J. Cell Sci. 33, 249-268 (1978)Printed in Great Britain © Company of Biologists Limited 1978

THE ULTRASTRUCTURE OF

NON-MEMBRANOUS NUCLEAR GHOSTS

D. E. RILEY* AND J. M. KELLERfDepartment of Biochemistry, University of Washington, Seattle, Wash. 98195, U.S.A.and ^Department of Biochemistry, University of Health Sciences, The Chicago MedicalSchool, 2020 West Ogden Avenue, Chicago, III. 60612, U.S.A.

SUMMARY

Non-membranous HeLa cell nuclear ghosts, representing non-membranous nuclear envelopeor 'skeletal' components, have been examined in whole-mount fashion by transmissionelectron microscopy. Major components of the ghosts include annuli with inner and outerdiameters of 43 and 90 nm, respectively, which are consistent in dimensions with nuclear porecomplexes. Also present are rod-like images (260 nm in length and 50 nm in width or diameter)representing either previously unobserved nuclear structures, or condensations of repeatingfunctional units not otherwise observable. The annular and rod-like images were also observedwhen various steps in the ghost isolation procedure, such as the use of detergents, 0-5 M MgCl,and polylysine attachment of the ghosts to electron-microscope grids, were circumvented.The annular and rod-like images are connected into linear and polygonal arrays by strands(15-30 nm in width) that are sensitive to DNase I and DNase II but resistant to nuclease Sj.Thus, although the non-membranous ghosts from HeLa cells are composed primarily ofprotein, enzymic dissection indicates that their gross integrity is markedly dependent ondouble-stranded DNA. Nuclear ghosts prepared from a wide range of species includingmammals, birds and plants, exhibited essentially the same components and organization.

INTRODUCTION

We and others have shown that isolated nuclei maintain their morphologicalintegrity after the removal of both the inner and outer nuclear membranes withdetergents (Aaronson & Blobel, 1974; Hancock, 1974; Riley, Keller & Byers 1975).A dense peripheral margin, in which the nuclear pore complexes are embedded,remains after membranes are removed (Aaronson & Blobel, 1974; Riley, et al. 1975;Scheer et al. 1976). It seems reasonable to propose that this non-membranous layer(sometimes mistaken for the inner nuclear membrane) could correspond to a structuralnuclear framework or 'skeleton* which confines and possibly organizes the inter-phase nuclear chromatin and confers a degree of mechanical rigidity on isolatednuclei. This skeletal role for the non-membranous margin is suggested by the obser-vation that spilling out of nuclear contents (when this occurs) takes place at the sitesof obvious breakage of the marginal layer of membrane-depleted nuclei (Hancock,1974; Holtzman, Smith & Penman, 1966; and our unpublished observations).

We have previously reported the isolation from HeLa cell nuclei of ghost-like

• Present address: Department of Biochemical Sciences, Princeton University, Princeton,N.J. 08540, U.S.A.

f Present address: To whom to address correspondence and reprint requests.

250 D. E. Riley and J. M. Keller

structures thought to be representative of this same non-membranous nuclear'skeletal* component (Riley et al. 1975; Keller & Riley, 1976; Riley & Keller, 19766,1977). These non-membranous nuclear ghosts are composed of 72% protein, 14%DNA, 10% phospholipid (not in the form of membrane) and 4% RNA. The isolatedghosts are substantially purified from intranuclear components, since 90% of thetotal nuclear protein, 95% of the DNA and 88% of the nuclear RNA are removedduring the isolation. Although the non-membranous ghosts are about 4 times largerin diameter than the nuclei from which they originate, individual ghost formationfrom individual nuclei can be directly observed (Riley et al. 1975). We believe thatthe larger diameter of the ghosts is due to a relaxation or expansion from the in vivostate of the proposed supportive or skeletal nuclear framework (Keller & Riley, 1976).

The non-membranous ghosts described above and the layer of material whichconfers mechanical stability on isolated nuclei probably corresponds, at least inpart, to ultrastructurally recognized non-membranous nuclear envelope components,such as the so-called fibrous lamina (Fawcett, 1966 a) or the envelope-associatedheterochromatin (Davies, 1967). Briefly, the fibrous lamina is a layer of variablethickness, distinguishable from chromatin and membrane, closely apposed to thenucleoplasmic aspect of the inner nuclear membrane. In a limited number of celltypes, particularly those with large nuclei, this layer is thick (60-280 nm) and manifestsa honeycomb appearance (Harris & James, 1952; Pappas, 1956; Beams, Tahmisian,Devine & Anderson, 1957; Coggeshall & Fawcett, 1964; Stevens, 1967; Feldherr,1968; Flickinger, 1970; Stelly, Stevens & Andre", 1970; Burr & West, 1971). Moretypically, the fibrous lamina is represented as a thinner (20-80 nm) layer (Fawcett,1966a, b; Kalifat, Bouteille & Delarue, 1967; Mazanec, 1967; Patrizi & Poger, 1967).For a number of years, the occurrence of this layer in other than an isolated numberof cell types was undetected, but several investigators are now proposing that it maybe much more universal than previously realized (Fawcett, 1966 a; Stevens & Andre\1969; Aaronson & Blobel, 1975). There is evidence that the fibrous lamina may beprimarily proteinaceous in composition (Stelly et al. 1970; Aaronson & Blobel, 1975).However, the precise chemical composition and substructural organization, with theexception of a ' fibrous' nature sometimes observed, are largely undetermined.

The heterochromatin layer of the nuclear envelope appears to consist of, at leastin several cases, alternating sheets of parallel heterochromatin fibres or 'threads'(Davies, 1967; Davies & Small, 1968). Sometimes only a single layer of bead-likeor rod-like structures, thought to be heterochromatin, is seen underlying the nuclearenvelope (for review, see Franke & Scheer, 1974). Both the fibrous lamina (Fawcett,1966a; Stevens & Andr6, 1969; Stelly et al. 1970; Riley et al. 1975) and the peripheralheterochromatin (Flickinger, 1970) have been postulated to serve as supportiveskeletons for nuclei.

One approach to exploring the substructural organization of these densely packedlayers is to expand their components over greater areas without disrupting all of thefundamental aspects of their organization. In fact, such an approach was found to bevery useful in the demonstration of nu bodies (Olins & Olins, 1974; Oudet, Gross-Bellard & Chambon, 1975) as well as nascent RNA transcripts (Miller & Beatty,

infrastructure of nuclear ghosts 251

1969). The non-membranous nuclear ghosts, which we have isolated, are expandedfrom their in vivo configuration, and most likely represent at least part of the fibrouslamina and/or peripheral heterochromatin layers. As with other 'spreading' orexpanding techniques, the extent to which the observed structures still representarrangements occurring in vivo must be carefully examined. That the non-mem-branous nuclear ghosts described here represent, with considerable fidelity, arrange-ments occurring in vivo is indicated by the finding that the ghost architecture undergoeselaborate and specific cell cycle-dependent changes which parallel known changesoccurring in the nuclei of intact cells (Riley & Keller, 1978). In the present reportboth the organizational logic or non-randomness of the ghost architecture, and theeffects on the final product of each individual step in the isolation procedure areexamined. A preliminary account of this work has been presented (Riley & Keller,1976 a).

MATERIALS AND METHODS

Cell and cell culture

The routine maintenance of suspension cultures of HeLa Sj cells used in this study hasbeen previously reported (Riley et al. 1975).

Isolation of nuclei and nuclear ghosts

The isolation of nuclei and nuclear ghosts has been described (Riley et al. 1975; Riley &Keller, 1977). Briefly, cells are swollen and homogenized in a hypotonic buffer in order torelease nuclei. The nuclei are collected by low-speed centrifugation (1800 rev/min for 30 s).Nuclear membranes are removed by a brief treatment with 0-87 % (v/v) Tween-40 and 0-43 %(w/v) sodium deoxycholate. The detergent-treated (membrane-free) nuclei are treated with0-5 M MgCl, which results in the release of the bulk of the nuclear constituents. The ghostsare separated from the released nuclear contents by velocity-sedimentation on sucrose gradients.

Preparation of non-membranous nuclear ghosts by an alkaline buffer-DNase I method, amodification of the method of Kay, Fraser & Johnston (1972) used to isolate whole nuclearenvelope, was performed by suspension of detergent-treated nuclei in an alkaline buffer(8 mM Tris-HCl (pH 8-8), o-i mM MgCla, 11 mM 2-mercaptoethanol, 0-25 M sucrose) followedby treatment with pancreatic DNase I (50 /tg/ml for 10 min at 22 °C). This treatment causedthe nuclei to lose much of their phase-contrast density when visualized by phase-contrastmicroscopy. The DNase I-treated nuclei were then chilled to 4 °C, pelleted, washed twice(by resuspension and pelleting) in the same buffer without DNase I, and prepared for electronmicroscopy.

The hen oviduct nuclei were a generous gift from Dr Richard Palmiter and colleagues(University of Washington). These nuclei were prepared by dounce-homogenization of henoviduct tissue in buffer (10 mM Tris-HCl (pH 8-o), 2-5 mM MgCli, 0-5 % Triton X-100,0-3 M sucrose) followed by filtration through glass wool and centrifugation through 2-4 Msucrose made up in buffer similar to that used for homogenization, but with the substitutionof o-i % Triton X-100 for 0-5 % Triton X-100. Ghosts were prepared from the hen oviductnuclei in a manner identical to that used to prepare HeLa cell nuclear ghosts.

To determine if a random mixture of nuclear components contained or produced annularand rod-like images (Results), detergent-treated HeLa cell nuclei were disrupted by sonication(Bronwill III sonicator, for 45 s at a setting of 30 using the red probe) and the resulting mixtureapplied to grids by the same method used to attach ghosts, as described below. Purified calfthymus DNA (Worthington, 0-3 mg/ml and serial 5 -fold dilutions thereof) was also attachedby this method (Results).

252 D. E. Riley andj. M. Keller

Electron microscopy

Nuclear ghosts and other fractions were attached to Formvar- and carbon-coated electron-microscope grids using poly-DL-lysine (Sigma), poly-D-lysine (Miles Laboratories, Inc.)or cytochrome c (equine heart, Calbiochem) with invariant results. A drop of o-1 % polylysine(Mazia, Shatten & Sale, 1975) or cytochrome c was allowed to sit on each grid for 3-5 min at22 °C. Excess polylysine or cytochrome c was removed by rinsing with 3 drops of distilledwater. Excess water was removed and the grids allowed to air dry. Samples were applied tothe grids and allowed to stand for 10 min at 4 °C. The supernatant was removed and immediate-ly (i.e., without drying) replaced by a drop of 0-3 % uranyl acetate, or, if enzymic digestionwas performed, by a drop of enzyme solution (see Results) followed after the indicated timesby replacement with 0-3 % uranyl acetate. After 1 min at 4 °C, the uranyl acetate was removed,the samples quickly rinsed with a small drop of distilled water and dried under a gentle streamof nitrogen. For negative staining, 0-5 % uranyl acetate was used and the final rinsing stepomitted. An alternative method of positive staining which yielded identical results was tofix the samples with 1 % glutaraldehyde, postfix and stain with 1 % OsO4, dehydrate in gradedethanol solutions and critical-point dry. The nuclear ghosts did not attach to glow-dischargedgrids, to grids without adherent polylysine or cytochrome c, or to grids treated with agedpolylysine solutions (apparently the polylysine becomes attached to the walls of the container).Observations were made with a Philips EM 201 transmission electron microscope.

Frequency distribution analysis of stainable ghost material (Fig. io, p. 263) was performedby inverted densitometric scanning of photographic negatives (Quick Scan, Helena LaboratoriesCorp.) in a manner similar to that used for scanning, without inversion, sodium dodecylsulphate-polyacrylamide slab gels (Riley & Keller, 1977). Frequency distributions determinedby actually counting the frequency of rod-like structures per unit area were essentially thesame as those obtained by scanning densitometry.

EnzymesEnzymic treatments of the non-membranous ghosts were performed either subsequent to

ghost attachment as indicated above, or in suspension prior to attachment (Results). Notethat the effects of DNase I (the disruption of connecting strands and organized linear sequences,the distortion of 'unravelling' of the rod-like structures, and finally the disappearance ofannular and rod-like images, see Results) were distinctly sequential effects produced byprogressively increasing levels of DNase I. It is important to note that the effective levels(Results) of DNase I treatment are substantially lower for ghosts treated in suspension, thanfor ghosts treated after attachment. We have not characterized rigorously the precise thresholdlevels of DNase I required to produce these effects. Also note that the effects of DNase I onthe rods and annuli themselves (but not the connecting strands) are conceivably due, not toDNase I, but to a protease, possibly contaminating the DNase I preparation (Burgess &Jendrisak, 1975). These considerations do not affect the conclusions presented. PancreaticDNase I, porcine spleen DNase II, and trypsin were obtained from Worthington (codesDPFF, HDAC and TRL, respectively). Nuclease Sx from Aspergillus oryzae was obtainedfrom Sigma (no. 5255). Pancreatic DNase and a-chymotrypsin were from Mann Laboratories(no. 377 and no. 4767, respectively).

RESULTS

Description of non-membranous nuclear ghost components

We have previously shown that non-membranous nuclear ghosts attached withpolylysine to electron-microscope grids are flattened 2-dimensional representations(determined by scanning electron microscopy) of the originally 3-dimensional ghosts(Riley & Keller, 19766). Fig. 1 shows an isolated non-membranous HeLa cell nuclearghost attached in whole-mount fashion with polylysine. Annular (43 and 90 nm,

Infrastructure of nuclear ghosts 253

inner and outer diameters, respectively) and rod-like images (260 nm in length and50 nm in diameter or width) are seen to be major constituents of the ghosts. Highermagnifications reveal the presence of thin strands (variable from 15 to 30 nm inwidth) that interconnect the annular and rod-like images into linear and polygonal

1

Fig. 1. Non-membranous HeLa cell nuclear ghost flattened and attached in whole-mount fashion with poly-D-lysine. Rod-like and annular structures with the dimensionsindicated in the text are widely distributed throughout the ghosts. This low magnifi-cation demonstrates the distribution of ghost components, but for more useful in-spection of the rod-like and annular structures refer to the higher magnifications ofFigs. 6 and 7. Small dense aggregates of material (ia) with radiating connected rodsequences are distributed throughout the ghost shown. Scale bar equals 5-0 fim.

arrays (Figs. 2, 3, 7). Also apparent are dense aggregates of material which are eitherlocalized centrally (Fig. 2) or scattered throughout the ghosts (Figs. 1, 5, 6). Finally,there is a fine 'meshwork' background or substratum in which many of the annularand rod-like structures appear to be embedded (Figs. 2, 5). Both the annular and

"ft CEL 33

D.E.Riley and J.M. Keller

rod-like images vary somewhat in apparent thickness. This may be due to variationin staining intensity or to the presence of more than one size class of annuli or rods.The annular and rod-like structures have also been observed by negative staining(Fig. 3) and after critical point drying of dehydrated samples fixed with glutaraldehyde

Fig. 2. Nuclear ghost from hen oviduct nuclei isolated as described in Methods.Double-headed arrows indicate the orientation of numerous marginally concentric'unconnected' linear rod sequences found near the margin and inward. The henoviduct nuclear ghosts were characterized by single large central aggregates ofmaterial. The numerous smaller dense aggregates found in most of the HeLa cellghosts (Fig. 6) are absent. The rod-like substructure of the strands radiating outwardfrom the central aggregate of the hen-oviduct nuclear ghost may not be apparent inthis photographic reproduction but was demonstrated for S-phase HeLa cell nuclearghosts (Riley & Keller, 1977). Scale bar equals 2-0 /im.

or osmium tetroxide (Methods). Nuclear ghosts with similar substructures have beenisolated from a variety of other eukaryotic cell types, e.g. hen oviduct cells (Fig. 2).

Focus variation using a phase-contrast microscope indicates that the ghost flatteningprocess occurs during the ghost attachment step prior to any drying (air or critical

Ultrastructure of nuclear ghosts 255

point drying; see Methods) of the sample. The tight attachment of the ghosts priorto drying probably explains the success of these methods in preserving the smoothcontour of the ghost outer margins (as compared to the extensive distortion andshrinkage often observed for whole mount preparations of particles the size ofnuclei (DuPraw, 1956, 1970; Hoeijmakers, Schel & Wanka, 1974)). Note that thedense aggregate material of the ghosts (Fig. 1), which one might initially ascribe toa non-specific coalescence phenomenon of drying, actually changes its organizationin a very extensive and specific manner during the cell cycle (Riley & Keller, 1977).

The possibility of artifact

Although the annular structures have the dimensions and occur with a frequencyconsistent with the dimensions and frequency of nuclear-pore complexes (Keller &Riley, 1976), rod-like structures comparable to those described here have not, to ourknowledge, been observed in intact cells or nuclei. For this reason, the possibleextent of macromolecular rearrangement during isolation of the ghosts was examined.In order to detect or rule out extensive artifact (e.g. by detergent, 0-5 M MgCl2 orpolylysine) individual steps in the ghost isolation procedure were circumvented.

Effects of detergents. To test for possible rearranging effects of detergents, ghostswere isolated from HeLa nuclei completely omitting detergent treatment. (Note thateven in the absence of detergents some, or even a large proportion, of the nuclearmembranes may be adventitiously removed during 0-5 M MgCl2 treatment andgradient centrifugation used to prepare ghosts (Riley et al. 1975).) Annular and rod-like structures, as well as the connecting strands, were readily observed in suchghosts by negative staining (Fig. 3). Thus, the appearance of these structures is notdependent on the use of detergents.

Effects of 0-5 M MgCl2. Ghost isolation was attempted under conditions indepen-dent of high ionic strength. Detergent-treated HeLa nuclei, prepared in the usualmanner, were suspended in an alkaline buffer (pH 8-8, Methods). Subsequenttreatment with DNase I caused the nuclei to lose much of their phase-contrastdensity when viewed by phase-contrast light microscopy. The resulting ghosts weremuch more compact (one-third to one-quarter the diameter) than the ghosts isolatedby the 0-5 M MgCl2 method. Numerous annular and rod-like structures (Fig. 4)were observed in ghosts isolated by the alkaline buffer-DNase method. The rod-likestructures were highly variable in length; the longest ones approaching the lengthof the rods isolated by the 0-5 M MgCl2 method. Possibly, some of this variation wasdue to variation in the 3-dimensional orientation of the rods, since the ghosts isolatedby this method were compact and exhibited dense centres (i.e. they probably have acertain amount of depth) whereas the ghosts isolated by the 0-5 M MgCl2 methodwere highly expanded and flattened (Riley & Keller, 19766).

Effects of polylysine used to attach ghosts. Polylysine has been shown to interactwith DNA under certain conditions to form so-called 'stem-like' and 'doughnut-like' structures (for the variable dimensions of those structures, see Haynes, Garrett& Gratzer, 1970; Olins & Olins, 1971; Laemmli, 1975) with dimensions somewhatcomparable to the annuli and rods of the present report. That polylysine is without

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256 D. E. Riley andj. M. Keller

Fig. 3. Portion of a HeLa cell nuclear ghost isolated without the use of detergents.The ghosts, in this case, were visualized by negative staining (Methods). Unconnectedrod sequences are not visible in this field, a, annuli; c, connecting strands; r, rod-like structures. Scale bar equals 0-3 fim.Fig. 4. Nuclear ghosts prepared from detergent-treated HeLa nuclei by the alkalinebuffer-DNase I method. Numerous rod-like structurea of variable length and theannuli are apparent, a, annuli; r, rod-like structures. Scale bar equals O'6 fim.Fig. 5. Non-membranous nuclear ghost from. HeLa cell nuclei attached with cyto-chrome c (Methods). Annular and rod-like structures, the connecting strands andsmall dense aggregates with radiating connected rod sequences are clearly apparent.Scale bar equals i-o fim.Fig. 6. Portion of a HeLa cell nuclear ghost showing small dense aggregates ofmaterial with radiating connecting rod sequences. Scale bar equals i-o /*m.

Ultrastructure of nuclear ghosts 257

effect on the final appearance of the ghosts is indicated by the following findings:(a) Purified calf thymus DNA attached with polylysine (Methods) by the samemethod used to attach ghosts did not result in any recognizable annular or rod-likestructures, (b) A random mixture of nuclear components prepared by sonicatingdetergent-treated nuclei (Methods) consisted of a variety of amorphous particles,but did not interact with the polylysine of the grids to produce any regular morpho-logical entities, (c) It was found that the cytochrome c effectively replaced polylysinein attaching and spreading the nuclear ghosts (Fig. 5). Although cytochrome c, itself abasic protein, has been widely used to attach or spread basic protein (e.g. histones orpolylysine) DNA complexes (Kleinschmidt, 1968), cytochrome c itself is not known to'collapse' DNA into recognizable particles as far as we are aware, (d) The annuli,rods and connecting strands of the cytochrome c-attached ghosts were indistinguish-able from those of polylysine-attached ghosts (compare Fig. 1 and Fig. 5). In contrast,the morphology and dimensions of particles formed by collapse of DNA with poly-ysine or histones under the appropriate conditions is sensitive to the kind of basic

protein used (Olins & Olins, 1971). (e) Treatment of ghosts with DNase I prior toattachment (see below) completely disrupts the ghost integrity but the annular androd-like structures still attach to the grids. This observation also eliminates thepossibility that the experiment described in (a) above failed to control for high localizedconcentrations of DNA which might occur in the ghosts.

Supporting our inability to attribute the observed ghost annular and rod-likestructures to any of the more harsh isolation conditions (i.e. detergents, 0-5 M MgCl2,and polylysine used for attachment), the same detergent mixture (Holtzman et al.1966; Riley et al. 1975), the 0-5 M MgCl2 (Monneron, Blobel & Palade, 1972; Rileyet al: 1975) and the low ionic strength conditions (Holtzman et al. 1966; Riley et al.1975) used for isolating nuclei (Methods) have all been used previously withoutobvious rearranging effects on the ultrastructure of non-membranous components ofthe nuclear surface. The effects of DNase I and proteases on, the rod-like and annularstructures (below) indicate that the rods and annuli are not adherent 'crystals' ofsome sort arising from non-cellular origin. Drying artifacts may also be excludedbecause of the diversity of methods used for preparing samples for electron micro-scopy (i.e. positive and negative staining and air or critical point drying all give similarresults; Methods). Therefore, the possibility must be considered that the annularand rod-like images represent either unaltered physiological structures or localizedcondensations (e.g. the possibility of a ghost-associated DNA-collapsing proteinhas not been excluded) of repeating structural units, perhaps not otherwise dis-cernible. That such possible localized condensations still reflect, with some fidelity,arrangements occurring in vivo is strongly suggested by the finding of (1) markedlynon-random ghost organizational properties (below) and (2) cell cycle stage dependentorganizational changes which parallel changes occurring in intact nuclei (Riley &Keller, 1977).

258 D. E. Riky andj. M. Keller

Infrastructure of nuclear ghosts 259

Organization

Connected linear sequences. A prominent class of linear rod-like structures exhibitsobvious connecting strands. These connected rod sequences can very often be tracedto an origin on a dense aggregate of material (e.g. Fig. 6). The densely aggregatedmaterial facultatively consists either of a number of small bodies distributed through-out the flattened ghosts (Figs. 1, 5, 6) or a single central aggregate (Fig. 2 and see the5-phase ghosts from HeLa cells (fig. 1 in Riley & Keller, 1977)). Interestingly,the connected rod sequences 'follow' these aggregates in that when the aggregates aresmall and widely distributed, the connected rod sequences radiate from them andthus extend in all directions (Fig. 1), but when a large central aggregate is observed,the smaller aggregates are absent, and the connected rod sequences have (for themost part) a particular orientation extending from the central aggregate outward(Fig. 2). For HeLa cells, the basis for these alternative morphologies (i.e. largecentral aggregate vs. numerous dispersed small aggregates and the accompanyingreorganization of the rod sequences) have been well characterized as cell cycle-stagedependent changes (Riley & Keller, 1977).

Polygonal arrays and networks. The connected linear rod sequences may exhibitinterstrand connexions so that elaborate polygonal networks are formed (Fig. 7).Several features suggest that these networks are not random arrays of connectedrod sequences and annuli.

Assume for the moment that the network of Fig. 7 is a product of random attach-

Fig. 7. A region of a non-membranous nuclear ghost from HeLa nuclei in which over-lap of connected sequences was low, allowing visualization of clearly defined inter-connexions of a network. Connected annuli (a) often form centres from which therod-like structures radiate. Small-double headed arrows indicate the angles (typicallynear 900) between pairs of connecting strands as they emerge from the ends ofrod-like structures or from bifurcations in a single connecting strand (see text), a,annuli; c, connecting strands; r, rod-like structures. Scale bar equals 05 fim.Fig. 8. Non-membranous nuclear ghosts were treated in suspension with DNase I(5 fig/ml for 1 min at 22 °C). The DNase I-treated ghost suspension was then appliedto an electron-microscope grid by the same method used to attach ghosts. No intactghosts were observed either by phase-contrast microscopy or by electron microscopyafter DNase I treatment in suspension. Somewhat distorted (see text) rod-like andannular structures were observed, randomly arranged and without interconnexions.Proteases also distort or 'unravel' the annular and rod-like images, but do not affectthe interconnexions or the background meshwork (see text). Small rod-like structures,which might be partially digested connecting strands, were also observed, r, rod-likestructures, which might be partially digested connecting strands, were also observed.a, annuli; r, rod-like structures; sr, small rod-like structures. Scale bar equals 0-5 fim.

Fig. 9. HeLa cell nuclear ghost attached and subsequently treated with DNase I(50 fig/ml for 10 min at 22 °C). Rod-like structures, annuli, and small dense aggre-gates of material remain, but interconnexions of the rod-like and annular structuresand the fine meshwork background are absent. Some of the rods exhibit tails whichare probably partially digested connecting strands. The dense aggregates (da) nolonger have radiating connected rod sequences. Both connected and unconnectedlinear rod sequences have been disrupted, a, annuli; da, dense aggregate. Scale barequals 1-5 fim.

260 D. E. Riley and J. M. Keller

ment of rod-like structures to one another by the connecting strands. Most of therods exhibit either one or two connecting strands emanating from each end (3 ateach end have been observed but with low frequency). Clearly, a rod with a total of4 (or 3) connecting strands emanating from it could be directly connected to a numberof neighbouring rods equal to or less than 4 (3). That is, connecting strands emanatingfrom a given rod could each attach to a different neighbouring rod, 2 of them couldattach to the same neighbouring rod, one could double back and attach to the originalrod, or any number of them could terminate without making further contact. Thus,assuming random connexions, a given rod could be directly connected to a number ofits neighbours equal to or less than the number of connecting strands emanatingfrom that rod. However, the only situation unambiguously and frequently observedis one in which the number of direct neighbour connexions precisely equals thenumber of connecting strands emanating from a given rod. Thus, an entire range ofpossibilities predicted on the basis of random interconnexion appear to be absent.

This departure from randomness can be illustrated in still another way. The anglebetween 2 connecting strands as they emanate from the end of a rod or from a bifur-cation in a single connecting strand has never been observed to be greater than 1800.Instead, this angle is typically close to 900 (Fig. 7). Thus, a minimum of 50% [(360 —i8o)/36o x 100] of the range of possibilities expected for random interconnexion areeither absent, or are so infrequent that they were never unambiguously observed.

Enzymic dissection of the non-membranous nuclear ghosts and the role of ghost DNA

Mild DNase I treatment of the non-membranous nuclear ghosts in suspensionresulted in the loss of all recognizable ghost images as visualized by phase contrast(5-phase ghost cores are an exception; Riley & Keller, 1977) or electron microscopy(Fig. 8). Although not shown, identical results were obtained with DNase II whereasno effect was observed with nuclease Sx. The products remaining after treatment withDNase I consist of a random mixture of annular and rod-like structures, some smalldense aggregates and some smaller rod-like structures which might be the remnantsof partially digested connecting strands (Fig. 8). Although the primary constituentof the non-membranous ghosts is protein (70-75% by weight (Riley et al. 1975)) itappears to be the ghost double-stranded DNA and not the ghost protein (see below)that is largely responsible for maintaining the integrity of the ghost particles.

In contrast to the non-membranous ghosts, the spherical integrity of nuclei them-selves (e.g. see the alkaline buffer - DNase I-treated nuclei of Fig. 4) and variouswhole nuclear envelope preparations (Discussion) are not sensitive to DNase I. Weinterpret this as indicating the presence at or near the nuclear surface, of a non-membranous DNase I-resistant component (removed from our DNase I-sensitiveghosts during isolation) which, in intact nuclei, is superimposed upon or acts inconcert with the DNase I-sensitive ghost material in maintaining the sphericalintegrity of intact nuclei. Rather strong support for this interpretation is providedby the ghosts prepared via alkaline buffer-DNase I treatment. Specifically, theconnecting strands which appear to hold the DNase I-sensitive ghosts together(Figs. 8, 9, Discussion) are absent from alkaline buffer-DNase I treatment of nuclei,

Ultrastructure of nuclear ghosts 261

and yet ghost-like images, consisting of unconnected annuli and rods embedded in aDNase I-resistant substratum, remain.

When the DNase I-sensitive ghosts were attached to the polylysine-coated gridsand subsequently treated with DNase I, ghost-like images remained, but connectingstrands between the rods and annuli were clearly either removed or broken (Fig. 9):Although rods without connecting strands are occasionally observed in DNase In-sensitive ghosts not treated with DNase, the connected rod sequences are the pre-dominant component of these undigested ghosts (Figs. 2, 5).

It is doubtful that the effects of DNase I were due to contaminating proteolytic(Burgess & Jendrisak, 1975) or RNase activities in the DNase preparation, sinceproteases and RNase failed to produce the same effects. High levels of chymotrypsin(1 mg/ml) and trypsin (1 mg/ml) tended to reduce the numbers of annuli and rods,but where these were still observed, the rods (although somewhat distorted), annuli,connecting strands and background meshworks were all intact (not shown). Althoughthese components were intact, proteases did have the effect of distorting or un-ravelling the rod-like structures in a manner similar to that sometimes observed forthe latter stages of DNase I treatment (Fig. 8). Therefore, we believe that of theeffects of DNase I, only this unravelling effect is conceivably due to a contaminatingproteolytic activity of the DNase I. Note that the rod-like and annular structures arenot observed after treatment with high levels of DNase I (e.g. o-i mg/ml for 10 min at22 °C). Whether the disappearance of the annuli and rods in this case is actuallydue to DNase I or to a contaminating activity is uncertain (Burgess & Jendrisak,1975). Treatment of the grid-attached ghosts with pancreatic RNase (1 mg/ml)had no detectable effect.

Possible interrelationship between pore complexes and ghost annular and rod-like structures

Observations suggesting that the annuli of the non-membranous ghosts are identifi-able as nuclear pore complexes include their frequency of occurrence per unit area(Keller & Riley, 1976), their dimensions of 43 and 90 nm for the inner and outerdiameters, respectively and their probable location at the ghost surfaces (annuliare found near the ghost outer margins and elsewhere, the outer margin being anunequivocal reference point for the ghost surface, Discussion). The annuli of thepresent report, in fact, resemble presumptive pore complexes visualized by otherwhole-mount techniques (DuPraw, 1956, 1970) and possible fibrous interconnexionsbetween pore complexes have been observed previously (Speth & Wunderlich, 1970;Franke & Scheer, 1970, 1974; Scheer et al. 1976). Uncertainties arise since the subunitstructure or 8-fold symmetry shown for pore complexes by a variety of techniques,and the differential architectural appearance of pore complexes observed by positiveand negative staining (Gall, 1964; Franke & Scheer, 1970, 1974; and referencestherein) is not apparent for the annuli described here. Possibly, the differences be-tween pore complexes observed by conventional methods and the ghost annuli canbe rationalized by the whole-mount visualization of the annuli, used here as opposedto sectioning, or by the absence of membrane, thought to play a role in the appearanceof pore complexes by negative staining (Maul, Price & Lieberman, 1971). (Conven-

262 D. E. Riley andj. M. Keller

tional methods of embedding and sectioning, which might be expected to resolve thepossible identity of these annuli with pore complexes, as well as the distinction be-tween ghost surface and interior, have been unsuccessful since the ghosts lose theirintegrity, possibly due to aggregation or disruption, under such conditions. However,structures consistent in dimensions with the rod-like structures, annuli and connectingstrands have been observed by these methods.)

DISCUSSION

Relationship to the nuclear surface

Several lines of evidence indicate that the non-random arrays which constitutethe DNase I-sensitive nuclear ghosts described above derive at least in part fromnon-membranous components of the nuclear surface. In particular, a relationshipto the nuclear surface is indicated by the retention in the ghosts of most of the residualphospholipid of membrane-depleted nuclei despite the loss of the bulk of intranuclearcomponents (Riley et al. 1975); the presence in ghosts of annular images which havethe same dimensions and frequency of occurrence per unit area as the nuclear porecomplexes of intact HeLa nuclei (Keller & Riley, 1976); the marked similarities inthe non-membranous ghost polypeptides and the polypeptides of the pore complex-lamina preparation of Aaronson & Blobel (Riley & Keller, 19766); and the apparentsac-like nature of the ghosts which can become highly flattened when compared tomore rigid spheres such as intact nuclei (Riley & Keller, 19766).

The DNase sensitivity of the non-membranous nuclear ghosts is, in one sense,not surprising since a firm association of heterochromatin with the inner aspectof the nuclear envelope has been widely observed (Introduction). However, it wassomewhat unexpected that that portion of the nuclear DNA which the ghosts contain(about 6 % of the total DNA) is not only separable during ghost isolation from thebulk of the DNA, but appears to be involved in maintaining the structure of a residual

Fig. 10. A, frequency distribution expected if most of the stainable material of a ghostwere on its surface before flattening. B, parabolic frequency distribution expected ifmost of the stainable material were uniformly distributed throughout the volume of aghost before flattening. C-E, frequency distribution found by inverted scanningdensitometry (or by direct counting, see Methods) of photographic negatives of GltGj and 5-phase ghosts (Riley & Keller, 1977) respectively, shown here to demon-strate variation in internal vs. surface components of the ghosts. Note that the steepparabola of B and the step function shown in A (and therefore the presence or absenceof a significant surface component) can be readily distinguished by examining thefrequency distributions from the background (regions A) to the ghost outer margins(B) and a short distance inward. That is, for a predominant surface component, thefrequency of occurrence of stainable material should not increase greatly from themargin inward (see region / and A), but for a predominant internal component,the frequency should rise steeply from the margin inward (region / in B). The largefluctuations in c and D are due to small dense aggregates (text). The 'inner' stepfunction in E is due to an internal component (smaller than the ghosts themselves)which is so dense as to produce a saturation effect. The distribution shown in c isconsistent with a predominance of surface components and distributions shown in0 and E with combinations of surface and internal components (Riley & Keller, 1977).

infrastructure of nuclear ghosts 263

sphere (i.e. the ghost particles). Further, whether the ghost rod-like structuresrepresent localized condensations or, intact, previously unobserved nuclear structures,the substantial proportion of the rod-like structures are oriented by DNase-sensitiveconnecting strands forming linear sequences. Cell cycle dependent variation (below)

264 D.E.Riley and J.M.Keller

in the organization of these sequences and the disintegrating effects of DNase (butnot protease or RNase) implicates the DNA as a key structural component of theghosts (as opposed to mere residual attachment of the DNA to a structure determinedby say, proteins). Certainly, in the intact cell other components of the nuclear en-velope (e.g. the fibrous lamina; Introduction) could be superimposed on the DNase-sensitive ghost components to impart the DNase I resistance observed for wholenuclei (e.g. Fig. 8; Results).

Distribution of components between ghost surface and interior

Isolation of an intranuclear matrix (Berezney & Coffey, 1974) and light-microscopestudies of intact cells (for review, see Riley & Keller, 1977) indicate both inter-connexions and a cell cycle dependent redistribution of structural components betweennuclear periphery and interior. Since the ghosts observed as whole-mounts areflattened on to the grid before any drying of the sample (collapse by drying of asphere would be expected to produce extensive shrinkage and distortion, see Methods)certain predictions regarding the arrangements in the 3-dimensional form canreasonably be made. For example, the outer margin of a flattened ghost must bederived from a portion of what was previously the surface of a 3-dimensional nuclearghost. Using the outer margin as a reference point for the surface we may ask whichof the components distant from the margin originally were on the surface beforeflattening and which were located internally. Proceeding from the background ofthe grid to the outer margin of a flattened ghost and inward, one would predict (ifthe material of the original ghost were primarily on the surface) that the frequencyof occurrence of the rod-like structures would follow a simple step function (Fig. 10 A).Alternatively, if the components of the 3-dimensional ghost were uniformly distri-buted throughout its volume a steep parabolic function would be predicted (Fig. 10 B).The frequency distribution shown in Fig. 10c is typical for ghosts isolated fromHeLa cells in the Gx phase of the cell cycle (Riley & Keller, 1977) and suggests thatthe majority of stainable material lies on the surface of the unflattened ghosts. Ghostsfrom other phases of the cell cycle (i.e. 5 and G2 phase) have, by these criteria,prominent internal as well as surface components (see the 5-phase ghosts of Riley &Keller (1977) in which an unequivocal reference point for the interior is established).

Clearly, analysis by frequency distribution may be an oversimplification, sincethere is no a priori reason to rule out non-uniform internal distribution or changesin distributions during the preparation of samples for electron microscopy. However,the utility of frequency distribution analysis is supported by its ability to distinguishsurface and internal components (identified by additional considerations) of 5-phaseghosts (Riley & Keller, 1977). Further, the difference between the 2 ghosts of Fig. 11,which were side by side in a single field, are difficult to explain except by variation,detectable by frequency distribution, in an internal component. Scanning electronmicroscopy also indicated a prominent ghost surface component (the interpretationof the high degree of flattening of the ghosts compared to nuclei similarly observed)as well as varying amounts of internal material (bulges) (Riley & Keller, 19766).Note that, although somewhat indirect, the process of frequency distribution analysis

Ultrastructure of nuclear ghosts 265

of whole mount preparations of ghosts may be the best presently available methodto study the organization of the non-membranous ghost components by electronmicroscopy.

Relationship to other residual nuclear structures

We have shown (Riley & Keller, 19766) that the non-membranous nuclear ghostsappear to have major polypeptides in common with both the pore complex laminapreparation of Aaronson & Blobel (1975) and the nuclear protein matrix of Berezney& Coffey (1974). Interestingly, it appears that a similar group of polypeptides may beinvolved in constraining the bulk DNA of mammalian cell nuclei in a supercoiledconfiguration after the majority of the histones and non-histones have been removed

Fig. i i . Two non-membranous HeLa cell nuclear ghosts side by side in a single field.Note the difference in the central densities. Scale bar equals 5-0 fim.

(Cook, Brazell & Jost, 1976; Ide, Nakane, Anzai & Andoh, 1975). The most con-spicuous difference between all of these preparations is the extent to which DNA isremoved from the residual structures. Although the bulk of the DNA has beenremoved from the non-membranous ghosts described here, the residual DNA appearsto have an important structural role. Isolation methods which involve nucleasedigestion (e.g. the nuclear protein matrix of Brezney & Coffey (1974) might be ex-pected to select for DNase resistant spheres such as the 5-phase ghost cores whichwe have described (Riley & Keller, 19766). Alternatively, the DNase-sensitiveghosts described here may be separated from DNase-resistance-conferring com-ponents (Results). The latter interpretation may be somewhat more likely since thespherical integrity of nuclei themselves is not destroyed by high levels of DNase

266 D. E. Riley andj. M. Keller

(Fig. 8). Clearly, much more work is required to clarify the interrelationships of thevarious isolates.

The isolation of nuclear ghosts from a wide range of higher eukaryotic cell types,human (HeLa), birds (chick oviduct) and plant (tobacco; unpublished observations),attests to the general occurrence of this structure. We have suggested that the integralinvolvement of DNA with the non-membranous nuclear envelope provides both aconvenient way to link the mitotic events of chromosome condensation and nuclearenvelope disruption in higher eukaryotes (as well as the reverse process) and a meansto organize interphase chromatin (Keller & Riley, 1976). As a result of this prediction,we would suggest that the ghost structure plays an important structural role in mitoticchromosomes. A direct test of this proposal is in progress. In addition, we are currentlyperforming biochemical analyses on the DNA present in the intact ghosts as well ason the proteins present in both the rods and annuli released from the ghosts by treat-ment with DNase I (see Results).

This work was supported by grants from NSF (PCM 76-82030), NCI (CA-23016) and theIllinois Division of the American Cancer Society. We thank Dr Byers, Department of Genetics,University of Washington, for the use of electron-microscope facilities, for technical adviceand for many valuable discussions.

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(Received 23 August 1977)