The role of lamin Llll in nuclear assembly and DNA replication, in cell-free

extracts of Xenopus eggs

J. MEEER1, K. H. S. CAMPBELL1, C. C. FORD2, R. STICK3 and C. J. HUTCHISON1'*

iThe Department of Biological Sciences, the University, Dundee DD1 4HN, ScotlandiThe Department of Genetics and Developmental Biology, The University of Sussex, Brighton BN1 9QG, England3Institut fur Genetik und Mikrobiologie, Ludwig-Maximillians Universitat Munchen, Maria-Ward-Strasse la, D-8000, Munchen 19,FRG

* Author for correspondence

Summary

Xenopus egg extracts, which support nuclear as-sembly and DNA replication, were functionallydepleted of lamin LIU by inoculating them withmonoclonal anti-lamin antibodies. Phase-contrastmicroscopy and electron-microscopy studies indi-cated that lamin-depleted extracts supported ef-ficient chromatin decondensation, and assembly ofdouble membrane structures and nuclear pores ondemembranated sperm heads. Immunofluorescencemicroscopy suggests that lamin-antibody complexesare transported across the nuclear membrane but donot assemble into a lamina. These findings were

confirmed by immunoblotting analysis of isolatednuclei. Metabolic labelling studies with either biotin-11-dUTP or [32P]dCTP, revealed that nuclei lacking alamina were unable to initiate DNA replication andthat, although such nuclei could import proteinsrequired for DNA replication (e.g. PCNA), theseproteins were apparently not organized into repliconclusters.

Key words: lamin T.TTT, nuclear assembly, DNA replication.

Introduction

S-phase is the period in the cell cycle during whichgenomic DNA is duplicated. To achieve this duplication,many thousands of initiation events must be co-ordinatedin time and space, implying a high degree of structuralorganization within the eukaryotic cell nucleus. Variousextraction procedures have demonstrated the presence of afilamentous substructure within the nucleus, termed thenucleoskeleton (Fey et al. 1986; Pouchelet et al. 1986;Jackson and Cook, 1988), which appears to be sub-dividiedinto three distinct compartments: the pore-complex lam-ina, intra-chromosomal filaments and inter-chromosomalfilaments (Aebi et al. 1986; Pouchelet et al. 1986). DuringS-phase, all nascent DNA appears to be associated withthe nucleoskeleton (Dijkwel etal. 1986; Pardoll etal. 1980;McCready et al. 1980; Smith and Berezney, 1982; Jacksonand Cook, 1986a). In addition, replicative polymerases arealso associated with the nucleoskeleton during S-phasebut not at other times during the cell cycle (Smith andBerezney, 1983; Jackson and Cook, 19866). These obser-vations have led to the view that replication complexes areimmobilized at the nucleoskeleton, and that replicationforks expand as DNA is threaded through the staticreplicase (Jackson and Cook, 1986a).

While fractionation of replicating nuclei has proveduseful for demonstrating an association between repli-cation forks and substructures within the nucleus, thisapproach provides no information as to how such associ-ations arise. In contrast, cell-free extracts of Xenopus eggsJournal of Cell Science 98, 271-279 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

that assemble nuclei capable of initiating DNA replicationin vitro (Blow and Laskey, 1986; Newport, 1987; Hutchisonet al. 1987) can be used to dissect those events involved innuclear assembly that permit DNA replication (Sheehanet al. 1988; Hutchison et al. 1988). Extracts prepared fromeither fertilized or unfertilized eggs of the frog Xenopuslaevis are able to assemble nuclear structures, includingbilayered membranes, nuclear pores and a lamina, arounda range of DNA templates, including demembranatedsperm heads and plasmid DNA (Lohka and Masui, 1983;Blow and Laskey, 1986; Newport, 1987; Sheehan et al.1988; Hutchison etal. 1988). Such nuclei are able to importnuclear proteins selectively (Newmeyer et al. 1986) andcan initiate and complete a single round of DNAreplication (Blow and Watson, 1987). Immunofluorescencestudies imply that in S-phase nuclei that have beenassembled in vitro, both replicons and replicases areorganized at multiple discrete sites, (Hutchison and Kill,1989; Hutchison et al. 1989; Mills et al. 1989), eachfluorescent spot representing a cluster of up to 300replicons (Mills et al. 1989). The organization of repliconsin clusters would again imply structural associations inorder to maintain each replication fork in close proximityto its neighbour.

Extracts that support extensive protein synthesis willalso induce periodic mitotic events in nuclei that havecompleted S-phase. Following each mitosis, nuclei re-formand are able to re-initiate DNA replication. Duringnuclear reconstitution at telophase, there is a strikingcorrelation in both time and space, between re-initiation of

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DNA replication and lamin polymerization, implying thatthe lamina may be involved in organizing S-phasechromatin (Hutchison et al. 1988; Hutchison et al. 1989).

One way of testing whether lamins participate in theorganization of replicon clusters would be to constructnuclei that lack lamin structures. This might be achievedby immunodepleting egg extracts of lamins prior tonuclear assembly. Similar experiments have beenattempted in extracts prepared from Chinese hamsterovary (CHO) cells. However, depletion of lamins in theseextracts at mitosis, prevents nuclear assembly at telo-phase (Burke and Gerace, 1986). Despite these findingsthere are good reasons for believing that removal of laminsfrom Xenopus egg extracts would not inhibit activitiesthat decondense chromosomes or assemble nuclear mem-branes and pore complexes around decondensed chroma-tin. In Xenopus, lamin TJTT (the only lamin type present inearly cleavage embryos) is freely soluble and does notsegregate with endoplasmic reticulum like membranesduring mitosis. Furthermore, nuclei at specific phases ofmeiosis lack a lamina structure but nevertheless havenuclear membranes and pore complexes (Stick andSchwarz, 1982, 1983; Stick and Hausen, 1985). Thus inorder to assess the relationship between lamin polymeriz-ation and DNA replication we have used monoclonalantibodies to functionally deplete lamin LJH from cell-freeextracts of Xenopus eggs. The results indicate that, whileT.TTT is not required for either chromatin decondensation ornuclear membrane assembly, it is essential for initiatingDNA replication and may be involved in targetingenzymes to the sites of DNA synthesis.

Materials and methods

Antibody reagentsAnti-lamin monoclonal antibodies were from the lines L6-8A7(Stick and Hausen, 1985) and L6-5D5 (Stick, 1988). Anti-vimentin monoclonal antibodies were from the line NZ11.C10(these were a generous gift from Dr M. OTarrell, University ofEssex). Rabbit polyclonal anti-mouse immunoglobulins werepurchased from ICN immunobiochemicals; FITC, TRTTC andperoxidase-conjugated antibodies were purchased from DAKO-PATTS.

Preparation of cell-free extractsCell-free extracts were prepared as previously described (Hutchi-son et al. 1988). Briefly, dejellied unfertilized eggs were washed inextraction buffer (100 HIM KC1, 20 mM Hepes, pH7.5, 5DIMMgCl2, lmM 2-mercaptoethanol) and then crushed by centrifu-gation at 10 000 g for 10 min. The soluble material was removedand treated with 50/igml"1 cytochalasin B before centrifugationfor a second time (10 000 g for 10 min) to remove debris.

Lamin depletionA 100/d sample of egg extract containing lO/Jgml"1 cyclohexi-mide (to prevent mitosis) was inoculated with 5 fd of a preparationof L6 5D5 monoclonal anti-lamin ascitic fluid that had beendiluted 1/10 in SuNaSp (0 .15 M sucrose, 75 mM NaCl, 0.5 mMspermidine, 0.15mM spermine; Gurdon, 1976) and incubated forl h at 21 °C; 106 demembranated sperm heads (Hutchison et al.1987) in 1 /d of SuNaSp were then inoculated into the extract,which was divided into 10 /A aliquots. At successive 10-minintervals, samples were either fixed for fluorescence microscopy(Hutchison et al. 1988) or for examination of nuclear membraneformation (Hutchison et al. 1987). Extensive controls wereperformed in which extracts were inoculated with monoclonalanti-vimentin ascites (to test for antibody specificity), rabbitpolyclonal antisera (to test for general effects of serum addition)and SuNaSp (to test for the effect of diluting the extract).

Immunoprecipitation with Staphlococcus aureus(Staph.aJParticulate material was removed from egg extracts by centrifu-gation at 100000g for l h at 4°C. The cytosolic fraction(containing 90 % of free lamins) was divided into 50 /d samplesand inoculated with either anti-lamin ascites, anti-vimentinascites fluid or SuNaSp, and incubated for l h at 21 °C. A 10//1sample of a suspension of Staph.a in SuNaSp was then inoculatedinto each extract and incubated for a further hour at 21 °C. TheStaph.a was then pelleted by centrifugation at 15000# for 5 minand the pellet and supernatant fractions were analysed byimmunoblotting following SDS-PAGE.

Direct immunofluorescence with FITC-conjugated L€8A7 monoclonal anti-lamin antibodiesA 100 /d sample of L6 8A7 ascites was precipitated with 40%saturated ammonium sulphate and resuspended in 0.25 M(pH9.0) carbonate-bicarbonate buffer (Hudson and Hay, 1980).Following dialysis in this buffer, fluorescein isothyocyanate(FITC, 0.05 mgmg"1 of total protein) was added and mixedovernight at 4°C. Conjugated protein was separated from freefluorochrome by gel filtration using a Sephadex G-25 columnequilibrated in phosphate-buffered saline. Direct immunofluor-escence was performed with this FITC-conjugated antibody asfollows: nuclei were fixed and spun onto glass coverslips aspreviously described (Hutchison et al. 1988). Each coverslip wasthen incubated with 30 /d of FITC-conjugated anti-lamin antibody(diluted 1/25 in PBS containing 1% foetal calf serum) andincubated overnight at 4"C. The coverslips were then washed inPBS and mounted in 50% PBS/glycerol containing 1/jgml"1

DAPI.

Electron microscopyA 10 /d sample of egg extract containing 104 nuclei was droppedinto lml of 0.2 M sodium cacodylate (pH7.4) containing 2.5%glutaraldehyde. After incubation for 2h on ice, the drop ofcytoplasm had fixed into a loose ball. This was washed twice inlml of 0.2M sodium cacodylate (pH7.4) before post-fixation inOsO4 for 1 h at 4°C. The sample was then dehydrated in a gradedethanol series (30 % to 100 %) and incubated overnight in 50 % LRWhites resin in 50 % ethanol at 4°C. The following day the samplewas transferred to 100 % LR Whites and incubated overnight at4°C. After a further change of resin and incubation for a further48h at 4°C the sample was polymerized at 60°C for 24 h. Thinsections were cut using a Reichert OM U3 microtome and post-stained on copper grids with uranyl acetate/lead citrate. Sectionswere viewed using a JOEL JEM 1200EX transmission electronmicroscope.

Isolation of nuclei and preparation of nucleoidsNuclei were isolated from egg extracts by the following procedure:200/d of extract containing 2xlOB nuclei were diluted in 2 ml ofnuclear isolation buffer (NIB: 60 mM KC1, 15 mM Tris-HCl,pH7.4, 15mM NaCl, lmM 2-mercaptoethanol, 0.5mM spermi-dine, 0.15 mM spermine). The diluted suspension of nuclei waslayered over a 60 % Percol cushion (1 ml) and subjected tocentrifugation at 3000 # for 10 min at 4°C. Nuclei were recoveredfrom the interface, diluted in 1 ml of NIB and pelleted bycentrifugation at 6000 g for 10 min. The nuclear pellet was eitherresuspended in SDS sample buffer or in NIB containing 0.5%Triton X-100 and 2 M NaCl. In the case of resuspension inmodified NIB, nuclei were incubated for 10 min on ice beforecentrifugation for a second time at 6000 # for 10 min. Both thepellet and supernatant were prepared for SDS-PAGE by theaddition of sample buffer.

ImmunoblottingProtein samples resolved on 10 % SDS-PAGE, were transfered tonitrocellulose (Schleicher and Schuell) by electrophoresis intransfer buffer (10 mM NaHCO3; 3mM Na2C03; 20% methanol).The nitrocellulose filter was blocked by incubation in BLOTTO(3% Marvel milk in blot rinse buffer; 10 mM Tris-HCl, pH7.4,

272 J. Meier et al.

1 mM EDTA, 75 nun NaCl, 0.1 % TWEEN 20) and then incubatedwith a 1/1000 dilution of L6 8A7 (in blot rinse buffer containing1 % FCS) overnight at 4°C. The filter was washed thoroughly inblot rinse buffer before incubation for 4h at 4°C with peroxidase-conjugated rabbit anti-mouse IgG (1/400 dilution DAKOPATTS).After a final wash the filter was developed by incubation in 24 mMcitric acid, 51mM Na2HP0* (pH5.0) lmgrnl"1 DAB and 0.3%H2O2.

Results

Absorbtion of free lamins to monoclonal antibody doesnot inhibit chromatin decondensation or nuclearmembrane assembly in Xenopus egg extracts

In order to establish that antibodies inoculated into eggextracts were absorbing all free lamins, the followingexperiment was performed. Extracts were incubated withmonoclonal anti-lamin antibodies (L6 5D5) for 1 h at 21 °C.The extracts were then inoculated with formaldehyde-fixed Staphlococcus aureus (Staph.a) and incubated for afurther 60min at 21°C. The resulting Staph.a-IgGcomplex was pelleted by centrifugation at 6000 g for10 min and the distribution of lamins between the pelletand the supernatant fractions was determined by SDS-PAGE followed by immunoblotting, using a secondmonoclonal anti-lamin antibody L6 8A7. Lamins wereonly detected in the pellet fraction following incubationswith L6 5D5 and Staph.a. This was in contrast toincubations of extracts with Staph.a only or with Staph.aplus an irrelevant antibody, where only small amounts oflamins co-precipitated with the pellet fraction while thebulk remained in the supernatant (results not shown).These data imply that essentially all lamins werespecifically bound to IgG upon inoculation of L6 5D5 intoegg extracts.

To examine the effect on nuclear morphology of theabsorbtion of free lamins to antibody, demembranatedsperm heads were added to extracts that had been pre-incubated with L6 5D5. In untreated extracts, or extractsthat had been inoculated with an irrelevant antibody,chromatin decondensation and nuclear membrane as-sembly occurred over a 20-min period (Fig. 1A and B).Nuclear assembly was slower in extracts pre-incubatedwith L6 5D5 antibodies, but nevertheless most (85 %)sperm heads had decondensed by 30-min and appearedidentical under phase-contrast optics to nuclei assembledin control extracts (Fig- IB and D).

To confirm that the structures observed under the lightmicroscope were indeed nuclei, samples were prepared fortransmission electron microscopy. In Fig. 2 typical struc-tures isolated from both lamin-depleted (Fig. 2A) andundepleted (Fig. 2B) extracts are compared. Both nucleiare large (>8jan) and are surrounded by double unitmembranes interupted by pore complexes (arrows). We didobserve more variation in the size of nuclei isolated fromlamin-depleted extracts (of 50 such nuclei counted themean diameter was 5 inn with variations between 2/anand 9 ftm) but all of these nuclei were otherwise identicalto controls.

The results described above contrast with previousstudies in which lamins had been depleted from somaticcells or somatic cell extracts during mitosis. The formerstudies demonstrated that the re-formation of interphasenuclei required lamins both for chromosome deconden-sation and membrane accumulation around decondensedchromosomes (Burke and Gerace, 1986). While previousdata have been obtained using polyclonal antisera, in

Fig. 1. Assembly of nuclei in vitro. Sperm heads wereinoculated into extracts that had been pre-incubated witheither L6 5D5 anti-lamin antibodies (C,D) or monoclonal anti-vimentin antibodies (A,B). Samples of each extract were fixed30 min after the addition of sperm as previously described(Hutchison et al. 1987) and viewed using phase contrast (B,D)or fluorescence optics fitted with a DAPI filter (A,C). Bar,10/an.

which the antibody-antigen complexes where removedfrom the extracts, in our experiments a monoclonalantibody was used and the antibody-antigen complexremained in the extract. Thus the contrast between theresults reported here and previous studies might beexplained if absorbtion of lamins to monoclonal antibodiesfailed to prevent either the accumulation of lamins withinnuclei or their subsequent polymerization. To test this, weisolated nuclei formed in extracts that had been inoculatedwith L6 5D5 and fixed them in preparation for directimmunofluorescence. Fixed nuclei were probed withTRITC-conjugated rabbit anti-mouse IgG to reveal thedistribution of L6 5D5 antibodies, and then with FITC-conjugated monoclonal anti-lamin antibodies (L6 8A7) toreveal independently the distribution of the bulk of thelamins.

TRITC-conjugated rabbit anti-mouse IgG stained nucleithat had been isolated from L6 5D5-treated extracts, withan intense punctate pattern (Fig. 3B). This fluorescencepattern indicated that L6 5D5 IgG was distributedthroughout the nucleoplasm with no particular associ-ation with the nuclear envelope. In contrast, TRITC-labelled rabbit anti-mouse IgG did not stain nuclei thatwere formed in extracts containing an irrelevant mouseIgG (Fig. 3e). These results imply that while IgG isnormally excluded from nuclei, L6 5D5 anti-laminantibodies were able to penetrate the nuclear envelope.Co-staining of these nuclei with FITC-conjugated L6 8A7(anti-lamin) monoclonal antibodies revealed that thedistribution of lamins in nuclei isolated from L6 5D5-treated extracts was identical to the distribution of L6 5D5IgG (compare Fig. 3B and 3C) and did not resemble thehalo-like pattern obtained with nuclei isolated fromcontrol extracts (Fig. 3F).

One interpretation of these data is that small amounts

Role of lamin in DNA replication 273

•

C -<=>-,

• • • . • T . - ^ - * * ^ -

*.S^* •bCV -

Fig. 2. Transmission electron micrographs of nuclei isolatedfrom lamin-depleted extracts. Sperm heads were incubatedeither in L6 5D5-treated extracts for 30min at 21 °C or inuntreated extracts for the same time. Samples (10 /JI) werethen prepared for transmission EM as described below. (A) Atypical nucleus assembled in a lamin-depleted extract viewedat X5000 magnification. (B) A nucleus formed in an untreatedextract at the same magnification. Arrowheads show theposition of structures resembling nuclear pores. Bars, 1 /an.

of free lamins in L6 5D5-treated extracts are able to enternuclei without assembling into a continuous lamina andthat, subsequently, during the fixation period free anti-body penetrates the nuclei and associates with suchlamins. To exclude this possibility we inoculated extractscontaining nuclei with L6 5D5 antibodies just beforefixation and stained them with TEITC-labelled rabbitanti-mouse IgG followed by FITC-labelled L6 8A7 anti-bodies. Only FTTC fluorescence was detectable in suchnuclei, implying that L6 5D5 antibodies had not pen-etrated the nuclei during the fixation period (Fig- 3H andI). Thus we infer that the presence of L6 5D5 in nucleiisolated from lamin-depleted extracts results from co-transport of antibody-lamin complex into those nuclei.Co-transport of antibody-nuclear antigen complexes hasbeen reported previously, and presumably occurs when theantibody does not cover the transport sequence on theantigen (Einck and Bustin, 1984; Borer et al. 1989).

Monoclonal anti-lamin antibodies prevent the formationof a Triton-insoluble lamina

While the immunofluorescence data imply that L6 5D5anti-lamin antibodies prevent the formation of a continu-ous lamina, the residual lamin structures that wereformed may be incorporated into a Triton-insolublenucleoskeleton, which could provide sites for the organiz-ation of interphase chromatin. To investigate this possi-bility, nuclei assembled in antibody-treated extracts wereisolated, by centrifugation onto Percol cushions, andextracted in 0.5% Triton X-100 and 2 M NaCl. Thedistribution of lamins between Triton-soluble and Triton-insoluble fractions was determined by immunoblottingfollowing gel electrophoresis on SDS-PAGE.

The results, illustrated in Fig. 4, show that monoclonalanti-lamin antibodies L6 8A7 detected three polypeptidesof MT 58xlO3, 60xl03 and 62xlO3 in nuclei isolated fromegg extracts (lane 1). Only one of these polypeptides(60xl03) was pelleted with bulk chromatin followingTriton/high-salt extraction (lane 2), while the other twopolypeptides were Triton/high-salt-soluble (lane 3). Pre-vious studies have demonstrated the presence of only asingle lamin species (lamin LJII) in lamina fractionsprepared from nuclei formed in Xenopus extracts and fromearly embryos as revealed by immunoblotting with eitherpolyclonal anti-lamin serum or monoclonal antibodyL6-8A7 (Stick and Hausen, 1985). The results illustratedin Fig. 4 lanes 2 and 8 are in agreement with theseobservations. A single band of 60xl03Afr (LET) waspelleted after Triton/high-salt extraction of nuclei iso-lated from extracts preincubated with either buffer alone(Fig. 4, lane 2) or an irrelevant antibody (Fig. 4, lane 8). Incontrast, no lamin proteins were detected in the Triton/high-salt-resistent fraction of nuclei isolated from extractspreincubated with anti-lamin monoclonal antibody L6-5D5 (Fig. 4, lane 5). In the latter case lamin proteins arepresent in the Triton/high-salt-soluble fraction (Fig. 4,lane 6) while the corresponding fractions in the controlexperiments have been largely depleted of T.TTT (Fig. 4,lanes 3 and 9). The monoclonal antibody L6-8A7recognizes, in addition to the lamin LIE, two otherpolypeptides in whole nuclei isolated from egg extracts.We do not know what these proteins are. However, theyare always found in the soluble fraction after extractionand thus do not contribute to the formation of the laminastructure. In conclusion, these results imply that inocu-lation of L6-5D5 monoclonal anti-lamin antibodies intoegg extracts functionally depletes the lamina; thuspreventing the formation of a lamina within pronuclei.

Lamin depletion with monoclonal antibodies inhibitsDNA replication and prevents the association ofreplication enzymes with the chromatinDNA replication is usually extremely efficient in nucleiincubated in Xenopus egg extracts. Since sperm heads thathad been inoculated into lamin-depleted extracts wereassembled into nuclei that lacked a lamina, we investi-gated whether such nuclei were able to replicate DNA.Extracts containing nuclei were either pulse labelled withbiotin-11-dUTP or labelled continuously with [32P]dCTP.Following biotin labelling nuclei were fixed and preparedfor fluorescence microscopy. Fig. 5A-C shows typicalnuclei isolated from lamin-depleted extracts; FITC-labelled anti-lamin antibody L6 8A7 detected only islandsof lamins with no particular association with the nuclearenvelope (Fig. 5B). Such nuclei did not incorporate biotin-

274 J. Meier et al.

Fig. 3. The distribution of lamins in nuclei formed in vitro. Nuclei assembled in extracts that had been inoculated with antibodywere fixed and prepared for fluorescence microscopy. (A, D and G) The distribution of DNA; (B, E and H) the distribution of IgG(detected by TRITC-conjugated rabbit anti mouse Ig); (C, F and I) the distribution of lamins (detected by FITC-conjugated L6 8A7anti-lamin antibodies). (A, B and C) Nuclei isolated from extracts pre-incubated with L6 5D5 anti-lamin antibodies. (D, E and F)Nuclei isolated from extracts which had been pre-incubated with anti-vimentin antibodies. (G, H and I) Nuclei isolated fromextracts to which L6 5D5 anti-lamin antibodies were added just prior to fixation. Bar, 10 /mi. All preparations were made 50 minafter the addition of sperm heads to extracts.

11-dUTP even after prolonged (2h) incubations (Fig. 5C).In contrast, nuclei isolated from extracts that had beeninoculated with an irrelevant IgG had a continuouslamina (Fig. 5E) and after only 30 min showed extensiveincorporation of biotin-11-dUTP (Fig. 5F). Fig. 6 illus-trates a typical time course of biotin-11-dUTP incorpor-ation in antibody-treated and control extracts. Up to 80 %of sperm heads had been assembled into nuclei that weresynthesizing DNA, 40 min after inoculation into either

control extracts or extracts treated with an irrelevant IgG.However, despite the fact that most sperm heads were stillassembled into nuclei in lamin-depleted extracts, less than10% of these nuclei incorporated biotin-11-dUTP. Fur-thermore, biotin incorporation in these nuclei was ex-tremely weak.

To estimate the amount of DNA replication in antibody-treated extracts, [32P]dCTP labelling was performed aspreviously described (Hutchison et al. 1988). Table 1 shows

Role of lamin in DNA replication 275

• 2 3 4 5 6 7 8 9 Fig. 4. Immunoblot analysis of isolatednuclei and nuclear matrix preparations.Nuclei were isolated from egg extracts andtreated with high salt and detergents asdescribed in Materials and methods. Eachlane contains the equivalent of 2.5 xlO6

nuclei. Lanes 1, 4 and 7 contain whole nuclei;lanes 2, 5 and 8 contain salt/detergent-insoluble material; and lanes 3, 6 and 9contain salt/detergent-soluble material.Lanes 1, 2 and 3 contain nuclear material

derived from extracts pre-incubated with SuNaSp. Lanes 4, 5 and 6 contain nuclear material derived from extracts pre-incubatedwith L6 5D5 anti-lamin antibodies. Lanes 7, 8 and 9 contain nuclear material derived from extracts pre-incubated with anti-vimentin antibodies. Mr values (xlO~3) are given on the left.

\

Fig. 5. Biotin labelling of nuclei. Eggextracts containing nuclei were pulselabelled for 10-min with 4/an biotin-11-dUTP (40-50 min after the addition ofsperm heads) before the nuclei werefixed in EGS (ethylene glycol-bis-succinic acid) and isolated for indirectimmunofluorescence. (A and D)Distribution of DNA determined byDAPI staining. (B and E) Distributionof lamins, determined by staining withFITC-labelled L6 8A7 anti-laminantibodies. (C and F) Biotinincorporation detected with Texasred-streptavidin. (A, B and C) Anucleus isolated from lamin-depleted(L6 5D5-treated) extracts; (D, E and F)A nucleus isolated from anti-vimentintreated extracts. Bar, 10 fan.

Table

Control

1. Reduction in DNADNA synthesized (ng)

Anti-vimentin

replication

Anti-lamin

40 36.5 2.4

ngDNA synthesized in 20/j] samples of extract containing 2xl(r*sperm heads. Extracts were pre-incubated with SuNaSp (control),monoclonal anti-vimentin ascites (anti-vementin) or L6 5D5 monoclonalanti-lamin ascites fluid (anti-lamin). Labelling periods were for 2 h at21 °C and the amount of DNA synthesized was determined as previouslydescribed (Hutchinson et al. 1987). The amounts of DNA synthesisshown represent the average of triplicate samples from one experiment.

that, whereas inoculation of an irrelevant antibody intoextracts resulted in a small (9%) reduction in the totalamount of DNA replication occurring over a 2 h period(compared to extracts without antibody), inoculation of L65D5 antibodies resulted in a 94 % reduction in the amountof DNA synthesized.

Several authors have suggested that intermediatefilaments within the nucleus provide sites for theorganization of the replication machinery (Jackson andCook, 1988; Nakayasu and Berezney, 1989). Furthermore,it now appears that this structural organization leads toclustering of replicons (Mills et al. 1989) and that the sitesof such replicon clusters can be detected with antibodiesrecognising proliferating cell nuclear antigen (PCNA)(Bravo and MacDonald-Bravo, 1987; Hutchison and Kill,

100

o

I 6 0I~ 40

3

z3s 20

40 60 80Incubation time (min)

100

Fig. 6. Percentage biotin incorporation in nuclei formed invitro. Egg extracts were pulse labelled with biotin-11-dUTP for10-min periods at 10-min intervals, before nuclei were fixedand isolated for fluorescence microscopy. Isolated nuclei werestained with Texas Red-streptavidin to detect biotinincorporation and a total of 200 nuclei were counted at eachtime point to determine the percentage synthesizing DNA.(•) Nuclei isolated from control (SuNaSp-treated) extracts;(•) nuclei isolated from anti-vimentin-treated extracts;(A) nuclei isolated from anti-lamin-treated extracts.

276 J. Meier et al.

Fig. 7. The distibution of PCNA in nuclei formed in vitro. S-phase nuclei were isolated from egg extracts, fixed and prepared forindirect immunofluorescence. (A-C) Nuclei isolated from lamin-depleted (L6 5D5-treated) extracts: (D-F) nuclei isolated from anti-vimentin-treated extracts; (A and D) shows the distribution of DNA; (B and E) distribution of lamins; and (C and F) distribution ofPCNA. Bar, 5/an.

1989). If lamins participate in the organization of repliconclusters, then lamin depletion may give rise to changes inthe distribution of PCNA. To test this we have stainednuclei that had been assembled in lamin-depleted ex-tracts, with anti-PCNA antibodies. Fig. 7F illustrates atypical distribution of PCNA in S-phase nuclei, in whichanti-PCNA antibodies co-localize with the chromatin(Fig. 7D) but not the lamina (Fig. 7E). The pattern of anti-PCNA staining in nuclei isolated from lamin-depletedextracts was clearly different, being diffuse and restrictedto areas of lamin fluorescence (Fig. 7B and C). Thus itappears that nuclei that are unable to form a lamina arealso unable to target replication enzymes correctly.

Discussion

Lamin-depleted extracts can decondense chromatin andassemble nuclear membranes and functional porecomplexesWe have used monoclonal antibodies to deplete laminsfunctionally from cell-free extracts of Xenopus eggs.Extracts depleted in this way are still able to decondensesperm DNA and to assemble double membranes and porestructures around that DNA. Furthermore, as nuclearproteins accumulate within such nuclei it seems likelythat the pore complexes are functional. These resultscontrast with previous studies in which lamins weredepleted from extracts of CHO cells, using polyclonalantisera (Burke and Gerace, 1986). Burke and Geracereported that lamin depletion in their extracts inhibitedboth chromatin decondensation at telophase and mem-brane assembly around telophase chromosomes. Similarstudies in which polyclonal anti-lamin antisera were

microinjected into mitotic cells, demonstrated that func-tional depletion of the lamins at mitosis preventedchromosome decondensation at telophase but did notcompletely inhibit the formation of membrane structuresaround telophase chromosomes (Benevante and Krohne,1986). Thus in somatic cells, it seems clear that lamins arerequired for chromosome decondensation, and may berequired for the assembly of the nuclear membrane.

Nuclear assembly in Xenopus egg extracts may differfrom nuclear assembly in somatic cell extracts. It hasalready been demonstrated that decondensation of spermDNA can be achieved independantly of nuclear envelopeassembly (Lohka and Masui, 1984; Sheehan et al. 1988).Other studies have shown that chromosome deconden-sation and membrane assembly occur before laminpolymerization, as extracts progress from mitosis tointerphase (Hutchison et al. 1988,1989). Furthermore, theassociation of structures resembling nuclear pores withthe chromatin also occurs independantly of envelopeassembly, implying that a lamina is not required for theorganization of pore complexes (Sheehan et al. 1988). Thusthere are considerable data that suggest that the keyprocesses of nuclear assembly in Xenopus egg extracts donot require lamins.

There are several reasons why Xenopus eggs and earlyembryos may differ from somatic cells of other species intheir requirement for lamins. Both Xenopus oocytes andeggs contain only a single lamin species LIU (Stick andHausen, 1986), which is structurally dissimilar to any ofthe two subtypes of lamins found in somatic cells (Stick,1988). Although lamin LUI seems to be more closelyrelated to B-type rather than A-type lamins, unlike theB-type lamins of somatic cells, lamin LUI is freely solublein the egg cytoplasm and does not segregate with

Role of lamin in DNA replication 277

membrane vesicles during metaphase in early cleavagestages. The segregation of B-type lamins in ER-likemembranes (Burke and Gerace, 1986; Stick et al. 1988) hasled to the suggestion that B-type lamins target theassociation of membranes with telophase chromosomes(Gerace et al. 1984). In early Xenopus embyros and oocytes,other membrane proteins may fulfil this role (Wilson andNewport, 1988). Furthermore, the existence of nucleiwithout a lamina structure is not without precedence.Nuclei at specific meiotic phases have well-defined doublemembranes and pore complexes despite the lack of anylamin structures (Stick and Schwarz, 1982, 1983). Thefinding that lamins are not required for chromosomedecondensation in Xenopus eggs could be explained by theunusually large stockpiles of enzymes required for DNAreplication and nuclear assembly in these cells. Inparticular, topoisomerase II has been implicated as beingrequired for chromatin decondensation in Xenopus eggextracts (Newport, 1987). In somatic cells, topoisomeraseII has been shown to be associated with the pore-complexlamina during interphase and with chromatin scaffoldsduring mitosis (Rottman et al. 1987; Earnshaw et al. 1985).Perhaps these structural associations are needed in orderto ensure local concentrations of the enzyme. In Xenopuseggs the higher levels of topoisomerase II may negate thisrequirement, thus allowing chromatin decondensation inthe absence of a lamina in Xenopus eggs but not in somaticcells.

The role of the lamina in DNA replicationStructural organization appears to be an essential featureof DNA replication in eukaryotes. Analyses of thedistribution of the sites of DNA replication in S-phasenuclei, by fluorescence microscopy, has indicated thatreplicons are clustered in groups of 100-300 (Nakamura etal. 1986; Mills et al. 1989). Furthermore, these repliconclusters are retained at the nuclear matrix followingextraction of biotin-11-dU TF-labelled nuclei with Tritonand high-salt (Nakayasu and Berezney, 1989). Whenlamins are depleted from cell-free extracts of Xenopus eggsusing monoclonal antibodies, DNA replication is inhibitedand PCNA is prevented from being distributed in thepunctate pattern that corresponds to replicon clusters.Inhibition of DNA replication, using aphidicolin, does notprevent the normal distribution of PCNA and DNApolymerase (Hutchison and Kill, 1989). Therefore, dolamins provide the structures at which replicon clustersare organized?

As it has already been shown that the assembly of anuclear envelope is essential for the initiation of DNAreplication (Blow and Laskey, 1986; Blow and Watson,1987; Sheehan et al. 1988), one trivial explanation of ourresults is that lamin-depleted extracts fail, to completenuclear membrane assembly around decondensed chroma-tin, thus inhibiting DNA replication. If this was so thenperhaps these nuclei could not maintain essential differ-ences in the concentration of replication factors betweenthe nucleoplasm and cytosol (Blow and Watson, 1987).However, electron-microscopy (EM) data indicate nodifference between the membrane structures surroundingnuclei in depleted and undepleted extracts. Furthermore,nuclear antigens accumulate within the nuclei assembledin depleted extracts. Thus it seems unlikely that DNAreplication is prevented as a result of the nuclearmembrane failing to act as a barrier.

In Xenopus egg extracts, lamin polymerization and there-initiation of DNA replication both appear to occur at

the same time and in the same place in nuclei that are re-forming during telophase (Hutchison et al. 1988). How-ever, in nuclei assembled from sperm heads in similarextracts, replicons are distributed throughout the nucleusthat have no particular association with the nuclearenvelope (Mills et al. 1989). Since studies using bothimmunofluorescence and EM indicate that the lamina isrestricted to the inner surface of the nuclear membrane(Gerace etal. 1978; Stick and Krohne, 1980; Brakenhoff etal. 1985; Aebi et al. 1986), it would appear that repliconsare not directly associated with the lamina. Moreover, it ispossible, by adding maturation promoting factor (MPF), todisrupt both the lamina and nuclear membrane ofreplicating Xenopus nuclei residing in egg extracts,without disrupting replicon clusters (Hutchison and Kill,1989). Thus, while the assembly of a lamina appears to bea pre-requisite for the initiation of DNA replication, itdoes not appear to be the structure at which replicons areorganized.

What then is the role of the lamina in DNA replication?Several authors have reported the existence of secondary'internal' matrices consisting of RNA and protein (Fey etal. 1984, 1986; Pouchelet et al. 1986). These matricesappear to consist of two clearly denned networks, an intra-chromosomal network and an inter-chromatin network(Pouchelet et al. 1986). Recent studies also indicate that,like the lamins, the internal matrix consists of intermedi-ate filaments that form a continuous lattice of fibres,communicating with cytoplasmic intermediate filamentsvia the pore complex-lamina (Jackson and Cook, 1988).Thus the lamina may function as a supporting sub-structure for the internal nuclear matrix and may alsoprovide continuity between the internal nuclear matrixand cytoskeletal structures. It has been suggested thatthis structural hierarchy provides a convenient highwayfor the transport of protein and RNA into and out of thenucleus (Jackson and Cook, 1988). Thus the abnormaldistribution of PCNA in lamin-depleted nuclei may resultfrom loss of structural integrity. Furthermore, withoutsuch structures to target replication proteins it may beimpossible for the nucleus to assemble all of thecomponents of the replication complex at the same timeand in the same place.

We thank Dr Ian Kill (University of Dundee) and Professor S.Shall (University of Sussex) for critical reading of the manuscriptand Dr John Newport (University of California at San Diego) forcommunicating to us his data on nuclear assembly in lamin-depleted egg extracts. This work was supported by grants fromthe Medical Research Council (C.J.H.), the Cancer ResearchCampaign (C.C.F). Dr Stick is a Heisenberg Stipendiate of theDeutsche Frechungsgemeinschaft.

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(.Received 24 September 1990 - Accepted 29 November 1990)

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