some observations on the nucleolus in spirogyra · j. cell sci. 4, 3-15 (1969) printed in great...

J. Cell Sci. 4, 3-15 (1969)Printed in Great Britain

SOME OBSERVATIONS ON THE NUCLEOLUS

IN SPIROGYRA

E.G.JORDAN* AND M. B. E. GODWARDDepartment of Botany, Queen Mary College, Mile End Road, London, E. 1, England

SUMMARYSeveral species of Spirogyra have been collected and fixed for electron microscopy in either

2 % osmium tetroxide or 5 % glutaraldehyde. Mitotic cells were selected with the light micro-scope from filaments of Spirogyra embedded in Epon.

Evidence is given for an interphase cycle in the nucleolus of Spirogyra. A clear associationbetween nucleolar material and chromosomes occurs at mitosis. The breakdown of the nucleolusis shown to occur in two stages, one as a consequence of the withdrawal of the nucleolarchromosomes, and the other after telophase in the new nucleus. The significance of thesefindings is discussed.

INTRODUCTION

The nucleolus is usually the only component which can be seen easily in the inter-phase nucleus by light microscopy.

For some time little was known about it apart from its association with a particularchromosome region (Heitz & Bauer, 1933; McClintock, 1934). It is only recently that itsmajor role in ribosome production has been clearly demonstrated (Brown & Gurdon,1964; Perry, 1966; Wallace & Birnstiel, 1966; Birnstiel, Wallace, Sirlin & Fischberg,1966). Now that this is known it is more meaningful to attempt explanations of its be-haviour at mitosis. It is commonly taught that the nucleolus disappears at nuclear divi-sion to reappear again in the new daughter nuclei. Studies of this phenomenon with theelectron microscope have been presented (Swift, 1959; Lafontaine & Chouinard, 1963).There are cases in which the nucleolus persists at mitosis (Brown & Emery, 1957;Hsu, Humphrey & Somers, 1964). It would seem better in the light of some studies(Hsu, Arrighi, Klevecz & BrinkJey, 1965) to refer to degrees of persistence or dis-appearance rather than to speak in absolute terms.

Spirogyra, although having specific variation, shows a very high degree of persistenceofthenucleoli(Geitler, 19350,6; Godward, 1950,1953; Godward & Newnham, 1965).The nucleoli do not persist throughout mitosis as spherical bodies, however, but show aremarkable redistribution of their material around the chromosomes. It is this featurewhich has been studied here by electron microscopy.

• Present address: Department of Biology, Queen Elizabeth College, Campden Hill, London,W. 8.

4 E. G. Jordan and M. B. E. Godward

MATERIALS AND METHODS

Spirogyra was collected from the field and grown in Godward's solution (Godward,1942) enriched with 10% soil extract. The mitotic cells were obtained by growing theSpirogyra in water-cooled culture chambers with a light/dark cycle of 18 h light and6 h dark. The cells were grown for 3-5 days in these conditions and fixed at a timewhen the mitotic index was highest. This time varied, since the material was never verywell synchronized, but about 2 h after the beginning of the dark period was generallyfound to be best. Preparations for the light microscope using the iron alum aceto-carmine technique (Godward, 1948) were used to check the mitotic index and find themost suitable time for fixation.

Fixations were made in 2% osmium tetroxide in veronal acetate buffer at pH 7-0or in 5 % glutaraldehyde with 5 % sucrose in cacodylate buffer at pH 7-0. Theglutaraldehyde solution was first neutralized with 1 N sodium hydroxide. The fixationwas carried out at 0-4 °C. The material fixed in glutaraldehyde was washed in bufferovernight and then treated with 2% osmium tetroxide. Alcohol dehydration wasfollowed by Epon embedding.

The resin was polymerized in shallow plastic dishes to give a thin disk which couldbe viewed subsequently with the light microscope. Cells in mitosis were identified andselected in this way. The microtomes used in this study were the Cambridge Huxleyand the LKB Ultratome. The cylindrical cells were oriented vertically to the knifeedge for cutting. This ensured that there was no sudden change in texture due tocutting the longitudinal cell walls because they were always in contact with the knifethroughout the cutting process. Sections were mounted on carbon-Formvar films ordirectly on to copper grids; 2% aqueous uranyl acetate was used routinely as a stain.Photographs were taken with Siemens Elmiskop 1 A, Zeiss EM 9, Jeol Jem 7, or AEIEM 6 electron microscopes.

EXPERIMENTAL RESULTS

The structure of the nucleolus is clearly related to the chromosome material whichhas been shown to be the organizing region of the nucleolar organizing chromosome(Godward & Jordan, 1965). The terms used to describe the two zones of the nucleolushave been well defined in the work of Bernhard (1966). The nucleolar material adjacentto the chromosome is the fibrillar region shown to be the first region labelled by iso-topic RNA precursors (Granboulan & Granboulan, 1965; Bernhard, 1966). The restof the spherical nucleolus is composed of the granular region characterized by 150-Agranules.

In Spirogyra the distinction between the fibrillar and the granular material of thenucleolus is much clearer in some nucleoli than others. There seems to be a correlationbetween smaller early interphase nucleoli and this sharper distinction between the twozones shown in Fig. 1. There is another feature which varies with the age of thenucleolus and that is the distribution of the chromosome within it. The two extremes ofthis are shown in Figs. 1 and 2. In the first, which is the smaller early interphase

Nucleolus of Spirogyra 5

nucleolus, the chromosome is restricted to more or less two regions and these are alsothe only places where the fibrillar material is seen. In Fig. 2, which is much larger andat a later stage of interphase, the chromosome material is distributed as smaller unitsthroughout the nucleolus and the fibrillar regions adjacent to it cannot be so clearlydefined as in early interphase. The chromosome region at this later stage would appearto be a thread of uniform thickness around 0-25 fi in diameter which meanders throughthe nucleolus (see Fig. 2, where both transverse and longitudinal sections of thischromosome region can be seen).

The fibrillar region which can be so clearly demonstrated at early interphase andwhich can still be defined, though not so easily, at the end of interphase, by prophasehas completely disappeared, at least as a separate definable zone.

After prophase and before metaphase all the chromosomes move into the nucleolus.This association of the material of the nucleolus with the chromosomes persists untiltelophase.

The nucleolar material around the chromosomes in mitosis is not demonstrablydifferent from the granular material of interphase (Figs. 2, 8). It has an overall reticularstructure with a thickness around o-i fi (Fig. 3). The reticular nature of the nucleolarmaterial becomes very clear as the initial chromosome component of the nucleolus,the organizing chromosomes, is withdrawn from the interstices (Fig. 3). This reticularstructure would be called the nucleolonema by some workers (Estable, 1966). Theorganizing chromosome can be identified in the prophase picture (Fig. 3) but in laterstages of mitosis it is not possible to distinguish it from the other chromosomes. Atprometaphase (Fig. 4) and all later stages all the chromosomes are included in thenucleolar material. This indicates that although the organizing chromosomes had aregion which was within the nucleolus, this is withdrawn when all the chromosomescondense, only to return to the nucleolus again with the other chromosomes later.There does not appear to be any structural continuity between the chromosomes andthe nucleolar material in this mitotic association. There is, however, a closer associationbetween them in anaphase than in metaphase (compare Figs. 6, 5).

The precise stage at which the anaphase separation starts has not been seen, but theearly anaphase picture (Fig. 6) shows the arrangement immediately after separation.The chromosomes are still surrounded by nucleolar material and it is not possible tosay which surfaces of the chromosomes were once in contact as chromatids because allthe surfaces of the chromosomes are covered with the nucleolar material and they arecompletely embedded in it.

In the telophase stage (Fig. 7) no chromosome can be seen. This may be due to thefact that they were missed by the plane of the sections. No pictures have yet beenobtained showing the condition at the time when the chromosomes leave the nucleolarmaterial. The nature of the nucleolar material of telophase is clearly the same as thatof the granular zone of interphase (Fig. 9). The new nuclear membranes are formedby the coalescence of vesicles around these two groups of nucleolar material (Figs. 7, 9).

The few pictures that have been obtained of post-telophase nuclei show that thenucleolar material breaks down completely prior to being reorganized into a nucleolus.It is clear that the granular material persists at least in part through mitosis and the


granules of this appear not to be destroyed at the time of the breakdown of nuclearmaterial which occurs in telophase (Fig. io). The nature of the organizing process hasnot yet been elucidated but the evidence of Fig. io points to the preliminary formationof a dense body resembling in structure the fibrillar zone of the interphase nucleolus.Figure 11 is a newly formed nucleolus of the same species as Fig. io and a chromosomebody, possibly a chromocentre, adjacent to the organizing region of the organizingchromosome can be seen. Fibrillar regions appear in the region of the organizingchromosome.

DISCUSSION

From the increase in size and change in distribution of components, particularlythe chromosome, there would appear to be an interphase cycle in the nucleolus ofSpirogyra. At the beginning of the cycle the nucleolus is smaller and the chromosomeregions are clumped together; later the chromosome material is more evenly distributedand the nucleolus is larger in size. The appearance of the fibrillar region is also asso-ciated with this cycle; at the start it is more distinct from the granular material thanat the end.

Among the most interesting aspects of this nucleolar behaviour are the two steps inthe breakdown process, the first at prophase consequent on the withdrawal of thenucleolar organizing chromosome; and the second at telophase, where a completedispersal precedes the formation of the new nucleolus. After the prophase withdrawalof chromosome the nucleolar material still has a definite structure showing clearly thatthe first breakdown process is not a complete one.

It is tempting to think that inside the sheets and strands of the nucleolar materialremaining after the first step in the breakdown process there are threads of DNAgiving them their structural continuity. The peripheral nucleoli of the newt oocytehave been shown to have a ring of DNA within them (Miller, 1964). Chromosomemodels have been proposed from considerations of lampbrush chromosome structureimplicating the production of detached copies of the genie DNA (Callan, 1967;Whitehouse, 1967). Such proposals fit well with our suggested interpretation of per-sistent nucleolar structure. The idea that there is a possible DNA backbone to thenucleolonema has been suggested by Lettre, Siebs & Poivelity (1966).

The second stage in the breakdown, the complete dispersal which takes place aftertelophase, presents two questions. First, why is it that the structure which clearlyexists throughout mitosis in the nucleolar material breaks down; and secondly, whyis it necessary that this material is reorganized into a nucleolus rather than remainingdispersed in the nucleus? This latter question is based on the assumption, which isdifficult to prove from photographs, that the material which is dispersed collects againat the site of the nucleolus. Whether this is a false assumption or not, it is clear that thenucleolus represents a large accumulation of material, some being RNA which wasproduced earlier at this particular site on the chromosome. It is possible that in inter-phase some control over the release of this material and, therefore, over proteinsynthesis, is exercised by virtue of the fact that it is held as an aggregate in the nucleolus.

Nucleolus of Spirogyra 7

An answer to the first question relating to the breakdown of the persistent nucleolusmaterial must await a clearer understanding of the basis of the structure which persistsinitially.

The remarkable appearance of the mitotic figures with all the chromosomes associatedwith the nucleolar material does not suggest any simple explanation. It is clear fromother organisms that this behaviour of the nucleolus is in no way necessary for mitosisand even here a normal spindle seems to be operating. It may be viewed as a mechanismfor the distribution of nucleolar material to the two new cells. Whatever the explanationit is difficult to envisage the nature of the forces which bring the chromosomes into thenucleolar material and which maintain this relationship through mitosis.

The nucleolar material which persists through mitosis is the granular zone materialof the nucleolus. The fate of the fibrillar zone is indicated by its changes in interphase,becoming progressively more indistinct from the granular zone and at prophase ceasingto exist as a separate definable region.

The prophase reticular structure, or nucleolonema (Estable, 1966), consists entirelyof granular zone material. But this not to say that the nucleolonema is always composedonly of granular material, since the structure which would most merit this name inearly interphase nuclei because of its reticular appearance is the fibrillar material.

The nucleolar material which persists through mitosis shows no sign of a distinctfibrillar region. The explanation for this absence of fibrillar material in mitosis couldquite easily be the inactivity of the nucleolus organizing region during this time. It hasbeen shown that this zone is the first to show any activity in labelling experimentswith RNA precursors (Granboulan & Granboulan, 1965; Bernhard, 1966), and thiswould seem to confirm that the fibrillar material is the first-formed material, and isvisible only if fixation coincides with its synthesis.

REFERENCES

BERNHARD, W. (1966). Ultrastructural aspects of normal and pathological nucleolus in mam-malian cells. Natn. Cancer Inst. Monogr. 23, 13-38.

BIRNSTIEL, M. L., WALLACE, H., SIRLIN, J. L. & FISCHBERC, M. (1966). Localization of theribosomal DNA complement in the nucleolar organizer region of Xenopus laevis. Natn.Cancer Inst. Monogr. 23, 431-447.

BROWN, D. D. & GURDON, J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolatemutant of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 51, 139—146.

BROWN, W. V. & EMERY, H. P. (1957). Persistent nucleoli and grass systematics. Am.J. Bot. 44,585-59°-

CALLAN, H. G. (1967). The organization of genetic units in chromosomes. J. Cell Sci. 2, 1—7.ESTABLE, C. (1966). Morphology, structure and dynamics of the nucleolonema. Natn. Cancer

Inst. Monogr. 23, 91-105.GEITLER, L. (1935a). Neue Untersuchungen iiber die Mitosen von Spirogyra. Arch. Protistenk.

85, 10-19.GEITLER, L. (19356). Untersuchungen iiber den Kernbau von Spirogyra mirtels feulgens

Nuklealfarbung. Ber. dt. bot. Ges. 53, 270-276.GODWARD, M. B.E. (1942). Life cycle of Stigeoclonium amoenum Kutz. New Phytol.41, 293-301.GODWARD, M. B. E. (1948). The iron alum acetocarmine method for algae. Nature, Lond. 161,

203.

GODWARD, M. B. E. (1950). On the nucleolus and the nucleolar-organising chromosomes ofSpirogyra. Ann. Bot. 14, 39-53.


GODWARD, M. B. E. (1953). Geitler's nucleolar substance in Spirogyra. Ann. Bot. 17, 403-416.GODWARD, M. B. E. & JORDAN, E. G. (1965). Electron microscopy of the nucleolus of Spirogyra

britannica and Spirogyra ellipsospora. Jl R. microsc. Soc. 84, 347—360.GODWARD, M. B. E. & NEWNHAM, R. E. (1965). Cytotaxonomy of Spirogyra. II . 5. neglecta

(Hass) Kutz., S. punctulata Jao, S. majuscular (Kutz.) Czurda emend., 5 . ellipsosporaTranseau, S. porticalis (Muller) Cleve. J. Linn. Soc. (Bot.) 59, 99-110.

GRANBOULAN, N. & GRANBOULAN, P. (1965). Cytochimie ultra-structurale du nucleole. II.Etudes des sites de synthese du RNA dans le nucleole et la noyau. Expl Cell Res. 38, 604—619.

HEITZ, E. & BAUER, H. (1933). Beweise filr die chromosomennatur der kernschleifen in denknauelkernen von Bibio hortulanus L. Z. Zellforsch mikrosk. Anat. 17, 67-82.

Hsu, T . C , ARRIGHI, F. E., KLEVECZ, R. R. & BRINKLEY, B. R. (1965). The nucleoli in mitoticdivisions of mammalian cells in vitro. J. Cell Biol. 26, 539-555.

Hsu, T . C , HUMPHREY, R. M. & SOMERS, C. E. (1964). Persistent nucleoli in animal cellsfollowing treatment with fluorodeoxyuridine and thymidine. Expl Cell Res. 33, 74-77.

LAFONTAINE, J. G. & CHOUINARD, L. A. (1963). A correlated light and electron microscopestudy of the nucleolar material during mitosis in Vicia faba. J. Cell Biol. 17, 167-201.

LETTRE, R., SIEBS, W. & POIVELETY, N. (1966). Morphological observations on the nucleolus ofcells in tissue culture, with special regard to its composition. Natn. Cancer Inst. Monogr. 23,107-123.

MCCLINTOCK, B. (1934). The relation of a particular chromosome element to the developmentof the nucleoli in Zea mays. Z. Zellforsch. mikrosk. Anat. 21, 294-328.

MILLER, O. L. JR. (1964). Extrachromosomal nucleolar DNA in amphibian oocytes. J. CellBiol. 23, 60A.

PERRY, R. P. (1966). On ribosome biogenesis. Natn. Cancer Inst. Monogr. 23, 527-545.SWIFT, H. H. (1959). Nucleolar function. In Symposium on Molecular Biology (ed. R. E. Zirkle),

pp. 266—303. Chicago: University of Chicago Press.WALLACE, H. & BIRNSTIEL, M. I. (1966). Ribosomal cistrons and the nucleolar organizer.

Biochim. biophys. Acta 114, 296—310.WHITEHOUSE, H. L. K. (1967). A cycloid model for the chromosome. J. Cell Set. 2, 9-22.

{Received 19 August 1967—Revised 5 June 1968)

Fig. 1. Spirogyra neglecta x 25000. Nucleolus at the start of interphase. c, chromo-some; / , fibrillar material; g, granular material.

Fig. 2. Spirogyra neglecta x 18500. Nucleolus at the end of interphase. The chromo-some, c, is distributed in the form of a meandering thread which has been cut in bothtransverse, t, and longitudinal, /, section. The fibrillar material, / , is not so strikinglydifferent from the granular, g, at this stage.

Nucleolus of Spirogyra

io E.G. Jordan and M. B. E. Godward

Fig. 3. Spirogyra britannica x 14000. Nucleolus at prophase. The chromosome region,c, has withdrawn and condensed from the nucleolus leaving it as a reticulum.

Fig. 4. Large unidentified species x 6000. Prometaphase condition. The chromosomes,c, have migrated into the nucleolar material, nm. For high power see Fig. 8.

Fig. 5. Spirogyra neglecta x 11000. Metaphase. The chromosomes, c, are aligned at theequator together with the nucleolar material, nm.

Fig. 6. Spirogyra neglecta x 8500. Anaphase. Compacting of the nucleolar materialoccurs as the two groups of chromosomes, c, and nucleolar material, nm, separate.

Fig. 7. Large unidentified species x 3000. The telophase nuclei are masses of nucleolarmaterial surrounded by the nuclear envelopes. Chromosomes do not appear in thissection. For high power see Fig. 9.


. V.

12 E.G. Jordan and M. B. E. God-ward

Fig. 8. Large unidentified species x 35000. Prometaphase. The association betweennucleolar material, nm, and chromosomes, c, does not interfere with normal spindleproduction, m, spindle microtubules.

Fig. 9. Large unidentified species x 30000. Telophase. The nuclear envelope, ne, isthe result of the coalescence of vesicles which collect on the surface of the nucleolarmaterial, nm.


Fig. 10. Spirogyra submargaritata x 30000. Telophase nucleus. The nucleolar materialwhich filled the whole nucleus earlier has been largely dispersed into ribosome-likegranules throughout the nucleus. The two arrowed regions are probably chromatin,and the darker structure could be a developing nucleolus.

Fig. 11. Spirogyra submargaritata x 24000. A young nucleolus in an early interphasecell. The nucleolus has fibrillar regions, / , associated with a chromosome region, c,which is adjacent to a chromocentre, cc.

some observations on the nucleolus in spirogyra · j. cell sci. 4, 3-15 (1969) printed in great...

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