J. Cell Set. 7 ) 35-48(1970) 35

Printed in Great Britain

ELECTRON-MICROSCOPE OBSERVATIONS ON

THE STRUCTURE OF CONDENSED CHRO-

MATIN: EVIDENCE FOR ORDERLY ARRAYS OF

UNIT THREADS ON THE SURFACE OF CHICKEN

ERYTHROCYTE NUCLEI

A. C. EVERID, J. V. SMALL* AND H. G. DAVIESMedical Research Council Biophysics Unit, Department of Biophysics, King'sCollege, 26-29 Drtiry Lane, London, W.C.z, England

SUMMARY

A unit thread has been identified by electron microscopy as the common structural element inthe condensed chromatin of a variety of cell nuclei. From the previous studies of thin sectionsnormal to the nuclear envelope it was concluded that the unit thread, of diameter about 17 nmbut varying somewhat depending on fixation, packed with spacings of about 28 nm on the sur-face of the nucleus to form one or more layers. Thin sections tangential to the nuclear envelope,described in this paper, reveal directly the degree of order within the surface layer; there aresmall areas or patches in which the units are regularly arranged. Units are also orderly arrangedaround the pores in the nuclear envelope. Unit threads are less easily visible in electron micro-graphs of mature erythrocytes than at earlier stages of development but the clarity with whichthey can be seen is increased by a brief treatment prior to fixation with sodium citrate.

INTRODUCTION

Recent electron-microscope studies show that the DNA and protein molecules inthe condensed chromatin, heterochromatin, of intact interphase nuclei from certainvertebrate cells are structurally organized at 2 levels (Davies, 1968 a, b\ Davies &Small, 1968), first, into thread-like units or unit threads. These units, which areabout 17 nm in diameter and are possibly tubular, pack about 28 nm apart, to formone or more layers on the surface of the nucleus in contact with the nuclear envelope.In the interior the unit threads appear to be more or less randomly arranged. Theseconclusions are based on micrographs of sections normal to the nuclear envelope.When sections tangential to the nucleus are examined, as in the present study onchicken erythrocytes, they directly reveal the arrangement of the unit threads withinthe surface layer. These face views will be related to the tangential sections (Davies &Small, 1968) through the nuclear envelope-limited sheets of chromatin which havebeen found particularly in the blood cells of many vertebrates and also other tissues(Mollo, Canese & Stramignoni, 1969). Certain factors governing the clarity withwhich the unit threads can be seen in the electron micrographs are also discussed.Thread-like structures intermediate in size between DNA molecules and chromosomes

• Present address: Institute of Biophysics, Aarhus University, Aarhus 8000, Denmark.

3-2

36 A. C. Everid, J. V. Small and H. G. Davies

are particularly evident in preparations of nuclei which have been disrupted and spreadon an air/water surface (Gall, 1966; Ris, 1967; DuPraw, 1965; Wolfe, 1968).

MATERIALS AND METHODS

Mature erythrocytes and reticulocytes were examined from circulating blood of chicks, 9, 12,16 and 17-day embryos, and 4 days and 3 weeks after hatching. The methods employed here forfixing, embedding and staining blood cells have been described in detail elsewhere (Achong &Epstein, 1965; J. V. Small & H. G. Davies, in preparation). Briefly, fixation of pellets formedby centnfuging blood taken up in narrow-bore tubes by capillary action was carried out inbuffered glutaraldehyde followed by OsO4: embedding was in Araldite: sections were cut withdiamond knives on a Cambridge-Huxley microtome and stained in uranyl acetate followed bylead citrate. Electron micrographs were taken on Siemens Elmiskops I and IA at 80 or 100 kV,objective aperture 50 /(m, at magnifications up to 40000. Cells were also treated prior to fixationwith a chelating agent, by mixing 1 part of whole blood with 3 parts of a solution containingo 4 M sucrose and 2 mM sodium citrate for periods of 30 min to 2 h. Section thicknesses weredetermined from measurements of the widths of folds (Small, 1968).

OBSERVATIONS

The clarity with which the structural units can be seen in chick erythrocyte nuclei,in sections either normal or tangential to the nuclear envelope, seems to depend on twofactors, first the maturation stage. The unit threads are more easily seen in late poly-chromatophil erythroblasts and reticulocytes than in mature erythrocytes, and seemto be more visible in the nuclei of mature erythrocytes from 17-day chick embryo,than in the, on average, older mature erythrocytes from 3-week chick. The stages inmaturation are characterized by the number of cytoplasmic ribosomes. In the cyto-plasm of polychromatophil erythroblasts there are numerous ribosomes, in reticulo-cytes few, and ribosomes are almost completely absent from mature erythrocytes.Second, the visibility of the units in the electron micrographs is increased by brieflytreating the erythrocytes with sodium citrate prior to fixation. This increase is mostmarked for the mature erythrocytes in the blood of 3-week chicks. The reasons forthese visibility changes with ageing and citrate treatment are not yet fully documentedand we hope to report on them in a later paper. They probably depend on severalfactors including changes in the degree of packing and coiling of the unit threads,alteration in the concentration of a matrix substance which may surround the threads(J. V. Small & H. G. Davies, in preparation) and their relative stainability. A pro-gressive decrease in nuclear volume during the maturation of erythrocytes, suggestingcloser packing, is well known from light-microscope observations (for example, seeLucas & Jamroz, 1961). The type of fixation also seems to have a role in determiningvisibility since threads were clearly distinguishable in the chromatin of mature ery-throcytes in adult chicken after fixation in osmium tetroxide only (Davies, 19680).This paper, therefore, is mainly concerned with presenting the electron micrographsshowing the arrangement of the structural units on the surface of the nucleus. So as tomake the data clear, previous nomenclature (Davies, 1968a) and findings on sectionsnormal to the nuclear envelope are presented first.

Unit threads of condensed chromatin 37

Sections perpendicular to the nuclear envelope

Figure 1 shows a section through a reticulocyte from a 3-week chick. The units arearranged on the surface of the nucleus so as to appear, in end-on view, as rows of'granules' (see arrow at a in Fig. 1) or in side-view as a band (see b2 in Figs. 2, 4). Asecond layer of unit threads is sometimes seen (see bj. in Figs. 2, 3) and these darkerbands are separated from each other and the interior membrane of the envelope byrelatively less electron-dense bands bj, bj, etc. (see Fig. 4). Throughout the condensedchromatin there are groups of granules (see untaded arrows in Fig. 1), indicating thatgroups of unit threads pack parallel to each other. When the units lie at an angle to theplane of the section they appear as short bands. Reticulocytes (Fig. 1) and erythro-cytes, after citrate treatment and when compared with controls, characteristicallyhave a less homogeneous distribution of haemoglobin throughout the cytoplasm.Furthermore small stain-free spaces appear within the intranuclear haemoglobin-containing regions surrounding the condensed chromatin. These effects suggest someswelling due to citrate treatment. That the cells do not lose haemoglobin was indicatedby its absence from the citrate solution after removal of the cells by centrifugation. Inthe cell shown in Fig. 1 the fine structure of the bulk of the chromatin clearly indicatesthe presence of the unit thread but there are small areas (around X in Fig. 1) whichseem to be more finely fibrous. Such regions appear in untreated reticulocytes and inmature erythrocytes from 17-day embryos and what they represent has yet to beascertained. In previous measurements (Davies & Small, 1968) on mature erythro-cytes from 17-day embryos, after fixation in glutaraldehyde plus OsO4, the averagediameter of the units was 17-4 nm (range 15-20 ran) and their separation, whenoriented on the envelope, was 28 ± 4 nm.

Sections tangential to the nuclear envelope

Untreated cells. Tangential sections through nuclei from reticulocytes in 4-daychick (Figs. 5, 7, 8) reveal the presence of many long threads, up to about 250 nm inlength. In one series of measurements on well-defined threads, the width was15-19 nm, average separation 30 nm (range 27-32 nm). Threads are frequently seen ingroups or patches: in each patch several threads, which may be gently curving orapproximately straight, lie parallel to each other. The angle at which the threads lievaries from one ordered patch to the other.

In the mature erythrocytes from 4-day chick blood the unit threads have con-siderably lowered visibility and are often difficult to distinguish at all (compareFigs. 8, 10 of the adjacent cells shown in Fig. 9). However, in those surface views wherethe units could be seen their widths, 15-19 nm, and separations, 29 nm average, wereapproximately equal to those of the reticulocytes. Figure 6 shows 2 ordered patches(at arrows) on the surface of a mature erythrocyte from a 17-day chick embryo.

Cells treated with citrate. In tangential sections from the nuclei of mature erythro-cytes in 3-week chick the unit threads were difficult to distinguish. However, aftercitrate treatment long threads could clearly be distinguished (Figs. 11-20). In oneseries of measurements the average width was 21 nm (range 17-24 nm), the average

38 A. C. Everid, J. V. Small and H. G. Davies

separation was 30-7 nm (range 29-8-31-5 nm) and lengths were up to 300 nm. Theelectron density of the individual units in the ordered patches varies from one patch tothe next (see a, b, in Fig. 16). The least-dense threads probably correspond to a singlelayer, denser threads arising by overlapping of threads in the 62, b^ layers, Thisinterpretation receives some support from the observation that denser threads are onaverage shorter, as might be expected since they acquire an additional condition,namely geometric overlap of the threads in the surface layer (b 2) and the one underit (b4).

Sometimes threads appear as more or less solid structures, sometimes electron-dense lines lie at an angle to their length, and frequently they consist of 2 parallellines, an appearance consistent with the earlier suggestion (Davies, 1968 a) that theymay be tubular. The projections, also reported earlier, traversing the spaces betweenthe threads can also be distinguished.

The nuclear pores in these tangential sections appear as semi-transparent, approxi-mately circular, spaces about 100 nm in diameter. Units are arranged around thenuclear pore on arcs of circles or parts of polygons centred on the pore centre (Fig. 11).

DISCUSSION

In sections perpendicular to the nuclear envelope the rows of equispaced granulesin bands £2 and 64 were shown (Davies, 1968 a) to be end-on views of groups ofthreads lying parallel to the inner component of the nuclear envelope. Tilting thesection through small angles of ±25° destroyed the granular appearance in the bandsnear the envelope as well as in the interior. The tangential sections described in thispaper directly reveal the presence of ordered patches on the surface of the chickerythrocyte nucleus, in which numbers of threads he parallel to each other and followstraight or gently curving lines. The regular disposition of threads around the nuclearpores indicates that the latter act as local centres for 'crystallization'. The fact thatthreads up to 300 nm or more in length can be seen in these tangential sections isdue to their lying on the gently curving plane of the nuclear envelope, which planecorresponds to that of the thin section. In the interior chromatin the unit threads arecoiled and hence only short segments are seen in thin sections.

The packing of the unit threads within each chromatin body appears to be whatcan be expected from physical considerations (Davies, 1968a; Bernal, 1964; Kavanagh,1965). In further model experiments, threads of spaghetti in liquid gelatin have beenshaken in a smooth-walled container. The crystalline patches on the surface, which canbe examined after the gel is set, are similar to the ordered patches of unit threads shownin our electron micrographs.

Electron micrographs of tangential sections through the envelope-limited sheets ofchromatin associated with the nuclei of heterophilic granulocytes of the lampreyshowed small groups of threads lying parallel to each other, diameter about 17 nm andcentre-to-centre separation about 30 nm (Davies & Small, 1968). It was suggestedthat these sheets of chromatin were monolayers of unit threads, confined on both sidesby nuclear envelope. They presumably arise by processes involving delamination from

Unit threads of condensed cliromatin 39

the main body of chromatin in the cell nucleus. The similarity in geometry betweenthe 2 sets of tangential sections further supports the hypothesis that sheets are mono-layers of the unit thread found in condensed chromatin.

Our results indicate that the diameter of the unit thread in citrate-treated matureerythrocytes, about 21 nm, is about 20% higher than in the untreated cells, where itis about 17 nm. If this result is confirmed and is not due, for example, to problems inmeasuring structures of differing relative electron density in the micrographs, then itwill be similar to that of Tokuyasu, Madden & Zeldis (1968). They reported an increasein the diameter of threads in the chromatin of human lymphocytes from about 15 nm, orless, in intact cells to about 20 nm or more, after disturbance due to mild lysis. Wolfe &Grim (1967) presented evidence which suggested that fibres in amphibian erythro-cytes changed from about 10 nm in the intact cell to about 25 nm after spreading on aLangmuir trough. These changes (Wolfe & Grim, 1967) upon disruption are muchlarger than the changes reported here or by Tokuyasu et al. (1968) and the dimensionsof the threads in the intact cell are smaller than those reported here in intact chickenerythrocytes or newt lymphocytes, where the diameter was 16-23 n m (Davies, 1968a).Subsequently Wolfe (1968) found that when newt erythrocytes were prefixed withformalin the diameter of the spread fibres was significantly reduced from about 25 nmto 12 nm. The reason for these changes in dimensions is not yet elucidated but, apartfrom the problems of accurate measurement, the small alteration in diameter and theincreased visibility of the threads after citrate treatment in our experiments might, wesuggest, be accounted for by transfer of substance to the unit thread from the relativelyless electron-dense regions which apparently surround them.

Hyde (1964) reported that when nuclei already isolated from pea seedlings in asolution containing sucrose and calcium chloride were further treated with sodiumcitrate, threads about 16 nm in diameter became visible. It was suggested that citrateremoved a substance surrounding the threads. Allfrey, Stern, Mirsky & Saetren(1952) reported the removal of much soluble protein during the isolation of nuclei in acitrate-containing medium. However, conditions in our experiments during citratetreatment are probably quite different from those reported above. Intact cells wereonly briefly treated and under conditions where there was no loss of cytoplasmicprotein, namely haemoglobin, although this does not preclude an intracellular re-distribution of material, nor chelation of positive ions from chromatin therebyincreasing the staining of the unit thread by uranyl ions. Hyde (1964) found no evi-dence for a regular arrangement of the 16-nm threads in chromatin from pea seedling.When interphase nuclei of mammalian cells in vitro were treated with hypotonicsolution swelling occurred (Brinkley, 1967) which 'resulted in decondensation ofchromatin along the inner surface of the nuclear envelope, revealing a regular shapedarray of 150-200 A particles'. We would assume that these particles are the end-onviews of threads and note that their dimensions are similar to those reported here.

Embryonic erythrocytes from 15-day chick embryos form heterokaryons withmouse A9 cells more readily and their nuclei are subsequently reactivated into meta-bolic activity more quickly than if adult hen erythrocytes are used (Harris, Side-bottom, Grace & Bramwell, 1969). At 15 days about 10% of the erythrocytes are at the

40 A. C. Ever id, J. V. Small and H. G. Davies

polychromatophil stage and a fraction of the remaining population are reticulocytes(J. V. Small, unpublished). Our electron micrographs indicate that the visibility of theunit threads diminishes as reticulocytes mature into erythrocytes and that they mayfurther diminish with ageing. We therefore suggest there may be a correlation betweenthe findings on the rate of reactivation of the erythrocyte nucleus and the morpho-logical observations reported in this paper.

We are pleased to thank Professor Sir John Randall, F.R.S., and Professor M. H. F. Wilkins,F.R.S., for continued support and interest. Two of us (A. C. E. and J. V. S.) acknowledgesupport from the Science Research Council. We are also indebted to Mrs F. Collier for photo-graphic assistance.

REFERENCES

ACHONG, B. G. & EPSTEIN, M. A. (1965). A method for preparing microsamples of suspendedcells for light and electron microscopy. Jl R. microsc. Soc. 84, 107-110.

ALLFREY, V., STERN, H., MIRSKY, A. E. & SAETREN, H. (1952). The isolation of cell nuclei innon-aqueous media. J. gen. Physwl. 35, 529-554.

BERNAL, J. D. (1964). The structure of liquids. Proc. R. Soc. A 280, 299-322.BRINKLEY, B. R. (1967). Effects of hypotonic culture media on the ultrastructure of mammalian

cells in vitro. J. Cell Btol. 35, 17 A.DAVIES, H. G. (1968a). Electron-microscope observations on the organization of heterochroma-

tin in certain cells. J. Cell Sci. 3, 129-150.DAVIES, H. G. (19686). Topographical observations on the envelope-limited sheets of chromatin

associated with the cell nucleus. In Proc. 4th Europ. Reg. Conf. Electron Microsc. vol. 2 (ed.D. S. Bocciarelh), pp. 175-176. Rome: Tipografia Poliglotta Vaticana.

DAVIES, H. G. & SMALL, J. V. (1968). Structural units in chromatin and their orientation onmembranes. Nature, Lond. 217, 1122-1125.

DUPRAW, E J. (1965). Macromolecular organisation of nuclei and chromosomes. A foldedfibre model based on whole mount electron microscopy. Nature, Lond. 206, 338-343.

GALL, J. G. (1966). Chromosome fibres studied by a spreading technique. Chromosoma 20,221-233.

HARRIS, H., SIDEBOTTOM, E., GRACE, D. M. & BRAMWELL, M. E. (1969). The expression ofgenetic information, a study with hybnd animal cells. J. Cell Sci 4, 499-525.

HYDE, B. B. (1964). A structural component of chromatin. In The Nucleohutones (ed. J. Bonner& P. Ts'o), pp. 163-166. San Francisco. Holden-Day.

KAVANAGH, J. L. (1965). Structure and Function in Biological Membranes, pp. 77-80. SanFrancisco: Holden-Day.

LUCAS, A M. & JAMROZ, C. (1961). Atlas of Avian Haematology. Agriculture Monograph No. 25.Washington D.C.: U.S. Department of Agriculture.

MOLLO, F., CANESE, M G. & STRAMIGNONI, A. (1969) Nuclear sheets in epithelial and con-nective tissue cells. Nature, Lond. 221, 869-870.

Ris, H. (1967). Ultrastructure of animal chromosomes. In Regulation of Nucleic Acid and ProteinBiosynthesis (ed. V. V. Komngsberger & L. Bosch), pp. 11-21. Amsterdam. Elsevier.

SMALL, J. V. (1968). Measurement of section thickness. In Proc. 4th Europ. Reg. Conf. ElectronMicrosc. vol 1 (ed. D. S. Bocciarelli), pp. 609—610. Rome: Tipografia Poliglotta Vaticana.

TOKUYASU, K., MADDEN, S. C. & ZELDIS, L. J. (1968). Fine structural alterations of interphasenuclei of lymphocytes stimulated to growth activity in vitro. J. Cell Bwl. 39, 630-660

WOLFE, S. L. (1968). The effect of prefixation on the diameter of chromosome fibers isolated bythe Langmuir trough-critical point method. J. Cell Bwl. 37, 610-620.

WOLFE, S. L. & GRIM, J. N. (1967). The relationship of isolated chromosome fibres to the fibresof the embedded nucleus. J. Ultrastruct. Res. 19, 382-397.

(Received 2 January 1970)

Unit threads of condensed chromatin 41

ABBREVIATIONS ON PLATES

bz, 64, relatively electron-dense bands om outer membrane61, 63, relatively less electron-dense bands p nuclear poreim inner membrane r nbosomesvi mitochondrion

All erythrocytes in Figs. 1-20 are of the definitive type, from the embryo and the chick.

42 A. C. Everid, J. V. Small and H. G. Davies

Fig. i. Electron micrograph of a citrate-treated reticulocyte from a 3-week chick.Arrows (untailed) indicate groups of unit threads seen in end-on view as granules,either on the envelope (arrow at a) or in the interior chromatin. The tailed arrowpoints to a fibril-free area in a haemoglobin-containing region surrounding the con-densed chromatin. Small regions in the chromatin around X are finely fibrillo-granular. Section thickness 73 nm, x 40000.

Figs. 2, 3. Electron micrographs of citrate-treated mature erythrocyte from 3-weekchick showing the band segments 62, 64 and granular end-on views of units (arrows)The hollow appearance of the granules is consistent with a tubular structure. 73 nm,x 129000.

Fig. 4. Electron micrograph of a mature erythrocyte from 17-day chick embryoshowing bands b 1, 62, 63 lying parallel to the nuclear envelope, x 90000.

Unit threads of condensed chromatin 43

w

44 A.C. Everid, J. V. Small and H. G. Davies

Figs 5, 7, 8. Electron micrographs of reticulocytes from 4-day chick showing unitthreads in surface view. 44 nm (Figs. 5, 8), 47 nm (Fig. 7), x 75000.

Fig. 6. Electron micrograph of a mature erythrocyte from a 17-day embyronic chick.Groups of oriented threads are arrowed. 58 nm, x 75000.

Fig. 9. Electron micrograph of a reticulocyte (S, see Fig. 8) and mature erythrocyte(10, see Fig. 10) from blood of 4-day chick. 44 nm, x 9000.

Fig 10. Electron micrograph of the mature erythrocyte marked 10 in Fig. 9. 44 nm,x 75000.

Unit threads of condensed chromatin 45

46 A. C. Everid, J. V. Small and H. G. Davies

Figs. 11-20. Electron micrographs of tangential sections through citrate-treatedmature erythrocytes from 3-week chick. For Fig. i6a, b see text. 75 nm (Figs. 11-13,JS> J6), 52 nm (Fig. 20). All x 75000 except Figs, n , 19, which are x 86000.

Unit threads of condensed cltromatin 47

A. C. Everid, J. V. Small and H. G. Davies

• • * , * < :

*

For legend see p. 46.