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Occurrence of nucleolar material in the cytoplasm of plant cells
cell Biology International
Repot-is, Vol. 3, AM. 3, -?979
215
OCCURRENCE OF NUCLEOLAR MATERIALIN THE CYTOPLASM OF PLANTCELLS, S. MORENO-DIAZ DE LA ESPINA!, M.E. FERNANDEZ-COMB2 and M.C. RISUEBO. Instituto de Biologfa Celular, C.S.I.C. Madrid. ABSTRACT This
paper deals with the inductionofcytoplasmic nucleolar bodies in meristematic Allium cepa L. cells after treatment with drugs which interfere with nucleolar functionality. The drugs which interfere with protein synthesis failed to produce these bodies.The ultrastructure originandphysiological significance of these bodies are discusas well as their relation with the sed here, mitotic prenucleolar bodies (Moreno-Dzaz de la Es pina et al., 1976).
INTRODUCTION The nucleolus is a constant component of the nucleus in thus, under normal conditions it can eukaryotic cells, find no material of nucleolar origin in the cytoplasm except for the finished ribosomes; but some authors(Ike uchi et al., have report&d the 1971; Bernhard, 1971) under existence of nucleolar bodies in the cytoplams extreme metabolic situations. We have tried to induce the appearance of these nucleolar structures in the cytoplasm by means of treatments with DNA-RNA and proteinsynthesis inhibitors in an attempt to clarifythephysiological significance of the appearance of bodies with a nucleolar origininthe cytoplasm.We have also studied the ultrastructure of these bodies as well as their relations with chromosomes and the nuclear envelope. MATERIAL AND'.METHODS used consisted of root-tips
The material cells from Allium
meristematic
cepaL.Bulbs grown intap water constant conditions of temperature and aeration, 0.5"C) and bubbling lo-20 cm3 air min-1. 1
Correspondence to Dr. S. Moreno-D$az de la Instituto de Biologfa tamento de Citologia, Velbzquez, 144. Madrid-6. Spain. I.C.), 0309-1651/79K)30216-1
l/$02.00/0
01979
Academic
Espina, Celular Press
Inc.
under (15 ?
Depar (C.S. (London)
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216
Ceil Biology International
Reports, Vol. 3, No. 3, 1979
For the light microscope the roots were prepared according to the silver impregnation technique (FernbndezGdmez et al., 1969): the roots were fixed ina 1:lmixture for 2 hr then of 10% formaldehyde and 1% hydroquinone and immersed in a solution of 2% silver washed in water, nitrate at 70°C for 15 hr in the dark. After washing in water they were treated for l-2 hr with formol-hydroquinone mixture and then treated immediately with kodak The squashing was afterwards done photographic fixative. with one or two drops or water. For the electron microsin 3% glutaraldehyde in a copy, the roots were fixed 0.025 M cacodylate buffer at pH 7 for 1 h at room and afterwards postfixed in 1% osmic acid temperature, for 1 h in the same buffer. The roots were dehydrated in ethanol series, passed through propylene oxide and embedded in Epon 812. The sections were then stained with uranyl acetate in a Michaelis buffer and lead citrate according to (Venable and Gogeshall, 1965). For detection of ribonucleoproteins, ultrathin sections glutaraldehyde-fixed Epon embedded roots were stained by uranyl acetate-EDTA-citrate sequence according to Bernhard, 1969. Treatments: The roots, while still attached to the bulbs were transferred to the drugs under the same conditions as were used for growing. The drugs used for different times at the following concentrations were : Ethidium Tomide lOO,ug.ml'l, 3'deoxyadenosine (Cordycepin) 10'4Mt -amanitin 10 ,ug.ml'l, Hydroxiurea 2OO,ug/ml, Anysomitin (1 Pg.ml'l), Cycloheximide (Ch) 1 pg.ml-1 and Iodoacetamide 10'2M. OBSERVATIONS Silver impregnation techniques have been widelly used for detection of different cellular materials, histones (Black and Ansley, 1964) o nuclei acids (Korson, 1951) DNA (Korson, 1964),nucleolar material (Tandler, 1959;Gon Das ard Alfert,1963;Eern&xdez-G&nez et al.,l96<) z6lez-Ramtiez,.l961; nucleolar organizer regions of chromosomes(Goodpasture and Bloom, 1975). We have used one of these techniques the silver impregnation for light (Ferndndez-Gdmez etal., 1969) and Electron Microscopy (Risuefio et al., 1973)which preferentially detects proteins of nucleolar origin, in order to check on the appearance of nucleolar material in the cytoplasm. This technique (Ferndndez-G6mez et al., 1969; Stockert et al., 1970) and also similar methods (Tandler, 1959; Gonzblez-Ramfrez, 1961; Das and Alfert, 1963; Paweletz et 1967) have been used as routine method for detection al., of nucleolar material (Ferndndez-G6mez et al., 1972;GimB nez-Martfn et al., 1974; Moreno-Dlaz de la Espina et al,,
Untreated aell under light and electron microscopes Fig.1 and'2.impregnated with silver. mp(micropuffs).No impregnated structures are seen in the cytoplasm. Fig. 3 to 7.- Cells treated with several drugs showing the nucleolus and the Cy.N.B. (arrows) preferentially stained by silver; 3 Ethidium bromide; 4 Cordycepin; 5 Eydroxiurea; 6 Iodoacetamide; 7'3(-amanitin. Fig. 8 and 8a.- Cells in late telophase stage in the presence of Ethidium bromide showing Cy.N.b. (Arrows) and prenucleolar bodies (pb) notfusingintheNOR.Fig.B.Glutaraldehyde osmium,lead citrate.Fig.8a.-glutaraldehyde EDTA.
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Cell Biology International
Reports, Vol. 3, No. 3, 1979
-1976; Risuefio et al.', 1976). Nucleolus and micropuffs are theonlyimpregnated ng clear structures in interphasic control cells (Risuefio et In mitotic cells onlythenucleolar al.,19781 (fig.l,2). material in all steps of structural organization appear clearly impregnated (Moreno-Diaz de la Espina et al., 1976). This material made of RNP and appears preferentially stained by EDTA (Bernhard, 1969; Moreno-Dfaz de 1976). We have never find impregnala Espina et al., tedstructures inthecytoplasm of control cells(fig.l,2). In cells treated for different times with Ethidium bromide, Cordycepin, <-amanitin, Hydroxiurea and Iodoace tamide one can observe bodies clearly impregnatedinthe cytoplasm (fig. 3 to 7). The number of "cytoplasmic nucleolar bodies", per cell for each drug and timeoftreat of cells whichhavethese ment, as well as the percentage bodies inthe meristematic population is shownin table I. The appearance of these bodies in the cytoplasm is different for those treatments. With Ethidium Bromide, Cordycepin and Iodoacetamide it can observemorecytoplas mic bodies in the first hours of treatments and their number decreases during the 6th hours of it, this fact is very obviousinthe caseofEthidium bromide. Nevertheless with Hydroxiurea the number of this cytoplasmic nucleolar bodies remains more or less constant throughout the trea& ment period while with o(-amanitinthereisalight increase of cytoplasmic bodies in the last hour.In the meristematic population the percentage of cells which have this bodies varies throughout the treatments. They are increas sing with o(-amanitin and Iodoacetamine butwiththe other drugs this percentage are more or less constant. Usually this bodies are present in interphasic cells but in some cases are present only in mitotic stages as occurs with Iodoacetamine at two hours of treatment. The maximum and minimum number of these bodiespercell and for the different treatment is also shown in Table I. We have failedtoinducetheappearanceofnucleolarbodies in the cytoplasmbymeansoftreatments with protein synthesis inhibitors (anysomicin and cycloheximide). Under the electron microscope(fig.8) the cytoplasmic nucleolar bodies appear as rough spherical structures preferentially contrasted by EDTA (fig. 8a). Their ultrastruc tureOis exclusively fibrillar,formed by fibrils of 90 to 100 A of diameter, homogeneous in their inner part,while in thg outer part there are fibres with a greater diameter (480 A) and higher contrast, which seem to correspond to central fibres spiralized within themselves (fig. 8 and 9).
219
Table Cytoglasmic nucleolar during cell drugs Treatlent
-s
X
bodies fCy.N.b.) inducedby several cycle in meristenatic cells.
% Cy.N.b. % cell with % Interphase par cell Cy.N.b, cell with Cy.N.b.
Ethidium bromide cordi-
cepin Ii$Sroaqurea
Iodoacetamide
Numbar of Cy.N.b. per cell maximummiriimum
2h
21
46
79
62
3
6h
5
43
83
20
1
2h
38
45
84
103
2
6h
12
50
W
28
1
2h
14
25
50
53
1
6h
15
67
115
1
2h
25
9
0
79
3
6h
17
21
49
60
1
-am&tin
2h
17
11
83
73
1
6h
22
54
90
100
1
AnysoIBiCin
2h 6h
22
0
0
0
0
0
Cyclehexfmide
2h
0
0
0
6h
0
0
0
4
-
These bodies show ne.ither surrounding membrane nor continuity with any cytoplasmic structures (fig. 10 and 12). The ultrastructure of the cytoplasmic nucleolar bodies is very similar to that of the mature prenucleolar bodies (which have arise a high degree of condensation) (fig.111 and of the fibrillar part of the nucleolus (Moreno-DSaz
Cell Biology international
Reports, Vol. 3, No. 3, 1979
Fig. 9 to 12.- Cells in presence of Ethidium bromide whose nuclear envelope does not break in prophase shoeing Cy.N.b. (arrows). !n.e.) structure of Fig. 9.- The fine these bodies is exclusively fibril lar, and in the outer part thereare fibres with a biger diameter than in the interior. Fig. 10 and ll.Extrusion of the nucleolar bo dies 'from the nucleus to the cyto Sometimes they are tr ,apped by the discontinuities plasm (arrows). of the n.e. Fig. 12.- The cytoplasmic nucleolar bodies show continuity with any other cytoplasmic structure (arrows).
no
de la Espina et al., 1976; Risuefio et 'al., 1976).We have found no significative ultrastructural differences among the cytoplasmic nucleolar bodies induced by the difHowever the sizeofthese bodies vaferent treatments. ries in the presence of the different drugs studied. So with Ethidium bromide and Cordycepin the cytoplasmic (0.55 to 0.65 )un nucleolar bodies have a great size of diameter) while with c(-amanitin and Hydroxyurea are smaller. We have observed the extrusion of nucleolar bodies from nucleus to cytoplasm under the electron microscope (fig. 10 to 12) in cells treated with EB and 3'AdR in which the nuclear envelope, does not breakdown in prophase, and mitosis goes on within the nucleus (Risueiio and Moreno-D$az de la Espina, 1972). The extrusion is observed from the anaphase to early Gl; during interphaseandother mitotic stages, thus these bodies do not appear to be associated with the nuclear envelope bodies after extrussion*During the outcome of nucleolar to the cytoplasm these appear trapped by the nuclear envelope which shows discontinuity in this area (fig. 11). DISCUSSION There are a lot of evidences to think thattheCy.n.b. have a nucleolar origin. 1) They both show the same cyto chemical behaviour by silver impregnation for nucleolar material and EDTA (Moreno-DZaz de la Espina et al., is similar to both that of 1976). 2) Their structure the mitotic prenucleolar bodies and of the fibrillar part of the interphasic nucleolus. The micropuff which are also strongly impregnated by silver show a very different structure and behaviour by EDTA (Risuefio et al., In the other hand we have never observed micro 1978). after treat-puff in the cytoplasm of these cells ments. The fibrillar bodies which appearinthe nucleusafter treatments with RNA and protein synthesis inhibitors and are no thatare. extruded occasionally to the cytoplasm, impregnated by silver (Moreno-Dlaz de la Espina and Risueilo, 1977). As nucleolar materialtheseCy.n.n.couldcorrespondto: 1) nucleolar fragments pro&ed by the action of the drug which would then be extended to the cytoplamn or 2) prenucleolar bodies which would not coalesce in the NOR (as in a normal situation) by the effect of the drug, and then pass throughtothe cytoplasm. The first hypothesis does not seem very likely, because the Cordicepin, which does not causeanynucleolar fragmentation (Gj&nez-Martfn
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et al., 1973), produces cells with a large numberofcyto The second case plasmic nucleolar bodies (see table I). (Eth& fits better with our results since the three drugs dium bromide, Cordycepin and Hydroxiurea) which produce these cytoplasmic bodies, interfere with nucleolar rezfganization, (Gimenez-Martin et al., 1974; Risuefio et 1976), and produce persistent prenucleolar bodies, thus producing whiih could then pass to the cytoplasm, These bodies are produced cytoplasmic nucleolar bodies. by treatments which strongly alter nucleolar functionality (either biosynthetic activity or capacity of reorganization in telophase); and for these reason, anysomicin and cycloheximide, which do not impede neither nucleolar activity (Ferngndez-G6mez et al., 1974) nor reorganization in the telophase, do not lead to the appearance of cytoplasmic nucleolar bodies, although they produce alte rations in the nucleolar ultrastructure (Gim&-iez-Martinet al., 1974; Ferndndez-G6mez et al., 197 ). FerndndezG6mez etal.,1978have studied thefluorodeoxyuridine effects on the nucleolar cycle and it alters nucleolar dispersion in prophase and produces persistent nucleoli which are eliminated to the cytoplasm and thus could be comparable in a certain degree to the cytoplasmic nucleolar bodies described, here. Ethidium bromide and Cordycepin impair both nucleolar biosynthesis activity and nucleolar reorganization in the telophase (Risuefio et al .,1972; 1976; Gimenez-Martfn,l973, has been described as a drug with a 1974). Hydroxiurea complex action, both on DNA and on protein synthesis by Sartorelly et al., 1969, which effects varies according to the cell cycle phase (Synclair, 1965) andalso strongly affecting nucleolar ultrastructure and activity (Bernhard, 1971). The nucleolar reorganization under this drugs is altered in the similar way as Cordycepin and Ethidium bromide do. .K-amanitine do not impedes the nucleolar reorganization (Gimgnez-Martin et al., 1974; Moreno-Dlaz de la Espina and Risuefio, 1976) so the cytoplasmic nucleolar bodies would produce during prometaphase as nucleolar remnants from the nucleolar dispersion whichcould then pass to the cytoplasm through the broken nuclear envelope. Iodoacetamide interferes with the nucleolar func ionality affecting the nucleolar dispersion in onion (uii published data) and producing the cytoplasmic nucleolarbodies during the mitotic stages. All these reasons give us to think that the main factor which leads to the production of cytoplasmic nucledar bodies is the persistence of prenucleolar bodies and of nucleolar fragments in the nucleoplasm. The alterations in the nuclear envelope formation or structure (as in the case with Ethidium bromide treatment (De la Torre and Risuefio, 1973) would favour the flow of these bodies to the
cytoplasm. In relation to the mechanism of f,ormation of the cytg plasmic prenucleolar bodies, two hypothesis may be con eluded. 1) Cytoplasmic bodies would form in the cytoplasm 2) The bodies would pass to the in a disorganized form. cytoplasm after the break-down of the nuclear envelope, either during mitosis or by the effect of the drugs. The first hypothesis does not appear likely because these cytoplasmic bodies are observed as discrete bodies passing through the nuclear envelope, although the possibility of the formation of a few of them by this mechanism may not be discarded. The second possibility seems the more accurate. The persistent nucleolar fragments would be liberated during prometaphase with the break-down of the nuclear envelope, and the persistent prenucleolar bodies formed in the telophase would then pass on to the cytoplasm through the discontinuities through the nuclear envelope in formation, or those produced by drugs. In short: the cytoplasmic nucleolar bodies which appear in meristematic Allium ce a L. after treatmentswith --+ eolar functionality, drugs which fnterferewithnuc seem to correspond to remnants of the fragmented nucleolus,or prenucleolar bodies which are not fused in the NOR bythe effect of the drug. They appear to pass to the cytoplasm when the nuclear envelope breaks-down. BIBLIOGRAPHY Bernhard, W. (1969). A new staining procedure for electron microscopical cytology. J. Ultrastruct. Res.27, 250-265. Bernhard, W. (1971). Drug-induced changes in the interphase nucleus. Advances Cytopharmacology I, 50-67. Black, M.M. and Ansley, H.R. (1964) Histone staining with ammoniacal silver. Science 143, 693-695. staining of a Das, N.K. and Alfert, H. (1963).Silver its origin and fate during the nucleolar fraction, mitotic cycle. Ann. Histochim. 8-, 109-114. DSez, J.L., Marfn, M.F., Esponda, P. and Stockert, J.C. (1971). Prenucleolar bodies in the cytoplasm of meristematic cells after thermal shock. Experientia -27, 266-267. Ferndndez-GCSmez, M.E., Stockert, J.C., L6pez-Sdez, J.F. and Gimenez-Martin, G. (1969). Staining plant cell fixnucleoli with AgN03 after formalin-hydroguinone ation. Stain Tech. 44, 48-49. Ferndndez-Gbmez, M.E. zd Stockert, J.C. (1970) Metallic impregnations of nucleoli in root-tip cells. The Nucleus. -13, 149-156.
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Ferndndez-Gbmez, M.E., Risuefio, M.C. and Gimenez-Martfn, G. (1973). Effect of cycloheximide on plant cell nucleoli. Arch. Biol. (Bruxelles) 84, 417-424. Ferndndez-Gbmez, M.E., Torre, C. de E and Gimgnez-Martin, Accelerated nucleolar reorganization with G. (1972). shortened anaphase and telophase during cycloheximide inhibition of protein synthesis in onion root cells. Cytobiologie, Vol. 5, n"2, 117-124. de la Espina, S. and Ferndndez-G6mez, M.E., Moreno-Dfaz of nucleolar disperRisuefio, M.C. (1978). Alt eration sion in prophase by 5-FUdR treatment. Cell Biol. Reports 2, 237-244. Inter. Gimgnez-Martin, G., Risuefio, M.C., Fernbndez-G6mez, M.E. and Ahmadian, P. (1973). Effect ofcordycepin on the fine structure ofinterpbasa nucleoli in plant cells. Cytobiologie 1, 181-192. Gimenez-Martin, G., Torre, C. de la, Fernbndez-G6mez, M. E. and Gonz%lez-Ferndndez, A. (1974). Experimental analysis of nucleolar reorganization. J. Cell Biol. 60, 502-507. Goodpasture, C. and Bloom, S.E. (1975). Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma (Ber1.)53, 37-50. Gonzalez-Ramfrez, J. (1961) Nucleolar physiomgy. III. Consideration about the meaning of nucleolar silver impregnation in cultured HeLa cells. Biol.Inst.Estud. Med. Biol. (Mex) 19, 195-206. Ikeuchi, T., Lanbe, M., Weinfeld, H. and Sandberg, A.A. (1971). Induction of nuclear envelope around metaphase chromosomes after fusion with interphase cells. J. Cell Biol. 51, 104-115. Korson, R. (1951).A differential stain for nucleic acids. Stain Technol. 26, 265-270. silver stain for deoxyrribonucleic Korson, R. (1964).x acic. J. Histochem. Cytochem. 12, 875-879. Moreno-DPaz de la Espina, S. and Risueiio, M.C. (1976).Effeet of oj-amanitine on the nucleolus of meristematic cells of Allium cepa in interphase and mitosis: an ultrastructural analysis. Cytobiologie 12, 175-188. Moreno-D$az de la Espina, S., Risueiio, M.C, FernbndezGbmez, M.E. and Tandler, C.J. (1976). Ultrastructural study of the nucleolar cycle in meristematic cells of Allium cepa. J.MicroscopieetBiol. Cell. 25, 265-278. M.E. (1977). Moreno-DPaz de la Espina, S. and Risuefio, Fibrillar bodies in Allium cepa : an ultrastructural and cytochemical study. Biol. Cell. 30, 93-102. Paweletz, N., Siebs, W. and Lettre, R. (1967). Untersuchungen zue argentaffinreaktion des nukleolus. Z. Zellforsch. 76, 577-605. -
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Risueiio, M.C., Fernbndez-G6mez, M.E., Gimenez-Martin, G. (1973). Nucleoli under the electron microscope by silver impregnation. Mikroskopie 29, 292-298. Risuefio, M.C., Fernbndez-Gbmez, M.E., De la Torre, C.and Gimenez-MartIn, G. (1972). Effect of ethidium bromide on the fine .structure of the nucleolus in plant cells. J. Ultrastruct. Res. 39, 163-172. Risuefio, M.C., Moreno-D1z de la Espina, S., FernbndezGbmez, M.E. and Gimenez-Martfn, G. (1976). Ultrastruc tural study of nucleolar material during plant mitosys in presence of inhibitors of RNA synthesis. J. Microscopie Biol. Cell. 26, 5-18. Risueiio, M.C., Moreno-Dlaz de la Espina, S., Fendndez-G6 mez, M.E. and GimBnez-MartXn, G. (1978). Nuclear micro puffs in Allium cepa cells. I. Quantitative, ultrastructural and cytochemical study. Cytobiologie 16,209223. Risuefio, M.C. and Moreno-D$az de la Espina, S. (1978). Analysis of the mitosis in presence of RNA synthesis inhibitors in meristematic cells.Caryologia31, 43-61. Sinclair, W.K. (1965). Hydroxiurea differentialethal effects on cultured mammalian cells during the cell cycle. Science 150, 1729-1731. Sartorelly, A.C. axcreasy, W.A. (1969). Cancer ch¬herapy. Ann. REV. Pharmacol. 2, 51-72. Ferndndez-Gbmez,-M.E., Gimhez-Martln,G. Stockert, J.C., and Lbpez-Sdez, J.F. (1970) Organization of argyrophi lit nucleolar material throughout the division cycle of meristematic cells. Protoplasma 69, 265-278. Tandler, C.J. (1959). The silver reducing property of the nucleolus and the formation of prenucleolar mate rial during mitosis. Exptl. Cell Res. 17, 560-564. Torre, C. de la , Risuefio, M.C. and Gin&n=-MartZn, G. (1973). Ethidium bromide and its effectsonthe nuclear membrane cycle. Protoplasma 76, 363-371. Venable, J.H. and Gogueshall, KT(1965) A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408.
Received: 1st August 1978 6th November 1978
Revised Version
Received:
Accepted: 20th November 1978
Repot-is, Vol. 3, AM. 3, -?979
215
OCCURRENCE OF NUCLEOLAR MATERIALIN THE CYTOPLASM OF PLANTCELLS, S. MORENO-DIAZ DE LA ESPINA!, M.E. FERNANDEZ-COMB2 and M.C. RISUEBO. Instituto de Biologfa Celular, C.S.I.C. Madrid. ABSTRACT This
paper deals with the inductionofcytoplasmic nucleolar bodies in meristematic Allium cepa L. cells after treatment with drugs which interfere with nucleolar functionality. The drugs which interfere with protein synthesis failed to produce these bodies.The ultrastructure originandphysiological significance of these bodies are discusas well as their relation with the sed here, mitotic prenucleolar bodies (Moreno-Dzaz de la Es pina et al., 1976).
INTRODUCTION The nucleolus is a constant component of the nucleus in thus, under normal conditions it can eukaryotic cells, find no material of nucleolar origin in the cytoplasm except for the finished ribosomes; but some authors(Ike uchi et al., have report&d the 1971; Bernhard, 1971) under existence of nucleolar bodies in the cytoplams extreme metabolic situations. We have tried to induce the appearance of these nucleolar structures in the cytoplasm by means of treatments with DNA-RNA and proteinsynthesis inhibitors in an attempt to clarifythephysiological significance of the appearance of bodies with a nucleolar origininthe cytoplasm.We have also studied the ultrastructure of these bodies as well as their relations with chromosomes and the nuclear envelope. MATERIAL AND'.METHODS used consisted of root-tips
The material cells from Allium
meristematic
cepaL.Bulbs grown intap water constant conditions of temperature and aeration, 0.5"C) and bubbling lo-20 cm3 air min-1. 1
Correspondence to Dr. S. Moreno-D$az de la Instituto de Biologfa tamento de Citologia, Velbzquez, 144. Madrid-6. Spain. I.C.), 0309-1651/79K)30216-1
l/$02.00/0
01979
Academic
Espina, Celular Press
Inc.
under (15 ?
Depar (C.S. (London)
Ltd.
216
Ceil Biology International
Reports, Vol. 3, No. 3, 1979
For the light microscope the roots were prepared according to the silver impregnation technique (FernbndezGdmez et al., 1969): the roots were fixed ina 1:lmixture for 2 hr then of 10% formaldehyde and 1% hydroquinone and immersed in a solution of 2% silver washed in water, nitrate at 70°C for 15 hr in the dark. After washing in water they were treated for l-2 hr with formol-hydroquinone mixture and then treated immediately with kodak The squashing was afterwards done photographic fixative. with one or two drops or water. For the electron microsin 3% glutaraldehyde in a copy, the roots were fixed 0.025 M cacodylate buffer at pH 7 for 1 h at room and afterwards postfixed in 1% osmic acid temperature, for 1 h in the same buffer. The roots were dehydrated in ethanol series, passed through propylene oxide and embedded in Epon 812. The sections were then stained with uranyl acetate in a Michaelis buffer and lead citrate according to (Venable and Gogeshall, 1965). For detection of ribonucleoproteins, ultrathin sections glutaraldehyde-fixed Epon embedded roots were stained by uranyl acetate-EDTA-citrate sequence according to Bernhard, 1969. Treatments: The roots, while still attached to the bulbs were transferred to the drugs under the same conditions as were used for growing. The drugs used for different times at the following concentrations were : Ethidium Tomide lOO,ug.ml'l, 3'deoxyadenosine (Cordycepin) 10'4Mt -amanitin 10 ,ug.ml'l, Hydroxiurea 2OO,ug/ml, Anysomitin (1 Pg.ml'l), Cycloheximide (Ch) 1 pg.ml-1 and Iodoacetamide 10'2M. OBSERVATIONS Silver impregnation techniques have been widelly used for detection of different cellular materials, histones (Black and Ansley, 1964) o nuclei acids (Korson, 1951) DNA (Korson, 1964),nucleolar material (Tandler, 1959;Gon Das ard Alfert,1963;Eern&xdez-G&nez et al.,l96<) z6lez-Ramtiez,.l961; nucleolar organizer regions of chromosomes(Goodpasture and Bloom, 1975). We have used one of these techniques the silver impregnation for light (Ferndndez-Gdmez etal., 1969) and Electron Microscopy (Risuefio et al., 1973)which preferentially detects proteins of nucleolar origin, in order to check on the appearance of nucleolar material in the cytoplasm. This technique (Ferndndez-G6mez et al., 1969; Stockert et al., 1970) and also similar methods (Tandler, 1959; Gonzblez-Ramfrez, 1961; Das and Alfert, 1963; Paweletz et 1967) have been used as routine method for detection al., of nucleolar material (Ferndndez-G6mez et al., 1972;GimB nez-Martfn et al., 1974; Moreno-Dlaz de la Espina et al,,
Untreated aell under light and electron microscopes Fig.1 and'2.impregnated with silver. mp(micropuffs).No impregnated structures are seen in the cytoplasm. Fig. 3 to 7.- Cells treated with several drugs showing the nucleolus and the Cy.N.B. (arrows) preferentially stained by silver; 3 Ethidium bromide; 4 Cordycepin; 5 Eydroxiurea; 6 Iodoacetamide; 7'3(-amanitin. Fig. 8 and 8a.- Cells in late telophase stage in the presence of Ethidium bromide showing Cy.N.b. (Arrows) and prenucleolar bodies (pb) notfusingintheNOR.Fig.B.Glutaraldehyde osmium,lead citrate.Fig.8a.-glutaraldehyde EDTA.
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-1976; Risuefio et al.', 1976). Nucleolus and micropuffs are theonlyimpregnated ng clear structures in interphasic control cells (Risuefio et In mitotic cells onlythenucleolar al.,19781 (fig.l,2). material in all steps of structural organization appear clearly impregnated (Moreno-Diaz de la Espina et al., 1976). This material made of RNP and appears preferentially stained by EDTA (Bernhard, 1969; Moreno-Dfaz de 1976). We have never find impregnala Espina et al., tedstructures inthecytoplasm of control cells(fig.l,2). In cells treated for different times with Ethidium bromide, Cordycepin, <-amanitin, Hydroxiurea and Iodoace tamide one can observe bodies clearly impregnatedinthe cytoplasm (fig. 3 to 7). The number of "cytoplasmic nucleolar bodies", per cell for each drug and timeoftreat of cells whichhavethese ment, as well as the percentage bodies inthe meristematic population is shownin table I. The appearance of these bodies in the cytoplasm is different for those treatments. With Ethidium Bromide, Cordycepin and Iodoacetamide it can observemorecytoplas mic bodies in the first hours of treatments and their number decreases during the 6th hours of it, this fact is very obviousinthe caseofEthidium bromide. Nevertheless with Hydroxiurea the number of this cytoplasmic nucleolar bodies remains more or less constant throughout the trea& ment period while with o(-amanitinthereisalight increase of cytoplasmic bodies in the last hour.In the meristematic population the percentage of cells which have this bodies varies throughout the treatments. They are increas sing with o(-amanitin and Iodoacetamine butwiththe other drugs this percentage are more or less constant. Usually this bodies are present in interphasic cells but in some cases are present only in mitotic stages as occurs with Iodoacetamine at two hours of treatment. The maximum and minimum number of these bodiespercell and for the different treatment is also shown in Table I. We have failedtoinducetheappearanceofnucleolarbodies in the cytoplasmbymeansoftreatments with protein synthesis inhibitors (anysomicin and cycloheximide). Under the electron microscope(fig.8) the cytoplasmic nucleolar bodies appear as rough spherical structures preferentially contrasted by EDTA (fig. 8a). Their ultrastruc tureOis exclusively fibrillar,formed by fibrils of 90 to 100 A of diameter, homogeneous in their inner part,while in thg outer part there are fibres with a greater diameter (480 A) and higher contrast, which seem to correspond to central fibres spiralized within themselves (fig. 8 and 9).
219
Table Cytoglasmic nucleolar during cell drugs Treatlent
-s
X
bodies fCy.N.b.) inducedby several cycle in meristenatic cells.
% Cy.N.b. % cell with % Interphase par cell Cy.N.b, cell with Cy.N.b.
Ethidium bromide cordi-
cepin Ii$Sroaqurea
Iodoacetamide
Numbar of Cy.N.b. per cell maximummiriimum
2h
21
46
79
62
3
6h
5
43
83
20
1
2h
38
45
84
103
2
6h
12
50
W
28
1
2h
14
25
50
53
1
6h
15
67
115
1
2h
25
9
0
79
3
6h
17
21
49
60
1
-am&tin
2h
17
11
83
73
1
6h
22
54
90
100
1
AnysoIBiCin
2h 6h
22
0
0
0
0
0
Cyclehexfmide
2h
0
0
0
6h
0
0
0
4
-
These bodies show ne.ither surrounding membrane nor continuity with any cytoplasmic structures (fig. 10 and 12). The ultrastructure of the cytoplasmic nucleolar bodies is very similar to that of the mature prenucleolar bodies (which have arise a high degree of condensation) (fig.111 and of the fibrillar part of the nucleolus (Moreno-DSaz
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Reports, Vol. 3, No. 3, 1979
Fig. 9 to 12.- Cells in presence of Ethidium bromide whose nuclear envelope does not break in prophase shoeing Cy.N.b. (arrows). !n.e.) structure of Fig. 9.- The fine these bodies is exclusively fibril lar, and in the outer part thereare fibres with a biger diameter than in the interior. Fig. 10 and ll.Extrusion of the nucleolar bo dies 'from the nucleus to the cyto Sometimes they are tr ,apped by the discontinuities plasm (arrows). of the n.e. Fig. 12.- The cytoplasmic nucleolar bodies show continuity with any other cytoplasmic structure (arrows).
no
de la Espina et al., 1976; Risuefio et 'al., 1976).We have found no significative ultrastructural differences among the cytoplasmic nucleolar bodies induced by the difHowever the sizeofthese bodies vaferent treatments. ries in the presence of the different drugs studied. So with Ethidium bromide and Cordycepin the cytoplasmic (0.55 to 0.65 )un nucleolar bodies have a great size of diameter) while with c(-amanitin and Hydroxyurea are smaller. We have observed the extrusion of nucleolar bodies from nucleus to cytoplasm under the electron microscope (fig. 10 to 12) in cells treated with EB and 3'AdR in which the nuclear envelope, does not breakdown in prophase, and mitosis goes on within the nucleus (Risueiio and Moreno-D$az de la Espina, 1972). The extrusion is observed from the anaphase to early Gl; during interphaseandother mitotic stages, thus these bodies do not appear to be associated with the nuclear envelope bodies after extrussion*During the outcome of nucleolar to the cytoplasm these appear trapped by the nuclear envelope which shows discontinuity in this area (fig. 11). DISCUSSION There are a lot of evidences to think thattheCy.n.b. have a nucleolar origin. 1) They both show the same cyto chemical behaviour by silver impregnation for nucleolar material and EDTA (Moreno-DZaz de la Espina et al., is similar to both that of 1976). 2) Their structure the mitotic prenucleolar bodies and of the fibrillar part of the interphasic nucleolus. The micropuff which are also strongly impregnated by silver show a very different structure and behaviour by EDTA (Risuefio et al., In the other hand we have never observed micro 1978). after treat-puff in the cytoplasm of these cells ments. The fibrillar bodies which appearinthe nucleusafter treatments with RNA and protein synthesis inhibitors and are no thatare. extruded occasionally to the cytoplasm, impregnated by silver (Moreno-Dlaz de la Espina and Risueilo, 1977). As nucleolar materialtheseCy.n.n.couldcorrespondto: 1) nucleolar fragments pro&ed by the action of the drug which would then be extended to the cytoplamn or 2) prenucleolar bodies which would not coalesce in the NOR (as in a normal situation) by the effect of the drug, and then pass throughtothe cytoplasm. The first hypothesis does not seem very likely, because the Cordicepin, which does not causeanynucleolar fragmentation (Gj&nez-Martfn
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et al., 1973), produces cells with a large numberofcyto The second case plasmic nucleolar bodies (see table I). (Eth& fits better with our results since the three drugs dium bromide, Cordycepin and Hydroxiurea) which produce these cytoplasmic bodies, interfere with nucleolar rezfganization, (Gimenez-Martin et al., 1974; Risuefio et 1976), and produce persistent prenucleolar bodies, thus producing whiih could then pass to the cytoplasm, These bodies are produced cytoplasmic nucleolar bodies. by treatments which strongly alter nucleolar functionality (either biosynthetic activity or capacity of reorganization in telophase); and for these reason, anysomicin and cycloheximide, which do not impede neither nucleolar activity (Ferngndez-G6mez et al., 1974) nor reorganization in the telophase, do not lead to the appearance of cytoplasmic nucleolar bodies, although they produce alte rations in the nucleolar ultrastructure (Gim&-iez-Martinet al., 1974; Ferndndez-G6mez et al., 197 ). FerndndezG6mez etal.,1978have studied thefluorodeoxyuridine effects on the nucleolar cycle and it alters nucleolar dispersion in prophase and produces persistent nucleoli which are eliminated to the cytoplasm and thus could be comparable in a certain degree to the cytoplasmic nucleolar bodies described, here. Ethidium bromide and Cordycepin impair both nucleolar biosynthesis activity and nucleolar reorganization in the telophase (Risuefio et al .,1972; 1976; Gimenez-Martfn,l973, has been described as a drug with a 1974). Hydroxiurea complex action, both on DNA and on protein synthesis by Sartorelly et al., 1969, which effects varies according to the cell cycle phase (Synclair, 1965) andalso strongly affecting nucleolar ultrastructure and activity (Bernhard, 1971). The nucleolar reorganization under this drugs is altered in the similar way as Cordycepin and Ethidium bromide do. .K-amanitine do not impedes the nucleolar reorganization (Gimgnez-Martin et al., 1974; Moreno-Dlaz de la Espina and Risuefio, 1976) so the cytoplasmic nucleolar bodies would produce during prometaphase as nucleolar remnants from the nucleolar dispersion whichcould then pass to the cytoplasm through the broken nuclear envelope. Iodoacetamide interferes with the nucleolar func ionality affecting the nucleolar dispersion in onion (uii published data) and producing the cytoplasmic nucleolarbodies during the mitotic stages. All these reasons give us to think that the main factor which leads to the production of cytoplasmic nucledar bodies is the persistence of prenucleolar bodies and of nucleolar fragments in the nucleoplasm. The alterations in the nuclear envelope formation or structure (as in the case with Ethidium bromide treatment (De la Torre and Risuefio, 1973) would favour the flow of these bodies to the
cytoplasm. In relation to the mechanism of f,ormation of the cytg plasmic prenucleolar bodies, two hypothesis may be con eluded. 1) Cytoplasmic bodies would form in the cytoplasm 2) The bodies would pass to the in a disorganized form. cytoplasm after the break-down of the nuclear envelope, either during mitosis or by the effect of the drugs. The first hypothesis does not appear likely because these cytoplasmic bodies are observed as discrete bodies passing through the nuclear envelope, although the possibility of the formation of a few of them by this mechanism may not be discarded. The second possibility seems the more accurate. The persistent nucleolar fragments would be liberated during prometaphase with the break-down of the nuclear envelope, and the persistent prenucleolar bodies formed in the telophase would then pass on to the cytoplasm through the discontinuities through the nuclear envelope in formation, or those produced by drugs. In short: the cytoplasmic nucleolar bodies which appear in meristematic Allium ce a L. after treatmentswith --+ eolar functionality, drugs which fnterferewithnuc seem to correspond to remnants of the fragmented nucleolus,or prenucleolar bodies which are not fused in the NOR bythe effect of the drug. They appear to pass to the cytoplasm when the nuclear envelope breaks-down. BIBLIOGRAPHY Bernhard, W. (1969). A new staining procedure for electron microscopical cytology. J. Ultrastruct. Res.27, 250-265. Bernhard, W. (1971). Drug-induced changes in the interphase nucleus. Advances Cytopharmacology I, 50-67. Black, M.M. and Ansley, H.R. (1964) Histone staining with ammoniacal silver. Science 143, 693-695. staining of a Das, N.K. and Alfert, H. (1963).Silver its origin and fate during the nucleolar fraction, mitotic cycle. Ann. Histochim. 8-, 109-114. DSez, J.L., Marfn, M.F., Esponda, P. and Stockert, J.C. (1971). Prenucleolar bodies in the cytoplasm of meristematic cells after thermal shock. Experientia -27, 266-267. Ferndndez-GCSmez, M.E., Stockert, J.C., L6pez-Sdez, J.F. and Gimenez-Martin, G. (1969). Staining plant cell fixnucleoli with AgN03 after formalin-hydroguinone ation. Stain Tech. 44, 48-49. Ferndndez-Gbmez, M.E. zd Stockert, J.C. (1970) Metallic impregnations of nucleoli in root-tip cells. The Nucleus. -13, 149-156.
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Ferndndez-Gbmez, M.E., Risuefio, M.C. and Gimenez-Martfn, G. (1973). Effect of cycloheximide on plant cell nucleoli. Arch. Biol. (Bruxelles) 84, 417-424. Ferndndez-Gbmez, M.E., Torre, C. de E and Gimgnez-Martin, Accelerated nucleolar reorganization with G. (1972). shortened anaphase and telophase during cycloheximide inhibition of protein synthesis in onion root cells. Cytobiologie, Vol. 5, n"2, 117-124. de la Espina, S. and Ferndndez-G6mez, M.E., Moreno-Dfaz of nucleolar disperRisuefio, M.C. (1978). Alt eration sion in prophase by 5-FUdR treatment. Cell Biol. Reports 2, 237-244. Inter. Gimgnez-Martin, G., Risuefio, M.C., Fernbndez-G6mez, M.E. and Ahmadian, P. (1973). Effect ofcordycepin on the fine structure ofinterpbasa nucleoli in plant cells. Cytobiologie 1, 181-192. Gimenez-Martin, G., Torre, C. de la, Fernbndez-G6mez, M. E. and Gonz%lez-Ferndndez, A. (1974). Experimental analysis of nucleolar reorganization. J. Cell Biol. 60, 502-507. Goodpasture, C. and Bloom, S.E. (1975). Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma (Ber1.)53, 37-50. Gonzalez-Ramfrez, J. (1961) Nucleolar physiomgy. III. Consideration about the meaning of nucleolar silver impregnation in cultured HeLa cells. Biol.Inst.Estud. Med. Biol. (Mex) 19, 195-206. Ikeuchi, T., Lanbe, M., Weinfeld, H. and Sandberg, A.A. (1971). Induction of nuclear envelope around metaphase chromosomes after fusion with interphase cells. J. Cell Biol. 51, 104-115. Korson, R. (1951).A differential stain for nucleic acids. Stain Technol. 26, 265-270. silver stain for deoxyrribonucleic Korson, R. (1964).x acic. J. Histochem. Cytochem. 12, 875-879. Moreno-DPaz de la Espina, S. and Risueiio, M.C. (1976).Effeet of oj-amanitine on the nucleolus of meristematic cells of Allium cepa in interphase and mitosis: an ultrastructural analysis. Cytobiologie 12, 175-188. Moreno-D$az de la Espina, S., Risueiio, M.C, FernbndezGbmez, M.E. and Tandler, C.J. (1976). Ultrastructural study of the nucleolar cycle in meristematic cells of Allium cepa. J.MicroscopieetBiol. Cell. 25, 265-278. M.E. (1977). Moreno-DPaz de la Espina, S. and Risuefio, Fibrillar bodies in Allium cepa : an ultrastructural and cytochemical study. Biol. Cell. 30, 93-102. Paweletz, N., Siebs, W. and Lettre, R. (1967). Untersuchungen zue argentaffinreaktion des nukleolus. Z. Zellforsch. 76, 577-605. -
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Risueiio, M.C., Fernbndez-G6mez, M.E., Gimenez-Martin, G. (1973). Nucleoli under the electron microscope by silver impregnation. Mikroskopie 29, 292-298. Risuefio, M.C., Fernbndez-Gbmez, M.E., De la Torre, C.and Gimenez-MartIn, G. (1972). Effect of ethidium bromide on the fine .structure of the nucleolus in plant cells. J. Ultrastruct. Res. 39, 163-172. Risuefio, M.C., Moreno-D1z de la Espina, S., FernbndezGbmez, M.E. and Gimenez-Martfn, G. (1976). Ultrastruc tural study of nucleolar material during plant mitosys in presence of inhibitors of RNA synthesis. J. Microscopie Biol. Cell. 26, 5-18. Risueiio, M.C., Moreno-Dlaz de la Espina, S., Fendndez-G6 mez, M.E. and GimBnez-MartXn, G. (1978). Nuclear micro puffs in Allium cepa cells. I. Quantitative, ultrastructural and cytochemical study. Cytobiologie 16,209223. Risuefio, M.C. and Moreno-D$az de la Espina, S. (1978). Analysis of the mitosis in presence of RNA synthesis inhibitors in meristematic cells.Caryologia31, 43-61. Sinclair, W.K. (1965). Hydroxiurea differentialethal effects on cultured mammalian cells during the cell cycle. Science 150, 1729-1731. Sartorelly, A.C. axcreasy, W.A. (1969). Cancer ch¬herapy. Ann. REV. Pharmacol. 2, 51-72. Ferndndez-Gbmez,-M.E., Gimhez-Martln,G. Stockert, J.C., and Lbpez-Sdez, J.F. (1970) Organization of argyrophi lit nucleolar material throughout the division cycle of meristematic cells. Protoplasma 69, 265-278. Tandler, C.J. (1959). The silver reducing property of the nucleolus and the formation of prenucleolar mate rial during mitosis. Exptl. Cell Res. 17, 560-564. Torre, C. de la , Risuefio, M.C. and Gin&n=-MartZn, G. (1973). Ethidium bromide and its effectsonthe nuclear membrane cycle. Protoplasma 76, 363-371. Venable, J.H. and Gogueshall, KT(1965) A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408.
Received: 1st August 1978 6th November 1978
Revised Version
Received:
Accepted: 20th November 1978