. The substance reacting with lead has not been identified. It is likely hat the lead-positive material of the centers differs from that of the associated fibrillar nucleolonema because of slight differences in resistance of the material of the two nucleolar zones to pepsin and ribonuclease digestion. Electron microscopic analysis of vertebrate cell nucleoli has shown that the nucleolar organization is complex. At least four principal components have been differentiated: the fibrillar and granular components, the nucleolar chromatin, and the amorphous matrix (1). In most nucleoli, the fibrillar and granular components form a filamentous network for which the term "nucleolonema" has been adopted (1, 4, 5). In the meshes of the nucleolonema, inclusions of condensed chromatin can be present that on occasions are seen to coalesce with the nucleolus-associated chromatin located on the nucleolar surface (6). Frequently, a fibrillar substance of loose texture is observed in the meshes of the nucleolonema that invariably is associated with the fibrillar component. Schoefl (15) referred to this substance as an amorphous material and noted that it stained less intensely after pepsin digestion and could no longer be distinguished after digestion with pepsin followed by ribonuclease. Terzakis (21) considered it a fibrillar substance with a moderately dense amorphous matrix, but differentiated it from the fibrillar nucleolonema that is associated with a dense amorphous matrix. In this laboratory, ultrastructural studies that included the use of various enzyme digestion procedures were carried out on human tissue culture cells. Particular atten1 This research was supported in part by the Albert Soiland Cancer Foundation. 1 -- 691837 J. Ultrastrueture.Research
2
RECHER~ WHITESCARVER,AND BRIGGS
tion was paid to this fibrillar substance and its association with the fibrillar nucleolonema. The results are summarized in this report.
MATERIALS A N D METHODS
Tissue culture. The rapidly growing CMP cell line (obtained from Dr. D. Rounds, Pasadena Foundation for Medical Research, Pasadena, California) has been used in most initial studies. The cell line derives from a human adenocarcinoma and has been propagated in our laboratory on Eagle's Minimum Essential Medium (MEM), supplemented with 20 % calf serum or 30 % fetal calf serum. In more recent studies, the cell line No. 180, derived from a metastatic carcinoma of the cervix, was employed. This cell line was established in our laboratory and is being propagated on Eagle's MEM supplemented with 30 % fetal calf serum. All cultures were grown in T-flasks. Electron microscopy. The cells were detached with a rubber policeman from the glass surface of the T-flasks, pelleted at 1000 g, fixed with cacodylate-buffered 2 % glutaraldehyde for 30 min and then washed twice with Millonig's buffer for 10 rain. Some cell pellets were immediately dehydrated, while others were posffixed with 1% osmium tetroxide in Millonig's buffer and then dehydrated in 70 % and 100 % ethyl alcohol. All specimens were embedded in Epon, by the method of Luft (10). For enzyme digestion studies, the cells were fixed with cacodylate buffered 2 % glutaraldehyde for 15-20 rain, washed twice with 0.01 M tris(hydroxymethyl)aminomethane (Tris), then digested for 1 or 2 hours at 37 ° with the following enzymes: ribonuclease (lyophilized, phosphate free, Worthington) or ribonuclease (5 x crystallized, Calbiochem) dissolved in 0.01 M Tris (1 mg/ml); pepsin (2 x crystallized, Worthington) dissolved in 0.1 N HC1 (0.1 mg/ml or 1 mg/ml); trypsin (1-300, Nutritional Biochemicals Corp.) dissolved in phosphatebuffered saline (2.5 mg/ml). After digestions, the cells were washed with 0.01 M Tris, pelleted in Millonig's buffer, postfixed with 1% osmium tetroxide in Millonig's buffer, and routinely processed. In some cases ribonuclease digestion was followed by incubation in 5 % trichloroacetic acid (TCA) for 10 rain at 4 °. Cell samples were incubated in 0.01 M T r i s or 0.1 N HC1 to serve as controls for ribonuclease and pepsin digestion. In a few instances, cells were suspended in the hypotonic buffer RSB (0.01 M NaC1, 0.01 M Tris, 0.0015 M MgCI~, pH 7.4) for 5-10 rain prior to processing for electron microscopy (11). Sections were cut on an LKB Ultrotome with a diamond knife at a thickness of 500-800 ~ . They were collected on Formvar-coated copper screens, stained for 5 rain with a saturated solution of uranyl acetate in 50 % ethanol, washed with distilled water, and then stained with lead citrate for 3 rain (13). If only lead citrate was used, the staining time was 5 rain. The sections were thinly coated with carbon and examined in a Siemens Elmiskop IA. FIG. 1. Nucleolus of No. 180 cell showing multiple fibrillar centers (FC) that are walled off by the dense fibrillar nucleolonema (FN). Fixation: glutaraldehyde. Stain: uranyl acetate and lead citrate. x 40,000. FIG. 2. Nucleolus of No. 180 cells showing one fibrillar center (FC) located at the periphery. Fixation: glutaraldehyde. Stain: uranyl acetate and lead citrate, x 60,000. Fla. 3. Nucleolus of CMP cell showing a vacuolar structure (V) that appears to have developed from a fibrillar center. Fixation: glutaraldehyde. Stain: uranyl acetate and lead citrate, x 40,000.
RECHER, WHITESCARVER,AND BRIGGS
RESULTS
General morphology The term "fibrillar center" has been adopted to designate the loose fibrillar substance associated with the fibrillar component of the nucleolonema (fibrillar nucleolonema). The term was chosen because of the central location of the substance within the fibrillar nucleolonema and because of its fibrillar fine structure. Fibrillar centers are consistent nucleolar components of cells of the CMP and the No. 180 cell lines. They are rounded structures of 300-900 m/z diameter that in all instances are associated with the fibrillar nucleolonema. The centers located within the nucleolar body are usually walled off by the fibrillar nucleolonema (Fig. 1). Those located at the periphery show association with the nucleolonema where they border on nucleolar substance (Fig. 2). Occasionally, fibrillar centers are found that have lost contact with the nucleolonema. Such separation ultimately seems to result in the formation of nucleolar vacuoles (Fig. 3). In general, the fibrillar centers have a low electron density that contrasts with the high electron density of the associated fibrillar nucleolonema. The difference in density of the two nucleolar zones is perhaps less evident in cells fixed with glutaraldehyde (Figs. 1 and 2) than it is in cells fixed with glutaraldehyde and osmic acid (Fig. 4). The fibrils of the centers and the associated nucleolonema may under certain circumstances appear extremely electron dense (Fig. 5). In Fig. 6, it can be seen that lead citrate staining is responsible for this effect. The intense staining of these fibrils is never observed in nucleoli of cells fixed with glutaraldehyde (Figs. 1 and 2). Postfixation with osmic acid is required. It can also be observed in cells fixed with osmic acid alone. The staining intensity by lead citrate varies among individual centers even though they may be part of the same nucleolus. Variations of staining intensity are also observed in the associated fibrillar nucleolonema. In most instances, a strongly lead-positive center is associated with a strongly lead-positive nucleolonema. In a series of experiments, tissue culture cells were briefly exposed to the hypotonic buffer RSB prior to processing for electron microscopy. Treatment of cells with RSB .causes shrinkage of the fibrillar centers and enhances their intense staining by lead citrate (Fig. 7). The centers partially separate from the associated nucleolonema, but few bridges remain that demonstrate the continuity of the two nucleolar zones. The centers often are Y-shaped and appear to be an integral part of the nucleolonema. Fro. 4. Nucleolus of CMP cell showing three fibrillar centers (FC)associated with extremely electron dense fibrillar nucleolonema. Fixation: glutaraldehyde and osmium tetroxide. Stain: uranyl acetate and lead citrate, x 40,000. Fro. 5. Nucleolus of CMP cell with two peripheral fibrillar centers (FC). Note the intense staining of individual fibrils of the centers and the associated fibrillar nucleolonema (FN). Fixation: glutaraldehyde and osmium tetroxide. Stain: uranyl acetate and lead citrate, x 60,000.
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RECHER, WHITESCARVER, AND BRIGGS
The texture of the lead positive material differs in the two nucleolar zones, the centers having a coarser texture than the fibrillar nucleolonema. The fibrillar centers and the fibrillar nucleolonema are not the only structures that show intense staining with lead citrate. A few strongly lead-positive granules are scattered throughout the granular portions of nucleoli. Interchromatin granules stain heavily with lead citrate. Extra nucleolar fibrillar bodies also have the same staining characteristic.
Enzyme digestion studies Ribonuclease. Ribonuclease digestion results in a loss of electron density of the nucleoli (Fig. 8). The fibrillar and granular components can still be differentiated, but lose their distinct appearance. The nucleolar chromatin has a higher electron density than other nucleolar components and is therefore more prominent. Ribonuclease digestion does not visibly change the fibrillar centers. It also does not seem to abolish the lead reaction of the centers. A weak lead reaction is occasionally observed in the associated fibrillar nucleolonema, but this is not a consistent finding. Pepsin. Pepsin appears to remove gradually the proteinaceous matrix of nucleoli, ultimately leaving behind a fibrillar framework. It clearly exposes the basic structure of the fibrillar centers (Fig. 9). The centers are composed of fine fibrils that average 50 ~ in thickness. These fibrils are interlaced and are not limited to the centers, but can often be traced deep into the fibrillar nucleolonema. They appear to be resistant to pepsin digestion even if the digestion time is 2 hours or longer and the concentration of the enzyme 1 mg/ml 0.1 N HC1 (Fig. 10). Prolonged pepsin digestion appears to eliminate the lead stainability of the fibrillar centers and nearly eliminate that of the fibrillar nucleolonema. Ribonuclease and pepsin. The fibrils of the fibrillar centers appear to resist double digestion by ribonuclease and pepsin, although they may be reduced in numbers or appear fragmented (Figs. 11 and 12). No lead-positive material can be detected following double digestion. Trypsin. Trypsin is not as effective as pepsin in removing the proteins of cells, but some proteolytic effect can be noted in the cytoplasm and the nucleus. The nucleoli appear generally pale; that apparently is due to some loss of proteinaceous matrix.
FIG. 6. Nucleolus of CMP cell with two fibrillar centers (FC). Note the intense staining of the fibrils of the centers and the fibrillar nucleolonema (FN) by lead citrate. Fixation: glutaraldehyde and osmium tetroxide. Stain: lead citrate (5 rain), x 80,000. FIG. 7. Nucleolus of CMP cell treated with RSB prior to processing for electron microscopy. The fibrillar centers (FC) appear shrunken and contain a strongly lead-positive coarse material. The associated fibrillar nucleolonema (FN) contains lead-positive material of a fine texture. Fixation: glutaraldehyde and osmium tetroxide. Stain: lead citrate, x 60,000.
8
RECHER, WHITESCARVER,AND BRIGGS
The lead stainability of the fibrillar centers a n d the fibrillar n u c l e o l o n e m a is not abolished by trypsin digestion (Fig. 13).
DISCUSSION Fibrillar centers are distinct nucleolar constituents. Considering their fine structure and composition, one feels justified to treat t h e m as a separate entity rather t h a n to include them i n one of the other n u c l e o l a r components. A l t h o u g h m a n y authors
(1, 3, 5, 7, 8, 14, 15, 18, 21, 22, 23)
have referred to struc-
tures resembling fibrillar centers u n d e r a variety of different names, relatively little is k n o w n a b o u t their nature. D u t t a et al. (3) described intranucleolar bodies ("nucleololi") in light a n d electron microscopic preparations of nerve cells of the m o n k e y cerebellar cortex. These nucleololi appear to be identical to the fibrillar centers described in this report. Cytochemical tests indicated that nucleo!oli c o n t a i n proteins a n d b o u n d lipids b u t n o D N A or R N A . The authors (3), therefore, suggested that these structures m a y be concerned with p r o t e i n synthesis. Schoefl (15), o n the basis of enzyme digestion studies, concluded that fibrillar centers are composed largely of proteins, b u t c o n t a i n also R N A . Y a s u z u m i a n d Sugihara (23) observed almost complete extraction of the filamentous elements of fibrillar centers (referred to as "clear zones" by these authors) after ribonuclease digestion. O u r enzyme digestion studies have shown that the fibrillar centers are primarily composed of pepsin digestible proteins. A fine fibrillar framework remains following
FIG. 8. Nucleolus of No. 180 cell that has been digested with ribonuclease (1 mg/ml) for 2 hours at 37°. The lead-positive material is still visible within the fibrillar center (FC). A few specks of leadpositive material are also seen in the associated nucleolonema (arrows). Fixation: glutaraldehyde prior to digestion, osmium tetroxide after digestion. Stain: uranyl acetate and lead citrate, x 60,000. FIG. 9. Nucleolus of No. 180 cell that has been digested with pepsin (0.1 mg/ml) for 1 hour at 37°. The fibrils of the fibrillar centers (FC) evidently resisted digestion. They average 50 ~ in thickness and appear to be continuous with fibrils of the associated nucleolonema. A few specks of lead-positive material are observed (arrows). Fixation: glutaraldehyde prior to digestion, osmium tetroxide after digestion. Stain: uranyl acetate and lead citrate, x 60,000. FIG. 10. Nucleolus of No. 180 cell that has been digested with pepsin (1 mg/ml) for 2 hours at 37°. The fibrils of the fibrillar centers (FC) are still recognized, although they may appear somewhat fragmented. Note the absence of lead-positive material. Fixation: glutaraldehyde prior to digestion, osmium tetroxide following digestion. Stain: uranyl acetate and lead citrate, x 60,000. FIG. 11. Nucleolus of No. 180 cell that has been digested with ribOnuclease (1 mg/ml) and pepsin (1 mg/ml) for 1 hour each at 37°. Although most of the nucleolar substance is removed, a fibrillar center (FC) with fragmented fibrils can be recognized. Fixation: glutaraldehyde prior to digestions, osmium tetroxide after digestions. Stain: uranyl acetate and lead citrate, x 30,000. FIG. 12. Same as Fig. 11 showing the fibrillar center at a higher magnification. The fibrils of the center resisted ribonuclease and pepsin digestion, x 80,000. FIG. 13. Nucleolus of No. 180 cell that has been digested with trypsin (2.5 mg/ml) for 1 hour at 37°. The lead-positive material resisted digestion. Note its distribution over almost the entire fibrillar nucleolonema. Fixation: glutaraldehyde prior to digestion, osmium tetroxide following digestion. Stain: uranyl acetate and lead citrate, x 40,000.
!iiiii,iiii~i!i~i~i~i!i~i, ~i~¸¸i~
'i
12
RECHER, WHITESCARVER, AND BRIGGS
pepsin digestion that shows continuity with a fibrillar network of the associated nucleolonema. This fibrillar framework appears to resist not only pepsin digestion, but also ribonuclease digestion. The question therefore is being raised whether these fibrils are composed of DNA. An attempt was made to digest cells with deoxyribonuclease but technical difficulties were encountered. Deoxyribonuclease was found to have no effect on glutaraldehyde-fixed cells, even if deoxyribonuclease digestion was preceded by pepsin digestion. The formalin fixative used by Swift (18)was tried, but the preservation of the nuclei was not good enough for proper evaluation of details. Further attempts are being made to develop a workable deoxyribonuclease digestion method. Incorporation of thymidine-~H with subsequent autoradiography will also be attempted. It is not believed that a negative Feulgen reaction (3) excludes the possibility of D N A being present in fibrillar centers, because the amount is likely to be too small to be detectable in the light microscope. The possibility that the fibrils of fibrillar centers contain RNA has to be considered. The RNA might be hybridized to D N A and thus be inaccessible for ribonuclease. Our findings, however, differ from those of Schoefl (15) and Yasuzumi and Sugihara (23) in that ribonuclease had no visible effect on fibrillar centers. The intense staining by lead citrate appears to be a characteristic feature of the fibritlar centers and the fibrillar nucleolonema. Little is known about the nature of this lead reaction. The lead-positive substance is found primarily along the fibrils of the two nucleolar zones. It is therefore possible that the component which reacts with lead is bound to or forms part of the macromolecules that compose these fibrils. It was hoped that enzyme digestion studies would point to a macromolecular species as being specifically involved in the lead reaction, but the digestion results are not conclusive. Pepsin appears to be most effective in abolishing the lead staining reaction, but whether this is due to the removal of a specific protein that can bind lead is uncertain. It is possible that peptic digestion masks the lead reaction or solubilizes and removes other nonproteinacious substances that may contain the lead positive material. Trypsin apparently has no effect on the lead stainability of either the fibrillar centers or the fibrillar nucleolonema. A weak lead reaction was occasionally observed in the fibrillar nucleolonema following ribonuclease digestion, but the finding was not consistent enough to allow any conclusions. Studinski and Love (16) demonstrated certain nucleolar constituents by lead precipitation in unfixed cultured cells. They referred to these constituents as nucleolini and described them as granules and hollow spherules. These nucleolini, the hollow spherules in particular, appear to have some resemblance to the fibrillar centers described in this report. It is of interest that both these structures can be demonstrated by lead precipitation. Studinski and Love (16) speculated that phosphates might be involved in the reaction. Tandler (19) described a similar lead precipitation reaction
NUCLEOLAR FINE STRUCTURES
13
to demonstrate nucleoli of unfixed cells. He identified the nucleolar precipitate as lead orthophosphate (20). The lead precipitation described by these authors (16, 19) succeeded only when unfixed cells were exposed to lead acetete. Any type of fixation abolished the reaction. The lead reaction described in this report occurs under somewhat different conditions. The cells are fixed with glutaraldehyde and osmic acid, dehydrated with ethyl alcohol, embedded in Epon and then exposed to lead citrate. But this difference may be more apparent than real, in principle the reaction may be the same. Phosphates may also be involved. The lead reaction in order to occur requires fixation with osmic acid. What role osmic acid could possibly play is unknown at the present time. It is likely that other conditions also may determine the outcome of the reaction because the lead precipitation did not always occur when it was expected. Further investigation is needed before the nature and significance of this reaction can be appreciated. Sankaranarayanan and Busch (14) described dense granules in nucleoli of Walker 256 carcinosarcoma cells. The granules were embedded in a fibrillar matrix in the "light regions" or "pars amorpha." It is likely that they are identical to the leadpositive material of the fibrillar centers described in this report because of their location. The authors (14) did use osmium tetroxide as a fixative and stained their sections with lead hydroxide. Nucleolar granules of high electron density have also been observed in plant cells by Lafontaine (9), Porter (12), and Sun (17) and in Amoeba proteus by Cohen (2). It is possible that these granules and the lead-positive material are of identical nature and are a regular component of nucleoli. The close structural relationship of the fibrillar centers and the fibrillar nudeolonema and the high affinity to lead suggests a close functional relationship of the two nucleolar zones. The fibrillar centers may be transitory structures that are being developed for a certain function and then regress. Variations of lead stainability may reflect different states of functional activity. Vacuoles as illustrated in Fig. 3 may be burned-out centers. REFERENCES
1 . BERNHARD,W. and GRANBOULAN,N., in DALTON, A. J. and HAGUENAU,F. (Eds.), Ultrastructure in Biological Systems, Vol. 3: The Nucleus, p. 81. Academic Press, New York, 1968. 2. COHEN,A. J., J. Biophys. Biochem. Cytol. 3, 859 (1957). 3. DUTTA, C. R., SIEGESMUND, K. A. and Fox, C. A., J. Ultrastruct. Res. 8, 542 (1963). 4. ESTABLE,C., SWIFT,H., BERNHARD,W., GALL,J., PERRY,R. and S1RLIN,J., Natl. Cancer Inst. Monograph 23, 573 (1966).
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RECHER, WHITESCARVER~AND BRIGGS
5. FAWCETT, D., The Cell. An Atlas of Fine Structure, p. 26. Saunders, Philadelphia, Pennsylvania, 1966. 6. GRAN~OULAN,N. and GRANBOULAN,P., Exptl. Cell Res. 34, 71 (1964). 7. HEINE, U., LANGLO~S,A. J. and BEARD, J. W., Cancer Res. 26, 1847 (1966). 8. JEZEQUEL,A. M. and BERNHARD,W., J. Microscopie 3, 279 (1964). 9. LAEONTA~NE,J. G., J. Biophys. Biochem. CytoI. 4, 229 (1958). 10. LUF~C,J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). 11. PENMAN,S., SMITH,I., HOLTZMAN,E. and GREE~ERG, H., Natl. Cancer Inst. Monograph 23, 489 (1966). 12. PORTER,K. R., Proc. 4th Intern. Congr. Electron Mikroscopy, Berlin, 1958 Vol. 2, p. 186. Springer, Berlin, 1960. 13. REYNOLDS,E. W., J. Cell Biol. 17, 208 (1963). 14. SANKARANARAYANAN,K. and BuscH, H., Exptl. Cell Res. 38, 434 (1965). 15. SCHOEFL,G. I., J. Ultrastruct. Res. 10, 224 (1964). 16. STUDINS~:~,G. P. and LovE, R., Stain Technol. 39, 397 (1964). 17. SUN, C. N., ExptL Cell Res. 25, 213 (1961). 18. SwiFt, H., Exptl. Cell Biol., Suppl. 9, 54 (1963). 19. TANDLER,C. J., J. Histochem. Cytochem. 4, 331 (1956). 20. - - - - ibid. 5, 489 (1957). 21. TERZArdS, J. A., J. Cell Biol. 27, 293 (1965). 22. TOKUYASU,K., MADDEN, S. C. and ZELD~S,L. J., J. Cell Biol. 39, 630 (1968). 23. YASUZUMI,G. and SUGIHARA,R., Exptl. Cell Res. 37, 207 (1965).