A gentle method for preparing cyto- and nucleoskeletons and

associated chromatin

D. A. JACKSON1, J. YUAN2 and P. R. COOK1*

'Sir William Dunn School of Pathology, South Parks Road, Oxford 0X1 3RE, UK and 2Laboratory of Cell Biology, Tite RockefellerUniversity, New York, NY 10021, USA

•Author for correspondence

Summary

We describe a method for permeabilizing andextracting cells that preserves both structure andfunction whilst allowing the cell derivatives to behandled freely. Cells are encapsulated in micro-beads of agarose; the coat of agarose, which isfreely permeable to small molecules, forms aprotective layer around fragile cell constituents.Cells are then permeabilized by the non-ionicdetergent Triton X-100 or antibody and comp-lement in a buffer whose ionic compositionmimics that of the cytoplasm. The resulting struc-tures have been characterized morphologically(by immunofluorescence and electron mi-croscopy) and biochemically. Lysis -with Tritonremoves both cell and nuclear membranes, andextracts most of the cytoplasm to leave chromatinsurrounded by cytoskeleton; nucleus and cyto-

plasm then become accessible to triphosphates,enzymes and antibodies. Lysis with complementpermeabilizes the cell membrane but leaves thenuclear membrane intact; triphosphates and re-striction enzymes, but not antibodies, can thenenter both nucleus and cytoplasm. Both types oflysis yield preparations whose chromatin tem-plate remains essentially intact, and which isreplicated and transcribed at rates close to, orgreater than, those found in vivo. Treatment ofcomplement-lysed cells with Triton reduces thevery efficient DNA synthesis, implying that thenuclear membrane is involved, directly or in-directly, in replication.

Key words: method, cytoskeleton, nucleoskeleton,permeabilize, chromatin.

Introduction

The skeleton of the cell is so very fragile that it isbroken by all but the gentlest fractionation procedure.For example, pelleting cells permeabilized by the non-ionic detergent Triton X-100 disrupts their cytoskel-eton, which then becomes further disrupted on resus-pension by pipetting. Cells are rarely lysed in bufferscontaining physiological concentrations of ions, sinceisolated nuclei and chromatin aggregate into anunworkable mess. Some procedures permeabilize cellmembranes using electric fields, detergents or abnor-mal ion concentrations, without allowing too muchcytoplasm to be lost (for reviews, see Miller et al. 1979;Knight & Scrutton, 1986; Otero & Carrasco, 1987;Beckers et al. 1987). Then cell remnants retain much ofthe original structure and can, in some cases, evenrepair the damaged membranes and resume growth;most importantly, the cell interior is accessible to smalltracer molecules. Other methods involve treating cells

Journal of Cell Science 90, 365-378 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

growing on coverslips, so allowing transfer from onesolution to another without too much shearing; suchcytoskeletons are usually well-preserved but they can-not be handled too vigorously without fixation.Methods for isolating nuclei in bulk almost invariablyinvolve swelling cells in hypotonic buffers, whichactivate nucleases and extract much of the activepolymerases (Jackson & Cook, 19856, 19866,c). Wetherefore felt that there was a need for another methodthat permitted the gentle deconstruction of the cell andalso preserved vital functions.

In developing a method, we have borne a number ofconsiderations in mind. First, we wished to permeabi-lize both the cell and nuclear membrane so that allmajor compartments were accessible to tracers andprobes (e.g. radiolabelled triphosphates, antibodies,enzymes and oligonucleotides). Second, we haveattempted to use 'mild' conditions. Third, and mostimportantly, we have made the preservation of nuclear

365

function our major priority.Our approach is an extension of a method originally

developed for isolating naked DNA from eukaryoticcells in a form that could be manipulated withoutbreaking it (Cook, 1984). Structure is preserved byproviding the fragile DNA with a protective coat ofagarose, by first encapsulating cells in agarose micro-beads. An aqueous phase containing living cells inmolten agarose is homogenized with an immisciblephase of liquid paraffin. On cooling, suspended agarosedroplets gel into microbeads of about 50^m in diam-eter. Protein complexes as large as l '5xlO8Mr, but notthe very much larger skeletal elements or chromosomalDNA, can diffuse through the agarose. Therefore ionicdetergents extract nearly all protein and RNA fromencapsulated cells, leaving naked DNA, which isnevertheless completely protected from shear. Tritonat a more physiological salt concentration extracts mostcytoplasmic proteins and RNA but leaves encapsulatedchromatin surrounded by insoluble cytoskeletal el-ements (Jackson & Cook, 1985a). Encapsulatedmaterial is completely protected from shear and can betransferred from one buffer to another simply bypelleting, yet it is also accessible to molecular probes.

We now have developed this approach further. Cellsare encapsulated, regrown in microbeads and then bothcell and nuclear membranes are disrupted using Tri-ton. Lysis and subsequent washing take place in abuffer whose composition is approximately cytoplas-mic. (For recent reviews, see Roos & Brown, 1985; Lev& Armstrong, 1975.) The buffer (pH7-4) contains22mM-Na+, 130mM-K+, lmM-Mg2+, <f>3jUM freeCa2+, 132mM-Cr, 11 mM-phosphate, 1 mM-ATP.1 mM-dithiothreitol is an optional addition. We chose aphosphate buffer to control pH to 7-4 and adjustedsodium, potassium and phosphate ions roughly tocellular levels. Levels of Cl~ are unphysiologicallyhigh. (In vivo proteins are the counterions.) Control-ling free Mg2+ levels posed a special problem. Currentconcern in our laboratory is with both the structure ofchromatin and the integrity of DNA, and in earlierexperiments we used EDTA to chelate any Mg2"1" thatmight activate nucleases; although this preserved DNAintegrity, it also decondensed heterochromatin (Jack-son & Cook, 1985a). We therefore decided to use amore physiological chelating agent, ATP. Using 1 mM-ATP and 1 mM-Mg2+ (levels roughly those found invivo) we find there is sufficient free Mg2+ to preserveheterochromatin structure but insufficient (because itis nearly all complexed with ATP) to activate nucleases.Fortunately, this formulation enables most ATP-utiliz-ing enzymes to function and ensures that contaminat-ing free Ca2+ is held at about 0-3 jUM, well below criticalactivating concentrations (Carafoh, 1987).

As we are also interested in the nuclear membrane,we developed a variant method that preserves its

structure. We permeabilize specifically the cell mem-brane using cytolytic antibodies and complement(Reid, 1986), whilst leaving the nuclear membraneintact. Of course, this has the disadvantage that we useundefined reagents (i.e. whole serum).

Using Triton or cytolytic antibodies, we lysed encap-sulated HeLa cells and removed soluble material bywashing in this isotonic buffer. Then we characterizedthe residual structures: the morphology of the cytoskel-eton and nucleus was well preserved, both nuclear andcytoplasmic compartments were accessible to proteinsand the DNA remained intact, and was replicated andtranscribed at rates close to those found in vivo.

Materials and methods

CellsHeLa cells were grown as suspension cultures in minimalessential medium plus 5 % newborn calf serum. In mostexperiments cells were grown for 18-24 h in [methyl-3H]thy-midine (0-05;tCiml"'; = 6 0 C i m m o r ' ) to label their DNAuniformly. This enabled corrections to be made subsequentlyfor any slight variations in cell numbers.

Cell encapsulation and lysisCells were encapsulated in 0 '5% agarose as described byCook (1984). Odd large beads that might block pipettes wereremoved by filtering a dilute solution through monofilamentnylon filters of 150(Urn mesh (R. Cadisch and Sons, LondonN3 2JW) using a Swinex filter. A variety of different lysisbuffers was used. (1) Triton, pH8-0. One vol. encapsulatedcells in phosphate-buffered saline was mixed with 3 vol. ice-cold lysis mixture containing 0-5% Triton X-100, 100 mM-KC1, SOmM-NaCl, lOmM-Tris-HC1 (pH80) , 1 mM-Na2EDTA, 1 mM-dithiothreitol; after IS min on ice they werewashed three times in ice-cold lOOmM-KCl, 50mM-NaCl,10mM-TrisHCl (pH8-0), 1 mM-Na2EDTA and 1 mM-di-thiothreitol (Jackson & Cook, 1985a). (2) Triton, pH7-4.Encapsulated cells were washed in ice-cold pH7-4 buffer,Triton X-100 was added to 0-5 % and, after 15 min on ice, thecells were washed three times in cold pH7-4 buffer. pH7-4buffer was made by adding 100 mM-KH2PO4 to 130mM-KCl,10mM-Na2HPO4, 1 mM-MgCl2, 1 mM-Na2ATP (Sigma typeII), 1 mM-dithiothreitol, to bring the pH to 7-4. As theacidity of the ATP varied from batch to batch, variousamounts of KH2PO4 must be added, never exceeding0-01 vol. and generally increasing K+ to 130-8 mM and PO4

3~to ll-6mM. The free Ca2+ levels (kindly measured by PeterGriffiths, Physiology' Department, Oxford) of this bufferwere below 0-3 fiM and can be clamped at 0-1 J.IM using 40ftM-EGTA. Note that many tissue-culture media contain milli-molar quantities of Ca2+, so that cells should be well washedif calcium levels are of concern. Note also that Ca2+ levelsmay rise transiently as cells are lysed, affecting cytoskeletalstructure (Schliwa el al. 1981). (3) Complement, pH 7-4.Antiserum directed against HeLa cell surface antigens wasobtained by injecting HeLa cells in phosphate-buffered salinesubcutaneously into rabbits (3 injections at weekly intervals)and collecting the serum 1 week later. Encapsulated cells

366 D. A. Jackson et al.

were incubated with 0*5 vol. heat-inactivated whole anti-serum for 30 min at 20°C, washed twice in pH 7-4 buffer andincubated for 10min at 20°C with 0 1 vol. rabbit comp-lement, which had a low toxicity for human lymphocytes(SeraLab), and then the lysed cells were washed three timesin ice-cold pH7-4 buffer. Note that serum, the source ofcomplement, contains millimolar amounts of free Caz+ andMg2"1" (as well as many other unknown factors) and that theseions are essential for lysis; therefore, the milieux during lysisby complement and by Triton in the pH 7-4 buffer aredifferent. (4) Complement, p H 8 0 . Cells were washed inlOOniM-KCl, SOmM-NaCl, 10mM-Tris-HCI (pH8-0),1 mM-Na2EDTA and 1 tnM-dithiothreitol, lysed by antibodyand complement as described above and rewashed in thesame buffer. (5) Triton, pH8-0 and 2M-NaCl. One vol. ofencapsulated cells in phosphate-buffered saline was mixedwith 3 vol. ice-cold lysis mixture that contained 0 5 % TritonX-100, 2-OM-NaCl, 10mM-Tris- HCI (pH80) , 100 mM-Na2EDTA; after 15 min on ice they were washed three timesin ice-cold 2-OM-NaCl, 10mM-Tris-HCI (pH80) , 1 mM-Na2EDTA (Cook el al. 1976).

Light microscopy and immunofluorescenceLysed encapsulated cells were generally washed once in theappropriate buffer minus dithiothreitol prior to incubations(45 min at 20°C or 2 h at 4CC) with primary antibodies, thenwashed three times with the same buffer, incubated (45 minat 20°C or 2 h at 4°C) with the appropriate second antibody,rewashed, viewed and photographed without fixation using aLeitz Orthoplan or a Zeiss Photomicroscope III fluorescencemicroscope fitted with the appropriate filters. A wide range ofantibodies has been used including: (1) 414 (kindly providedby Dr L. I. Davis), a mouse monoclonal directed against aprotein of 62xlO3/V/r in the rat liver nuclear pore complex(Davis & Blobel, 1986). The supernatant from the culturedmonoclonal cell line was used at a ratio of 1 : 1 and found tocross-react with the human antigen. Rhodamine-conjugatedaffinity-purified goat anti-mouse IgG (Cappel) diluted 1:25with the appropriate buffer supplemented with 1 % bovineserum albumin, 5 % Triton was used to visualize the primaryantibody. (2) Anti-Sm mouse monoclonal (Cappel), used(dilution 1 in 50) with thodamine-conjugated affinity-puri-fied goat anti-mouse IgG (Cappel; dilution 1 in 100). Amonoclonal directed against a glial fibrillar acidic protein(Amersham; dilution 1 in 50) served as a control firstantibody. (3) Anti-human vimentin mouse monoclonal(Dako; dilution 1 in 50), used with fluorescein-conjugatedaffinity-purified rabbit anti-mouse IgG (dilution 1 in 100) oncells pre-fixed in 3 % formaldehyde in phosphate-bufferedsaline.

Lysed cells were also incubated with and without cytochal-asin B (lO/Ugml"', 30min, 37°C), then with rhodamine-conjugated phalloidin (3 units ml" ' , 10 min, 4°C, MolecularProbes Inc) or 3,3'-dihexyloxacarbocyanine ( l j igmP1,5 min, 20cC) and washed as above.

Macromolecular recoveriesMacromolecular recoveries were determined by measuringthe amount of label associated with encapsulated cells beforeand after lysis and washing (Cook, 1984). Protein recoverieswere determined using cells grown overnight in medium

lacking leucine or methionine and supplemented with 2%normal medium, 5% dialvsed foetal calf serum and L-[U-HC]leucine (01 jiCi ml" ' ; 350 mCi minor1) or L-[3SS]meth-ionine (1 or 0-lf/Ciml"1, 800mCimmor ' ) . RNA wasestimated using cells labelled in growth medium plus [5,6-3H]uridine (53Cimm O r ' ) for 2min (SO/iCiml"1) or 24h(OSjuCiml"'); in the former case cells were encapsulatedprior to labelling and in the latter, values were corrected forincorporation (21 %) of uridine into DNA. DNA recoverieswere determined by labelling for 24h with [methyl-3\i]thy~midine (47Cimmor ' ; 0-5fiCiml"').

The protein content of extracted cells was analysed using12% to 20% polyacrylamide gradient gels (see Fig. 4A).Cells, prelabelled with L-[35S]methionine, were encapsulated(2-5X 106ml~'), lysed and washed in buffers supplementedwith 1 mM-phenylmethylsulphonyl fluoride and 25 ;/M-leupeptin, pepstatin and L-l-chloro-3-(4-tosylamido)-4-phenyl-2-butanone, proteins dissolved in sodium dodecylsulphate, ~50000ctsmin~' loaded per lane and subjected toelectrophoresis and autoradiography (2 weeks). For Fig. 4Bany soluble proteins remaining after washing were electro-eluted from beads under isotonic conditions using slots in a0-8 % agarose gel (4 h at 4 V cm" ' ; buffer was recirculated toprevent pH drift). The buffering capacity of the pH 7-4buffer was increased for this by increasing the phosphate ionconcentration at the expense of the chloride ion so that theelectrophoresis buffer contained 806mM-KCl, lOmM-Na2HPO4, 20mM-K2HPO4, 9-4mM-KH2PO4, 1 mM-MgCI2,1 mM-dithiothreitol and 1 mM-Na2ATP. Beads were re-covered, washed in pH7-4 buffer, treated in various ways asdescribed in the Fig. legend, rewashed in pH 7-4 buffer,samples were applied to a gel and autoradiographs (2 or 14days) prepared as described above.

Electmn microscopyBeads were fixed in 2-5% glutaraldehyde, post-fixed in 2 %osmium tetroxide, stained in 0 5 % uranyl acetate andembedded either in Epon-Araldite or the removable embed-ding compound diethylene glycol distearate (Capco el al.1984), using the procedure described in detail by Fey el al.(1986). Samples were also critical-point dried, taking care toensure samples were free of water (Ris, 1985).

Digestion with nucleasesCells, prelabelled with [3H]thymidine, were encapsulated(5xl06ml~') , lysed, washed and incubated in an equal vol.buffer at 37 °C for 0-80 min with 500 or 2500 units ml" '£coRI or 20 or 200uni tsmr ' HaeUl. In Fig. 5, units referto the product of units ml"1 and time (h). After incubation,samples were removed and split. DNA fragment sizes wereanalysed in half the sample after addition of sodium dodecylsulphate to 0 2 % by gel electrophoresis (0-7 V cm" ' ; 16 h)through 0-8% agarose in 40mM-Tris*acetic acid (pH8-3),2 mM-EDTA and 20 mM-sodium acetate; after electrophoresisRNA was solubilized by soaking the gel in ribonuclease(20ftgml~'; 30min, 20°C) and the gel was stained withethidium and photographed. The other half was used todetermine how much chromatin could be electroeluted frombeads under isotonic conditions (using 0-8% agarose gel andthe buffer with extra phosphate described above, 15 h,2Vcm~ ). The amount of 3H remaining in beads was

Cvto- and nucleo-skeletons 367

determined by mixing 100-j«l samples with 250^1 2% sodiumdodecyl sulphate. More than 2h later three 100-/<l sampleswere spotted onto GF/C discs and the discs were extractedsuccessively with 5 % trichloroacetic acid, ethanol and ether,then dried and their radioactivity was estimated using aPackard 300 CD scintillation counter.

Penneability of beads to DNAThe permeability of beads to DNA fragments of differentsizes was determined by encapsulating bacteriophage A DNAcut with / / /ndl l l , quickly washing in 100vol. 50mM-NaCl,10mM-Tris'HC1, 1 mM-EDTA in a microcentrifuge, thenincubating the beads plus DNA at 20°C and at various timesdetermining the amount of DNA remaining in beads byquantitative densitometry after quickly washing beads, sub-jecting them to gel electrophoresis, staining with ethidiumbromide and photography (Cook & Brazell, 1978).

Replication and transcription assaysGeneral methods for assays have been described by Jackson &Cook (19856, 1986a). Replication assays were conducted at37°C in appropriate buffers supplemented with 250 jtM-dATP, dCTP and dGTP plus 125/iM-dTTP and lmCiml"1

[32P]dTTP (=3000Cimmor ' ) plus 100fJM-CTP, GTP andUTP plus 5 mM-potassium phosphate (pH7-4) to ensuretriphosphates were neutralized, plus 1 mM-ATP and 5 mM-MgCI2. Transcription assays included 250f<M-CTP and GTPplus 125/iM-UTP and l m C i m r 1 [32P]UTP (=3000Cimmol"') plus 1 mM-ATP plus 5 mM-MgC^. Initial rates weremeasured over the first 5min. Encapsulated and lysed cells( 5 x l 0 6 m r ' beads; 2-5X106 ml"1 solution) and freshly made10 times concentrated solution of supplements were incu-bated separately on ice, then at 37°C for 5 min before mixingto start the reaction. Reactions were stopped by removing100- tl samples and mixing them with 250jtt! 2% sodiumdodecyl sulphate. More than 2h later three 100-/il sampleswere spotted onto GF/C discs and the discs were extractedsuccessively with 5 % trichloroacetic acid, ethanol and ether,then dried and their radioactivity was estimated using aPackard 300 CD scintillation counter.

FluowmettyThe ethidium-binding capacity of encapsulated and lysedcells was determined on ice-cold samples as described (Cook& Brazell, 1978; Jackson & Cook, 1985a). Every experimentinvolved a comparison of the fluorescence of y-irradiated(9-6Jkg~') and non-irradiated samples. Encapsulated andlysed cells were incubated in pH 7-4 buffer at 37°C and aftervarious times EDTA, NaCl and ethidium bromide wereadded to final concentrations of lOmM, 2M and 8^gml~',respectively. Half were y-irradiated and the fluorescence ofboth halves was measured. After subtraction of appropriateblanks, the fluorescence of dye bound to non-irradiated beadswas divided by that bound to irradiated beads, effectivelynormalizing all non-irradiated values to those of equalamounts of fully relaxed DNA. This ratio reflects theproportion of nicked loops: at the ethidium bromide concen-tration used it is insensitive to changes in degree of super-coiling. To some extent it reflects the amount of RNA, whichalso binds ethidium, remaining after extraction with 2 M-NaCI and as this differs for samples lysed differently, relative

changes occurring on incubation are more informative thanabsolute values.

Results

Morphology

Fig. 1A illustrates HeLa cells encapsulated in 0-5 %agarose microbeads. On resuspension in warm me-dium, they resume growth. Addition of Triton lysescells and within seconds most soluble proteins diffuseout of beads (Cook, 1984), leaving a well-preservednucleus surrounded by the Triton-insoluble cyto-skeleton (Fig. IB). (The various buffers are describedby the lytic agent used (Triton or complement) and pH(80 or 7-4), the pH8-0 buffer containing Tris andEDTA and the pH7-4 buffer being closer to thephysiological.) Cells lysed with Triton at pH 8-0 retainthe major nuclear functions (Jackson & Cook, 1975a,b,1986a,b); for example, they replicate DNA in a cell-cycle-dependent manner at a rate in vitro that is at least85 % of the rate found in vivo. However, despite suchexcellent preservation of function, their structure is notso well-preserved, since some heterochromatin decon-denses (see below). Therefore, we explored lysis withTriton and our pH 7-4 buffer that more closely mimicsthe ionic content of the living cell. The light micro-scope reveals an excellently preserved nucleus sur-rounded by cytoplasmic remnants (Fig. 1C). Lysiswith complement rather than Triton swells the cellsand induces some cytoplasmic loss (Fig. ID). (HeLaare particularly resistant to lysis by complement,presumably because their cytoskeleton is so well devel-oped. Other cells, for example lymphocytes and theirderivative, are lysed more easily, with almost completeloss of cytoplasm.) This loss can be visualized afterstaining with 3,3'-dihexyloxacarbocyanine, a lipophilicand fluorescent dye (Terasaki et al. 1984); Tritonextracts all membranes (cf. Fig. IE and G), whereascomplement lysis leaves the endoplasmic reticulum andthe nuclear membranes essentiallv intact (cf. Fig. IEand F).

Electron micrographs of typical conventional thinsections of these derivatives are illustrated in Fig. 2.(Note that heterochromatin of unlysed cells variesgreatly and this is naturally reflected in lysed deriva-tives; we therefore present typical cases.) As expected,Triton removes both cell and nuclear membranes andextracts most cytoplasm (cf. Fig. 2B and C with A). Inmany cases the cytoskeleton contracts from the sur-rounding agarose coat. The improved buffer preservesnuclear structure slightly better than the pH 8-0 bufferthat we used earlier; for example, there are moreperipheral clumps of heterochromatin and typically thechromatin is less 'washed-out' (cf. Fig. 2B and C).Nevertheless, much heterochromatin has decon-

368 D. A. Jackson et al.

Fig. 1. The morphology of encapsulated HeLa cells and their derivatives lysed with Triton or complement. A—D. Phase-contrast micrographs: A, unlysed; B, lysed, Triton/pH 8 0 ; C, lysed, Triton/pH 7-4; D, lysed, complement/pH 7 4 .E-G. Fluorescence micrographs using the lipid stain, 3,3'-dihexyloxacarbocyanine: E, unlysed; F, lysed,complement/pH 7-4; G, lysed, Triton/pH 7-4. H—I. Fluorescence micrographs using rhodamine-conjugated phalloidin:H, lysed, complement/pH 7-4; I, lysed, complement/pH 7-4 and then incubated with cytochalasin B.J-N. Immunofluorescence using various first antibodies: J, lysed, Triton/pH 7 4 , anti-nuclear pore complex; K, lysed,Triton/pH 74 , anti-Sm; L, lysed, Triton/pH 7-4, anti-ghal protein (i.e. control); M, lysed, Triton/pH 7-4, fixed, anti-vimentin; N, lysed, complement/pH 7-4, fixed, anti-vimentin. A-B and C-N are at the same magnification, respectively.Bars: 50 and lO^tm.

densed. (As expected, spermine and spermidine lessendecondensation (results not shown) but probably haveother effects.) Nuclear structure is excellently pre-served on complement-mediated lysis (Fig. 2D). Theholes punched by the attack complex of complement inthe cell membrane are too small to be seen at this

magnification, but in consequence the cytoplasmusually becomes vacuolar (this is not well illustrated inFig. 2D; but see Fig. 3D). Our improved buffer alsomaintains the morphology of internal membranes,including the nuclear membranes, and those of mito-chondria, organelles that are most sensitive to abnor-

Cvto- and nncleo-skeletons 369

2A

Fig. 2. Electron micrographs of thin sections of encapsulated HeLa cells. A. Unlysed; B, lysed, Triton/pH7-4; C, lysed,Triton/pH 8; D, lysed, complement/pH 7-4. All are at the same magnification. Bar, 2-5/*m.

mal ionic concentrations.Conventional sections used for electron microscopy

are too thin (about 50 nm, with only the top few nmbeing accessible to stain) to reveal much detail ofstructures many fim long; therefore, skeletal structuresare better seen in thicker (resinless) sections (Capco etal. 1984). Fig. 3 illustrates such 250 nm thick sections,where the surrounding filaments of agarose can beclearly seen. These thicker sections also give a betterimpression of the average density of material inextracted cells and appear quite different from thinnersections; for example, the peripheral clumps of hetero-chromatin become more difficult to see. On lysis inTriton (Fig. 3B and C) the cytoskeleton collapses awayfrom the agarose, whereas complement vacuolates andswells the cytoplasm (Fig. 3D).

Whether skeletal networks like the microtrabecularlattice (Wolosewick & Porter, 1979) and nuclear matrix

(Agutter & Richardson, 1980), which are visible in theelectron microscope only after fixation or exposure toabnormal salt concentrations, also exist in vivo iscontroversial. (See, for example Ris, 1985.) Therefore,we have taken care to use fixation and drying pro-cedures that minimize the artefactual creation of fila-mentous structures. Thus, we fixed freshly lysed cellsin the (Ca2+-free) pH7-4 buffer with glutaraldehyde,conditions that best preserve nuclear morphology(Skaer & Whytock, 1977) and prevent ribonucleo-protein aggregating into filaments (Lothstein et al.1985). We also critically point dried them prior tosectioning, using conditions that both permitted(Capco et al. 1984; Wolosewick & Porter, 1979) andprevented (Ris, 1985) subsequent visualization of amicrotrabecular lattice; at the magnification used inFig. 3 no gross differences could be seen, but at highermagnifications the different methods yielded marked

370 D. A. Jackson et al.

• 1 U

3A

V

B

Fig. 3. Electron micrographs of thick sections of cells. A. Unlysed; B, lysed, Triton/pH 7-4; C, lysed, Triton/pH8; 0,lysed, complement/pH 7-4. All are at the same magnification. Bar, 2-5 im.

differences in detail (D. A. Jackson & P. R. Cook,unpublished data).

Macromolecular recoveries

Table 1 and Fig. 4A illustrate the macromolecularcontent of cells lysed in different ways. They allcontain all the DNA and nascent RNA, and variableamounts of long-lived (i.e. cytoplasmic) RNA andprotein; complement-lysed cells retain the most. Tri-ton selectively removes certain proteins, unlike comp-lement and antibody (Fig. 4A; note that twice thenumber of cell equivalents were loaded in lanes 4 and5). There was no obvious effect of pH.

When cells are heat-shocked by temperatures3-5 deg. C above normal, some proteins associate withkaryoskeletal elements. This may happen with nucleiisolated by conventional procedures, but it is thentriggered by physiological temperatures; isolation ofnuclei sensitizes them. The effect is especially notice-

Table 1. Macromolecular recoveries of encapsulatedcells lysed in different ways

% Remaining after lysis

RNA

Lvsis conditions

Complement, pH7-4Triton, pH7-4Complement, pH 80Triton, pH80

Nascent RNA andlabelling for 2min or

Protein

67246523

Nascent

95989497

Long-lived

62285820

long-lived RNA were determined a24 h.

DNA

%99

101100

ftcr

able after the heat-shocked nuclei are treated withdeoxyribonuclease and extracted with 2M-NaCl (Evan& Hancock, 1985; Littlewood et al. 1987; McConnellet al. 1987). This has led to skepticism as to whether

Cvto- and ftucleo-skeletorts 371

Lane 1 2 3 4 5 1 2 3 4 5 6 7 8

I

w * = sFig. 4. Autoradiographs of gels containing proteins fromcells, prelabelled with [35S]methionine, and extracted invarious ways (A) or extracted and heat-treated (B).Arrowheads indicate the positions of size markers of 93, 69,46, 30, 21 and 14(X 103).1/r. A. Encapsulated cells, unlysed(lane 1) or lysed and washed using complement/pH 8 0(lane 2), complement/pH 7-4 (lane 3), Triton/pH 8-0 (lane4) and Triton/pH 7-4 (lane 5). Lanes 1-3 contain 10s cellequivalents and lanes 4 and 5, 2xlO5. B. Encapsulatedcells, lysed in Triton/pH 7*4 were washed, residual solubleprotein was removed electrophoretically under isotonicconditions and beads treated for 20min at 0°C (lanes 1,2),30°C (lanes 3, 4), 37°C (lanes 5, 6) and 43°C (lanes 7, 8).After treatment, some of each sample was applied directlyto the gel (odd-numbered lanes) and the remaining beadswere extracted with 2M-NaCI, 10mM-Tris-HC1 (pH8-0),1 mM-EDTA and washed thoroughly before applying to thegel (even-numbered lanes). Autoradiography was for 2weeks; the inset below shows a 2-day exposure of theoverexposed region at the bottom of the gel that containsthe histones.

structures seen only after pretreatment of nuclei at37°C, like scaffolds (Mirkovitch et al. 1984), havecounterparts in vivo (McConnell et al. 1987). Wetherefore repeated the experiments of Littlewood et al.(1987) to see whether heat-shocking our preparationsinduced a similar set of proteins to become associatedwith the karyoskeleton. Cells were prelabelled with[35S]methionine, encapsulated, lysed, washed, incu-bated at different temperatures and any residual sol-uble proteins were removed electrophoretically; thenproteins remaining associated with beads were analysedby gel electrophoresis (Fig. 4B, odd-numbered lanes).No proteins associate with our preparations on incu-bation at 43°C /;/ vitro, nor do they do so afterextraction with 2M-NaCl (Fig. 4B, even-numberedlanes; a shorter exposure is shown in the inset below

and shows that most histones have been extracted).(Treatment with deoxyribonuclease (10|*gmi~';30min at 0°C or 30°C) prior to extraction with 2 M-NaCl gave similar results (results not shown).) As weshall see, functional studies also show that our prep-arations have not been sensitized to heat, since repli-cation and transcription, which are shut down whenliving cells are heat-shocked, continue efficiently at37°C. Perhaps nuclei prepared by conventional pro-cedures become sensitized to heat when they areexposed to the hypotonic conditions used during celllysis.

Permeability

Before they can reach all cell compartments, addedprobes must pass successively through the agarose coatand however much of the cell membrane, cytoplasm,nuclear membranes and chromatin that remains. Theagarose coat offered no barrier to the probes that weused. Theory suggests 0-5 % agarose is permeable toglobular proteins of <2xl08iV/r and 120 nm diameter,and practice shows that nucleoprotein complexes ofl-5Xl08iV/r pass through it (Jackson & Cook, 1985ft).As a result, enzymes equilibrate throughout beadswithin seconds. For example, encapsulating DNAdelays maximal transcription by added RNA polym-erase from E. coli, one of the largest enzymes known(i.e. 4-8xl05iV/r), by less than 2min (results notshown). However, the diffusion of larger DNA mol-ecules is limited. Thus, the half-times for escape of AHindlU fragments of 23-7, 9-5, 6-7, 4-3 and 2-3 kbfrom 0-5% beads are 200, 80, 35, 20 and lOmin,respectively (see Materials and methods). In contrast,DNA molecules the size of yeast chromosomes(>250kb) remain trapped for at least 6 months (resultsnot shown).

We next determined how accessible the variouscellular compartments were to various probes.Although it was originally thought that Tritonextracted only the cell and outer, but not inner, nuclearmembrane, it is now known to remove all three(Aaronson & Blobel, 1974). Therefore, Triton-extracted encapsulated cells are accessible to a varietyof molecular probes (e.g. restriction enzymes, polym-erases and antibodies). Fig. 1J-M illustrates such cellsstained with various antibodies (i.e. those to thenuclear pore complex on the nuclear periphery(Fig. 1J), Sm antigens inside the nucleus (Fig. IK)and cytoplasmic vimentin fibres that have collapsed onlysis into a dense mass on the nuclear surface(Fig. 1M). An antibody directed against a protein notfound in HeLa cells provides a control (Fig. 1L). Notethat unlike most other immunofluorescence images,Fig. 1J and K are from unfixed cells, i.e. unfixed in thesense that many enzymic activities remain (see below).

In contrast to the permeability of most compart-

372 D. A. Jackson et al.

Lane M

200Enzyme (units)

300 3000

Fig. 5. Accessibility to restriction enzymes of chromatin in encapsulated cells lysed using Triton or complement at pH 7-4.Cells were labelled with [3H]thymidine, encapsulated, lysed, treated with various amounts of Haelll (A,C) or KcoRl(B,D) and samples split. A,B. The size distribution of DNA was determined in one half. DNA was purified, subjected togel electrophoresis, and the gels stained with ethidium and photographed. A. Haelll digestion. Lane M, marker A DNAcut with /-///idIII; lane 1, uncut sample; lanes 2—4, complement-lysed cells; and lanes 5—7, Triton-lysed cells incubatedwith 16, 66 and 270 units, respectively. B. EcoRl digestion. Lane M, marker DNA; lane 1, uncut sample; lanes 2-4,complement-lysed cells; and lanes 5-7, Triton-lysed cells incubated with 42, 167 and 666 units (product of units ml"1 andtime (h)), respectively. C,D. The percentage of chromatin that resisted electroelution from beads was determined using theother half. Beads were subjected to electrophoresis, recovered and the percentage of 3H (i.e. chromatin) remaining withinthem determined. Digestion with Hae\\\ (C) or EcoRA (D). ( • ) Triton-lysed cells; ( • ) complement-lysed cells.

ments of Triton-lysed cells to antibodies, the cellmembrane of complement-lysed cells is impermeable(results not shown); antibodies are too bulky to passthrough the 10 nm pores generated by the attackcomplex (Muller-Eberhard, 1986). (Note that this istrue only for 'robust' cells like HeLa. The precisemechanism by which complement lyses nucleated cellsremains obscure, but the membrane of some cells (e.g.lymphocytes) may tear on lysis, so that antibodies enterthem (results not shown).) Of course, complement-lysed cells become permeable to antibodies after fix-ation; Fig. IN illustrates an excellently preservednetwork of vimentin fibres. However, unfixed butcomplement-lysed HeLa cells are permeable to smallermolecules like rhodamine-conjugated phalloidin(Fig. 1H). The actin filaments in the cytoplasm areseen as a fine three-dimensional network, which is notreproduced well at the exposure chosen in Fig. 1H. Ontreatment after lysis with cytochalasin these filamentsaggregated; the resulting thicker filaments fluoresced

brightly (Fig. II ; a similar exposure was used forFig. 1H and I). Note that images of cytoskelctalnetworks are usually obtained using well-flattened cellsattached to coverslips; here we used round cells. Inaddition, cytochalasin can only effect a rearrangementafter lysis as the material is 'unfixed'.

We next explored the accessibility of chromatin inTriton-lysed cells to the restriction enzymes, Haellland EcoRl (Fig. 5). (EcoRl is composed of twosubunits of 29xlO3/V/r (Modrich & Zabel, 1976); wehave been unable to discover the size of Haelll.) Beadswere incubated with the enzymes, then their DNA wassubjected to electrophoresis. DNA from the zero timepoint is of such a high molecular weight it barely entersthe gel (Fig. 5, lane 1; the slight cutting seen at 'zero'time occurred during the inevitable lag between ad-dition of enzyme and stopping the reaction). Asexpected, treatment with Haelll cut most of the DNAin Triton-lysed cells into pieces of <20kb, with somebeing cut to nucleosomal size (Fig. 5A, lane 7). EcoRl,

Cyto- and iiucleo-skelelons 373

which cuts less frequently, generated larger fragments(Fig. 5B, lane 7). The enzymes also cut chromatin incomplement-lyscd cells, albeit to a lesser extent(Fig. 5A and B, lanes 2-4). Clearly, they pass not onlythrough the pores generated by the attack complex butalso those in the nuclear membrane. Hindll(75Xl03iV/r) is the largest enzyme that we have foundthat enters the nucleus (results not shown). (Both setsof pores are about 10 nm in diameter, slightly largerthan that of 'ideal' spherical proteins the size ofHindll.) If beads containing lysed cells are subjectedto electrophoresis in isotonic conditions after digestion,chromatin fragments escape (Fig. 5C and D), presum-ably through these pores. (We suspect that electrophor-esis does not tear membranes, as both nucleus andcytoplasm remain inaccessible to antibodies.) Essen-tially all chromatin can be electroeluted after Haellltreatment of Triton-lysed cells (Fig. 5C). The kineticsof removal are biphasic and probably reflect a complexinterplay between an initial accessibility of hetero- andeu-chromatin and then differential electrophoreticmovement of variously sized particles through residualskeletons and pores.

These results mean that the nuclear interior of bothTriton- and complement-lysed preparations will prob-ably be accessible to most enzymes currently used inmolecular biology. Note also that nearly all the RNAand DNA polymerase activities of the cell are recoveredin beads after cutting and electroelution (Jackson &Cook, 19856, 1986fl). (We have repeated these earlierexperiments using the pH7-4 buffer, with essentiallysimilar results (not shown; see below).)

DNA integrity

The presence of nicks or breaks in encapsulated nuclearDNA can be detected with great sensitivity by fluor-ometry using the intercalating dye, ethidium bromide(Cook & Brazell, 1978; Cook, 1984). Treating lysedand encapsulated cells with 2M-NaCl removes mostproteins, leaving naked and superhelical DNA loopedby attachment to a nuclear cage or matrix. One nickanywhere within a loop of about 100 kb releases allsupercoiling from that loop and this loss of supercoilingincreases the amount of ethidium bound to the DNA.As the fluorescence of ethidium is enhanced when itbinds, binding, and hence integrity, can be con-veniently monitored by fluorometry. After addition ofethidium, each sample was divided, half y-irradiatedwith a dose (9-6 J kg"1) sufficient to relax DNA fullyand the fluorescence of both was measured. Aftersubtraction of appropriate blanks, the fluorescence ofdye bound to non-irradiated beads was divided by thatbound to irradiated beads, effectively normalizing allnon-irradiated values to those of equal amounts of fullyrelaxed DNA. Therefore, this ratio reflects the pro-portion of loops that are nicked. Ratios from encapsu-

30 60Time (min)

Fig. 6. Nicking DNA during incubation. Cells wereencapsulated, lysed with Triton ( • ) or complement (A) atpH7-4, washed and incubated at 37°C. At different times,2M-NaCl was added to remove histones and generate loopsof superhelical DNA. The ratio was determinedfluorometncally and reflects the percentage of nicked loops,the higher the ratio, the more breaks. ( • ) Encapsulatedcells lysed directly in Triton and 2M-NaCl.

lated cells lysed directly in 2M-NaCl and their y-irradiated counterparts provide fully superhelical andfully relaxed DNA for comparison (with ratios of 0-86and 1-0, respectively).

Most loops in encapsulated cells lysed in Triton atpH 7-4 are supercoiled, whereas some of those lysed incomplement are nicked. On incubation at 37°C theDNA of both becomes progressively nicked (Fig. 6).Nicking occurs more rapidly if the Mgz +/ATP ratio isincreased (Jackson & Cook, 1985a; results not shown).Since this assay is so sensitive, this means that netnucleolytic activity is effectively suppressed and that,at least for the first few minutes, these beads containessentially intact DNA. It also means that lysing cellswith detergents or complement does not grosslydamage DNA (Li & Kaminskas, 1987), as may cell-mediated cytolysis (Duke et al. 1983).

Efficiency of replication and transcriptionTable 2 illustrates the efficiency of replication andtranscription in these preparations using optimal con-centrations of triphosphates. (We would stress that weassay activities due to polymerase halted at lysis, andwhich then continue synthesis /// vitm without re-initiation (Jackson & Cook, 19856, 19866). For theseexperiments, extra Mg2+ and, of course, triphosphateswere added: if maintained in equimolar amounts,nicking then occurs at roughly the rates indicated inFig. 6.) Cells lysed in Triton replicate their chromatintemplate in a cell-cycle-dependent manner at >75 % ofthe rate in vivo at pH7-75, the reaction being verysensitive to pH and Mg2"*" concentrations (Jackson &Cook, 19866). Consequently, Triton-lysed cells repli-

374 D. A. Jackson et at.

Table 2. The efficiency of replication and transcription

Lysis conditions

ReplicationTranscription

Rate Efficiency rate(pmol/106 cells per mm) (%) (pmol/106 cells per mm)

Complement, pH7-4Triton, pH7-4Complement, pH80Triton, pH8-0

6-42-0903-1

8527

12041

9-514-617-519-2

The efficiency of replication is determined assuming that an average nucleus contains 12pg DNA and that one cell cycle takes 22h(Jackson & Cook, 1986o).

cate slightly less efficiently at pH7-4 and 8-0. Incontrast, complement-lysed cells replicate more ef-ficiently, replicating faster in vitro at pH 8-0 than invivo. Nuclei prepared conventionally (without Triton)by homogenizing cells swollen in a hypotonic buffer(for example, our pH7-4 buffer minus KC1) provide areference level of replication for comparison; theyreplicate under identical conditions at only 70 % of therate found with cells extracted with Triton at pH7-4(results not shown).

We explored the basis for this difference in efficiencyof replication (assayed under identical conditions) ofTriton- and complement-lysed cells but are unable asyet to explain it. The difference is also obtained with[ P]dCTP (results not shown). It is strongly depen-dent on deoxynucleotide triphosphate concentration,being about threefold at optimal concentrations (i.e.125/IM; Table 2); lower concentrations reduce the rateof Triton-lysed cells more than complement-lysedcells, so that the difference increases to about 15-fold at2jUM (Fig. 7) and to 30-fold at lower concentrations.The difference in rate is directly attributable to Tritonas addition of detergent to complement-lysed cellsimmediately reduces their rate (Fig. 7). This reducedrate was not due to a non-specific loss of activity onincubation, since addition of Triton at the beginning ofthe reaction reduced the rate to the same low level(results not shown). The activity in complement-lysedcells is sensitive to aphidicolin (Fig. 7), as are the majoractivities in vivo and in Triton-lysed cells (Jackson &Cook, 1986a,6).

Transcription is equally efficient (Table 2). As we donot know the absolute rate of transcription in vivo wecannot estimate relative efficiencies; however, we knowof no preparation that transcribes more efficiently.

Screening for lytic agents that selectivelypenneabilize the cell membrane

The observation that Triton reduces the efficiency ofreplication of complement-lysed cells, presumably bypermeabilizing the nuclear membrane, allowed us toscreen lytic agents for thos'e that might permeabilize

20 30Time (min)

40

Fig. 7. The kinetics of replication in Triton- andcomplement-lysed cells. Replication assays were conductedas described in Materials and methods except that thedTTP concentration was reduced to 2-5 [IM.( • ) Complement-lysed cells. After IS min Triton wasadded to 0-25 %. (D) Complement lysed cells,preincubated for 30min with lOgml"1 aphidicolin.( • ) Triton-lysed cells; preincubation with aphidicolinreduces the initial rate 15 times (results not shown) andaddition of Triton to 0'25 % during the reaction has noeffect.

the cell, but not the nuclear, membrane. RadiolabelleddTTP cannot pass through the cell membrane, soaddition of such a specific agent should increaseincorporation of label to that of complement-lysedcells; non-specific agents should increase the level onlyto that of Triton-lysed cells. We tested various non-ionic detergents, the best of which, Tween 20, gave theresults illustrated in Table 3; we found that manycould be diluted to a concentration where most cells inthe population became permeable to Trypan Blue, butthis never led to replication at the rate of complement-

Cyto- and nucleo-skeletons 375

Table 3. The relative efficiency of replication afterlysing cells in the pH 7-4 buffer and various agents,

measured using 2-5jUM-dTTP

Lytic agent

Triton X-100, 0-5%Tween 20, 0-5 %Mclittin, 25 fig ml 'Mclittin 250//g ml 'Complement

Values are expressed relative to cells

Relative efficiencyof replication

102-41-70-46-5

lysed using Triton.

lysed cells; higher concentrations always reduced rates.Lysis was also variable and critically depended on cellnumber and type. Similar results were obtained withionic detergents (e.g. SDS and lysolecithin; results notshown). Lysis by the protein, melittin, was alsovariable, high levels inhibiting replication (Table 3).

Discussion

We set out to develop a method for gently permeabiliz-ing cells with a number of aims in mind. We hoped touse as 'physiological' a buffer as possible and topermeabilize selectively the various cellular compart-ments to most of the probes currently used in cellbiology. We have partly achieved these aims.

Buffers

The cationic, but not anionic, constitution of ourpH 7-4 buffer is roughly that of the cytoplasm. Proteinsare the dominant anions in vivo but their inclusion in abuffer for routine use is inconvenient. However, theiraddition might serve a useful purpose. Although ourbuffer preserves heterochromatin better than most,some still decondenses when the nuclear membrane isremoved (cf. Triton- and complement-lysed cells),presumably because nuclear proteins diffuse out withinseconds, a loss that can be made slower by highconcentrations of protein (Paine et al. 1983). Despitethese shortcomings, we believe that our formulationprovides a suitable base that can be modified as moreinformation about the precise cellular environmentbecomes available.

Lytic agents

We tested various lytic agents (Table 3) to see whetherany preferentially permeabilized the plasma mem-brane, but found none as efficient as antibody andcomplement. (But see, for example, Schindler et al.(1985), who selectively removed the outer nuclearmembrane with citraconic anhydride.) Antibodies pro-vide the advantage of specificity but the disadvantagethat complement (i.e. whole serum) is ill-defined and

so might have unexpected effects. For example, inter-mediate filaments contain high-affinity sites for the Fcpart of IgG and so fix complement (Hansson et al.1987). It also has the disadvantage that although itsperforations (about 10 nm) permit ingress of restrictionenzymes they are too small for antibodies. (Note thatthe membrane of cells with less well-developedcytoskeletons than HeLa may tear on complement-mediated lysis so that their interiors do become access-ible.) We are currently exploring whether the nuclearmembrane of complement-lysed cells is functionallyintact. We also permeabilized the plasma membraneusing strong electric fields, but found that this is bestachieved in buffers of low ionic strength, which reducethe rates of transcription and replication (Knight &Scrutton, 1986). (Note that by virtue of its largerradius, the cell membrane becomes permeable at lowerfields than those of intracellular organelles (Knight &Scrutton, 1986).)

Preservation of structure

The agarose coat protects the fragile contents of cellsvery effectively. For example, the three-dimensionalstructure of the cytoskeleton survives repeated pellet-ing and resuspension, as does the fragile DNA. Thecoat also provides the added benefit of packagingviscous DNA, which, if released, makes handlingdifficult. Most of the experiments that we describewould be impossible using nuclei prepared usingconventional procedures, since they aggregate andjellify at physiological salt concentrations.

Preservation of function

A major concern in our laboratory has been withnuclear function, replication and transcription. Wehave shown elsewhere that after lysis in Triton atpH8'0 the encapsulated cells replicate their DNA /'//vitro at 85 % of the rate found in vivo in a cell-cycle-dependent manner (Jackson & Cook, 19866) and theytranscribe more efficiently than nuclei prepared con-ventionally (Jackson & Cook, 19856). The rates on lysiswith Triton in our improved buffer are similarly high.Surprisingly, complement-lysed cells replicate underidentical conditions about three times more efficientlythan their Triton-lysed counterparts (Table 2) and it isperhaps premature to speculate on the basis of thisinteresting phenomenon. However, it is interestingthat efficient replication in extracts of Xenopus oocvtesis preceded by the re-formation of an intact nuclearmembrane (Blow & Laskey, 1986; Blow & Watson,1987; Newport, 1987), so that membrane integrity maybe a prerequisite for efficient replication.

We have not explored cytoplasmic function in anydetail, but we would assume that as complement-lysispreserves cytoplasmic morphology quite well, manyfunctions in addition to the cvtochalasin-induced col-

376 D. A. Jackson et al.

lapse of the actin ne twork (F ig . I I ) may also bepreserved .

Accessibility

The agarose pores are so large in comparison with thesize of the average protein that antibodies and enzymesequilibrate throughout beads within seconds. Afterlysis with Triton, both cytoplasm and nucleus becomefreely accessible to antibodies and, to a lesser extent, tolarger nucleic acids (results not shown). After comp-lement-mediated lysis, the pores generated by theattack complex (Muller-Eberhard, 1986) do not permitingress of large proteins like antibodies unless themembrane is torn, but their chromatin is accessible torestriction enzymes and so probably to most enzymesused in molecular biology.

We hope that these encapsulated derivatives of cells,well-preserved and containing intact DNA, cytoskel-eton and nucleoskeleton, accessible, yet expressing invitro the major nuclear functions at rates found in vivo,provide a useful experimental material for studies onthe relations between the various structures in the celland their functions.

We thank the Cancer Research Campaign (to P.R.C.), andThe Rockefeller University and the Lucille P. MarkeyCharitable Trust, Miami, Florida (to J.Y.) for support; DrL. 1. Davis for providing the monoclonal antibody, Dr P.Griffiths for measuring Ca2+ levels and Mary Bergin-Cart-wright for help with electron microscopy.

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{Received 8 February 1988 - Accepted 25 March 1988)

378 D. A. Jackson et al.