Isolation and partial characterization of the nuclear shell of HeLa cells

446 4. 5. 6. 7. 8. 9.

Preliminary

notes

Nannev. D L; J orotozool4 (1957) 89. Butzel; Jr, H M,-J protozool’20 (1973) 140: Koizumi, S, Genetics 68, suppl. (1971) 34, Sonneborn, T M, Methods ckil physiol4 (1970) 241. Koizumi, S, Exp cell res 88 (1974) 74. Koizumi, S & Kobayashi, S, J cell sci 30 (1978) 187.

Received July 22, 1980 Revised version received September 23, 1980 Accepted September 29, 1980

Copyrqht 0 19x1 h) Academic PI?\\. Inc. All right\ ol’ reproduction in any fivm rexned ~XII4-4X~7iXl/02~144h-o79o?.~K)/ll

Isolation and partial characterization of the nuclear shell of I-IeLa cells J. HUBERT, D. BOUVIER, M. BOUTEILLE, Laboratoire

J. ARNOULT

and

de Puthologie Cellulaire, Institut Biome’dical des Cordeliers, 15, rue de I’Ecole de Mbdecine, 75270 Paris Cedex 06, France

We recently described the spontaneous dissociation from isolated HeLa cell chromatin of a nuclear shell consisting of the outermost layer of peripheral chromatin and of fibrous lamina material [l-3]. The present paper reports our attempts to isolate and purify this chromatin subfraction. Preliminary results of electron microscopy and sucrose gradient centrifugation indicate that nuclear shell DNA accounts for 0.8-l% of total nuclear DNA, and is presumably constrained in highly stable supranucleosomes by a specific protein environment. Small variable amounts of RNA are also found in the nuclear shells but do not seem to be involved in maintaining shell structure. Further studies of the isolated nuclear shells should shed some light on the specific organization of the chromatin adjacent to the inner nuclear membrane in the nucleus in situ.

Summary.

In nuclei isolated from many cell types, cortical chromatin, i.e. the outermost layer of dense chromatin, is resistant to dispersal in low-salt media, whereas more internal chromatin rapidly unfolds under the same conditions [4-g]. In cortical chromatin, DNA is resistant to DNases when the internal chromatin is digested, and only becomes accessible to the enzymes after deproteinization [9-l I]. This stability is probably due to interactions between cortical DNA and a specific subset of non-histone proteins and even proteins of the inner nuclear membrane surface. The study of Exp Cell RPS 131 (1981)

such interactions requires the isolation of a subnuclear fraction in which cortical chromatin retains its native superstructure ‘and remains associated with membrane proteins. This was achieved in our laboratory and we recently reported the spontaneous release from isolated and membrane-depleted HeLa cell nuclei of a nuclear shell containing both cortical chromatin and remnants of the nuclear pore-lamina layer [l-3]. This paper deals with a reproducible method of nuclear shell isolation allowing further biochemical and electron microscopy investigations of this new subnuclear fraction. Materials

and Methods

HeLa cells were grown as monolavers in Eagle’s MEM supplemented with 8% calf s&urn containing 2 % tylocine. For DNA labelhng, 3 X 106 cells were plated in 14 ml of medium and cultured as above. One &i/ml of 13H-methvllthvmidine (The Radiochemical Centre, kmershatk-43 Ci/mmolk) was added 20 h after plating, and cells were allowed to grow for 48 h. For RNA labelling, 10’ exponentially growing cells were treated with 0.04 pg/ml of actinomycin D in 10 ml for 30 min, and r3H]uridine (Radiochemical Centre; 23 Ci/mmole) was added to a final concentration of 50 &i/ml. Incubation lasted for 30-60 min. Cells were detached bv mild trvnsinization. resuspended in fresh culture-medium akd washed twice in PBS containing 5 mM EDTA and 10 &ml of sovbean trypsin inhibitor. Interphase chroma& was preoared bv hvnotonic cell lvsis. using Nonidet P40 [7]. The chrdmatin pellet was’resuspended and allowed to dissociate at 4°C in 20 mM Tris-HCl (DH 8.0) as previously reported [l-3]. The chromatin”suspen: sion was subseauentlv adiusted to 0.1 mM CaCl,. and 30 U/ml of~micrbco&al nuclease (Worthington Biochemicals, NFCP grade) were added after preheating the sample at 37°C for 3 min. Digestion proceeded for another 3 min at 37°C. aDd was StODDed by addition of 50 ~1 of cold 0.1 M EDTA to 1 s of chromatin digest. After standing for 15 min in ice, the digest was layered on a 0.1 M sucrose underlay in 0.2 mM phosphate buffer and 0.2 mM EDTA, and snun at 800 e for 20 min. Nuclear shells were purified from the &Ring low-speed pellet by centrifugation at 50000 g for 1 h through a 1540% sucrose gradient in 0.2 mM phosphate buffer (pH 7.2) and 0.2 mM EDTA. All stens in the isolation nrocedure are summed up in fig. 1: 200 pg/rnl alpha chymotrypsin, and a mixture of 100 &ml RNase A and 10 U/ml RNase Tl were used for enzymatic digestion of the low-speed pellets. Prinled

m Sweden

Preliminary notes In each case, the sedimentation properties of the shells were tested in the 1540% sucrose gradient described above. Protein depletion of the shells was performed in 10 mM Tris-HCI (oH 8.0) and 0.2 mM MeCh containing 2 M NaCI. ~* ’ The electron microscopic appearance of the nuclear shells after micrococcal nuclease digestion was examined according to Miller & Bakken [12]. Proteindepleted nuclear shells were spread on distilled water in the presence of cytochrome c according to Comings & Okada [13]. Grids were rotary shadowed with platinum, and observed with a Philips EM 300 electron microscope. I

447

Chromatin

dissociated in 20 mM Tris-HCl (pH 8.0) 15 min at 4°C

-

Results When chromatin was isolated according to Hancock, it resembled swollen and membrane-depleted spheres, corresponding to the initial interphase nuclei, and consisted of a homogeneous network of unravelled chromatin fibrils bound by a more compactly structured layer: the nuclear shell. The first step in the isolation procedure was the spontaneous dissociation of the nuclear shell from the inner chromatin material. When chromatin was transferred from the hypotonic isolation medium to 20 mM Tris-HCl (pH 8.0), the shells ruptured and contracted and the innermost masses of chromatin were expelled [l-3]. The second step consisted of discarding the internal chromatin by micrococcal nuclease digestion at low ionic strength. Under the conditions used here, 18% of the total nuclear DNA was rendered acid-soluble after 3 min digestion of the Tris-dissociated chromatin. Fig. 2 illustrates the nuclease-resistant material which sedimented at low speed onto an electron microscope grid through an 0.1 M sucrose underlay. The shells, each corresponding to one starting nucleus, were abundant and consisted of a tight network of knobby fibres, with thicknesses varying between 27 and 30 nm. Their folded appearance after flattening onto the grids clearly recalled their initial spherical shape. The third step consisted of purifying the

t Dissociated

chromatin

+O. 1 mM CaCl, +30 U/ml micrococcal nuclease 3 min at 37°C +50 pi/ml 0.1 M EDTA 15 min at 0°C I 800 g 10 min I

Low-speed pellet

+0.2 mM EDTA (pH 7.2) t 50000 g 60 min through a 1540% sucrose gradient t Isolated

nuclear shells

Fig. I. Scheme for HeLa cell chromatin subfractionation, yielding a nuclear shell fraction.

shells from the nuclease digest. In a first series of experiments, we attempted to purify the shells from the digested internal chromatin by direct centrifugation of the EDTA-arrested digest through a linear sucrose gradient. The shells then formed a faint band which was generally difficult to distinguish from the heavily labelled chromatin peak. We therefore eliminated 98% of the digested internal chromatin by centrifuging the digest through an 0.1 M sucrose underlay. The low-speed pellet, corresponding to the material shown in fig. 2, was highly enriched in nuclear shells and was used for further shell purification by sucrose gradient sedimentation. The sedimentation pattern of this material through a 1540% linear sucrose gradient is shown in fig. 3: shells were recovered in a fast migrating peak which accounted for 0.8-l % of the initial nuclear radioactivity after [3H]thymidine labelling, whereas the small

448

Preliminary notes

Preliminary notes

449

3. Sedimentation of [3H]thymidine-labelled nuclear shells from low-speed pellets. 03, Untreated; *- *, RNase-treated; Cl-Cl, alpha-chymotrypsintreated; O-a, 2 M NaCltreated. The sucrose concentrations varied from 15 (lefr) to 40% (right).

Fig.

Fraction no.

amounts of contaminating chromatin monoFig. 5 is an illustration of the organizaand oligomers did not enter the gradient. tion of partly protein-depleted shells obPreliminary attempts to characterize the served after spreading from a 2 M NaCl purified shell fractions were made. First of epiphase. The poorly dissociated shells reall, centrifugation of enzymatically and salt- sembled lattices of melted chromatin fibres. extracted low-speed pellets revealed that In the well-dispersed areas surrounding the the sedimentation properties of the shells lattices, long DNA fibres were attached remained unaffected by RNases but were at one or both ends onto the lattices in abolished by chymotrypsin and 2 M sodium a garland-like fashion. chloride (fig. 3). After partial nucleolar synthesis inhibi- Discussion tion, less than 0.2% of the total nuclear The resistance to low-ionic strength decon[3H]uridine labelling was recovered into densation of cortical chromatin, in striking shell fractions for incubation periods of 30 contrast to internal chromatin, is certainand 60 min. This labelling was totally sensi- ly the most original feature of the nuclear tive to RNases as was RNA in the internal shell. As previously illustrated, this feature explains the maintenance of the shape of chromatin (fig. 4). isolated chromatin structures [7], the spontaneous dissociation of the shells from the Fig. 2. Nuclear shells directly spread by centrifugation internal chromatin ,[ 1, 21, and the preservaof the nuclease-resistant material onto electron microscope grids, and rotary shadowed with platinum. tion of the structural integrity of the shells (a) Survey electron micrograph of a representative during nuclease treatment, unlike the largegrid square; (b) typical appearance of the spread shells; and (c) details of the fine shell structure with ly unfolded chromatin, which is uniformly knobby fibres (double arrows) made up of 27-30 nm digested [3]. Under our visualization condibeads (arrows). (a) x2500; (b) X15000; CC) x6OooO. Exp Cell Rcs I31 f/981)

450

Preliminary

notes

l

:

l

\

\

l\ ‘k-t -. -, *,-*-*-*~-*-*-*4~~~~~~~~~~~-~~*~*~*-*-5

15

10 Fraction

/*

I

t-• -*

Fip. 4. Sedimentation of

)

[3H]uridine-labelled nuclear shells from low-speed pellets after incubation of cells for 60 min. f- *, Untreated; O-0, RNase-treated. Same conditions as in fig. 3.

20

no.

tions, the isolated nuclear shells therefore appear as monolayers of broad, compact beaded fibres, closely resembling the outermost layer of dense peripheral chromatin in the nucleus in situ [ 14-161, in isolated chromatin [7], and in nuclear spreads [17, 181. Some kind of chromatin superstructure may be present in the isolated nuclear shell and, especially in the case of cortical DNA, may correspond to one of the first orders of supranucleosome coiling [19-211. These ultrastructural features, and the identification of the nuclear subfraction isolated here as chromatin allow to distinguish between the nuclear shell and the non-membranous nuclear ghosts obtained frbm HeLa cells by Riley & & Keller [22]. The latter structures remain after extraction of most chromatin proteins in high salts, so that the superstructural folding of DNA attached to the fibrous lamina is probably lost. Moreover, electron microscope and PAGE analyses of nuclear ghosts suggest that not only the fibrous lamina, but also residual nucleoli and internal matrix material are present. Exp Cell RPS 131 (1981)

I

I

The molecular basis for the original property of cortical chromatin should primarly be sought in its link with membrane proteins, which display a high affinity for certain peripheral DNA sequences [23]. Such a predominance by proteins is suggested by the loss of nuclear shell integrity after protease treatment. The involvement of proteins associated with, or forming part of the inner nuclear membrane is supported by the previously noted association of cortical chromatin with a tibrillar material presumably issued from the fibrous lamina [2, 31, and by the immunocytochemical study of Krohne et al. [24]. In addition, a possible role of non-histone chromatin proteins is currently under investigation. Although the abolition of ionic bonds between proteins and DNA in 2 M NaCl Fig. 5. Water spread of a histone-depleted and pla-

tinum-shadowed nuclear shell, showing (a) the lattice of undissociated material (L) and the surrounding areas containing dispersed material (da); (b) details of a da area with long, undigested DNA strands radiating from fibrillo-granular material (fg). (n) x2OooO; (6) x55ooo.

Preliminary notes

45 1

452

Preliminary

notes

Busch) vol. 1, p. 219. Academic Press, New York strongly affects the sedimentation proper(1974). ties of the shells, it is not sufficient to dis9. Dwyer, N & Blobel, G, J cell biol70 (1976) 581. organize the shell structure. This suggests 10. Herman, R, Weymouth, L & Penman, S, J cell biol 78 (1978) 663. that other types of protein-DNA interac- 11. Aaronson, R P, Methods in cell biology (ed D M Prescott) vol. 16, p. 337. Academic Press, New tions may be involved in maintaining shell York (1978). integrity. 12. Miller, 0 L & Bakken, A H, Acta endocrinol 188 (1972) 155. Lastly, it has been suggested that RNA 13. Comings, D E & Okada, T A, Exp cell res 103 may help to maintain a folded DNA con(1976) 341. 14. i)avi&, H G, J cell sci 3 (1968) 129. figuration [lo]. However, this is unlikely A C. Small. J V & Davies. H G. J cell sci 7 in the case of nuclear shells, since the small 15. Everid. (1970) j5. and variable amounts of RNA which they 16. Olins, A L & Olins, E D, J cell biol81 (1979) 260. 17. Franke, W W. Scheer, U, Trendelenburg, M F, contain are easily removed by RNases withSpring, H & Zentgraf, H, Cytobiologie 13 (1976) 401. out modification of the shell compactness. 18. Franke, W W, Zentgraf, H & Scheer, U, Electron From this preliminary characterization, microscopy (ed J M Sturges) p. 573. Microscope Society of Canada, Toronto (1978). we may reasonably conclude that nuclear 19. Kiryanov, G I, Manamshjan, T A, Polyakov, U Y, shells represent the superstructural folding Fais, D & Chentsov, J S, FEBS lett 67 (1976) 323. 20. Hozier, J, Renz, M & Nehls, P, Chromosoma 62 of membrane-bound chromatin within (1977) 301. chains of superbeads whose sensitivity to 21. Stratling, W H, Miiller, U & Zentgraf, H, Exp cell res 117(1978) 301. micrococcal nuclease is slight. This folding 22. Riley, D E & Keller, J M, J cell sci 32 (1978) 249. is maintained throughout a wide range of 23. Franke, W W, Deumling, B, Zentgraf, H, Falk, H & Rae, PM M, Exp cell res 81 (1973) 365. ionic strength conditions by protein-protein Krohne, G, Franke, W W, Ely, S, D’Arcy, A & or protein-DNA interactions which allow 24. Jost, E, Cytobiologie 18 (1978) 22. the nuclear shells to remain structurally inReceived July 3 I, 1980 tact under our conditions of isolation. The Revised version received October 6, 1980 nature of the proteins responsible for this Accepted October 21, 1980 folding, and the involvement of membrane proteins in such interactions remain to be determined. The isolation procedure deCopyright 0 1981 hy Academic Press, Inc. All rights of reproduction m any form reserved veloped here provides a tool for this purlK)l4-4827/8110204.(2-03602.00/U pose. Rapid effects of nerve growth factor on This work was supported by CNRS (ER 189 and the Na+,K+-pump in rat pheochromoGRECO 130023) and INSERM (U 183and SCN 18). cytoma cells

References

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1. Hubert, J, Bouvier, D & Bouteille, M, Biol cell 36 (1979) 87. ternational Embryological Institute, Uppsalalaan 8, 2. Bouvier, D, Hubert, J & Bouteille, M, J cell biol83 3584 CT Utrecht, The Netherlands (1979) 171a. 3. - J ultrastruct res. In press. Summary. Nerve growth factor (NGF) induces neu4. Franke, W W & Schinko, W, J cell biol 42 (1969) ronal differentiation of rat pheochromocytoma cells 326. (PC12). Here we show that NGF causes a stimulation 5. Brasch, K, Seligy, V L & SetterfIeld, G, Exp cell of Na+,K+-pump mediated K+ influx, with a maxires 65 (1971) 61. mum at 30 min after addition of NGF. The stimula6. Barton, A D, Kisielesky, W E, Wassermann, E & tion of the Na+,K+-pump is completely blocked by the Mackevicius, F, 2 zellforsch mikr anat 115 (1971) Na+-flux inhibitor amiloride (0.2 mM) and can be 299. mimicked by the Na+ ionophore monensin. These re7. Hancock, R, J mol biol86 (1974) 649. sults suggest that NGF causes a rapid enhancement 8. Franke, W W & Scheer, U, The cell nucleus (ed H of Na+ influx leading to an activation of the Na+,K+Exp Cell Ras 131 (1981)

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