The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs

S-phase is the period in the cell cycle during which genomic DNA is duplicated. To achieve this duplication, many thousands of initiation events must be co-ordinated in time and space, implying a high degree of structural organization within the eukaryotic cell nucleus. Various extraction procedures have demonstrated the presence of a filamentous substructure within the nucleus, termed the nucleoskeleton (Fey et al. 1986; Pouchelet et al. 1986; Jackson and Cook, 1988), which appears to be sub-dividied into three distinct compartments: the pore-complex lamina, intra-chromosomal filaments and inter-chromosomal filaments (Aebi et al. 1986; Pouchelet et al. 1986). During S-phase, all nascent DNA appears to be associated with the nucleoskeleton (Dijkwel et al. 1986; Pardoll et al. 1980; McCready et al. 1980; Smith and Berezney, 1982; Jackson and Cook, 1986a). In addition, replicative polymerases are also associated with the nucleoskeleton during S-phase but not at other times during the cell cycle (Smith and Berezney, 1983; Jackson and Cook, 1986b). These observations have led to the view that replication complexes are immobilized at the nucleoskeleton, and that replication forks expand as DNA is threaded through the static replicase (Jackson and Cook, 1986a).

While fractionation of replicating nuclei has proved useful for demonstrating an association between replication forks and substructures within the nucleus, this approach provides no information as to how such associations arise. In contrast, cell-free extracts of Xenopus eggs that assemble nuclei capable of initiating DNA replication in vitro (Blow and Laskey, 1986; Newport, 1987; Hutchison et al. 1987) can be used to dissect those events involved in nuclear assembly that permit DNA replication (Sheehan et al. 1988; Hutchison et al. 1988). Extracts prepared from either fertilized or unfertilized eggs of the frog Xenopus laevis are able to assemble nuclear structures, including bilayered membranes, nuclear pores and a lamina, around a range of DNA templates, including demembranated sperm heads and plasmid DNA (Lohka and Masui, 1983; Blow and Laskey, 1986; Newport, 1987; Sheehan et al. 1988; Hutchison et al. 1988). Such nuclei are able to import nuclear proteins selectively (Newmeyer et al. 1986) and can initiate and complete a single round of DNA replication (Blow and Watson, 1987). Immunofluorescence studies imply that in S-phase nuclei that have been assembled in vitro, both replicons and replicases are organized at multiple discrete sites, (Hutchison and Kill, 1989; Hutchison et al. 1989; Mills et al. 1989), each fluorescent spot representing a cluster of up to 300 replicons (Mills et al. 1989). The organization of replicons in clusters would again imply structural associations in order to maintain each replication fork in close proximity to its neighbour.

Extracts that support extensive protein synthesis will also induce periodic mitotic events in nuclei that have completed S-phase. Following each mitosis, nuclei re-form and are able to re-initiate DNA replication. During nuclear reconstitution at telophase, there is a striking correlation in both time and space, between re-initiation of DNA replication and lamin polymerization, implying that the lamina may be involved in organizing S-phase chromatin (Hutchison et al. 1988; Hutchison et al. 1989).

One way of testing whether lamins participate in the organization of replicon clusters would be to construct nuclei that lack lamin structures. This might be achieved by immunodepleting egg extracts of lamins prior to nuclear assembly. Similar experiments have been attempted in extracts prepared from Chinese hamster ovary (CHO) cells. However, depletion of lamins in these extracts at mitosis, prevents nuclear assembly at telophase (Burke and Gerace, 1986). Despite these findings there are good reasons for believing that removal of lamins from Xenopus egg extracts would not inhibit activities that decondense chromosomes or assemble nuclear membranes and pore complexes around decondensed chromatin. In Xenopus, lamin LITT (the only lamin type present in early cleavage embryos) is freely soluble and does not segregate with endoplasmic reticulum like membranes during mitosis. Furthermore, nuclei at specific phases of meiosis lack a lamina structure but nevertheless have nuclear membranes and pore complexes (Stick and Schwarz, 1982, 1983; Stick and Hausen, 1985). Thus in order to assess the relationship between lamin polymerization and DNA replication we have used monoclonal antibodies to functionally deplete lamin LIU from cell-free extracts of Xenopus eggs. The results indicate that, while LIU is not required for either chromatin decondensation or nuclear membrane assembly, it is essential for initiating DNA replication and may be involved in targeting enzymes to the sites of DNA synthesis.

Anti-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 of Essex). Rabbit polyclonal anti-mouse immunoglobulins were purchased from ICN immunobiochemicals; FITO, TRITC and peroxidase-conjugated antibodies were purchased from DAKO-PATTS.

Cell-free extracts were prepared as previously described (Hutchison et al. 1988). Briefly, dejellied unfertilized eggs were washed in extraction buffer (100 mM KC1, 20 mM Hepes, pH 7.5, 5mM MgCL, ImM 2-mercaptoethanol) and then crushed by centrifugation at 10 000 g for 10 min. The soluble material was removed and treated with 50 μg ml⁻¹ cytochalasin B before centrifugation for a second time (10 000 g for 10 min) to remove debris.

A 100 μ1 sample of egg extract containing 10 μg ml⁻¹ cycloheximide (to prevent mitosis) was inoculated with 5 μl of a preparation of L6 5D5 monoclonal anti-lamin ascitic fluid that had been diluted 1/10 in SuNaSp (0.15 M sucrose, 75 mM NaCl, 0.5 mM spermidine, 0.15mM spermine; Gurdon, 1976) and incubated for lh at 21 °C; 10⁶ demembranated sperm heads (Hutchison et al. 1987) in 1 μl of SuNaSp were then inoculated into the extract, which was divided into 10 pl aliquots. At successive 10-min intervals, samples were either fixed for fluorescence microscopy (Hutchison et al. 1988) or for examination of nuclear membrane formation (Hutchison et al. 1987). Extensive controls were performed in which extracts were inoculated with monoclonal anti-vimentin ascites (to test for antibody specificity), rabbit polyclonal antisera (to test for general effects of serum addition) and SuNaSp (to test for the effect of diluting the extract).

Particulate material was removed from egg extracts by centrifugation at 100000g for 1h at 4 °C. The cytosolic fraction (containing 90% of free lamins) was divided into 50 μl samples and inoculated with either anti-lamin ascites, anti-vimentin ascites fluid or SuNaSp, and incubated for 1h at 21 °C. A 10 μl sample of a suspension of Staph.a in SuNaSp was then inoculated into each extract and incubated for a further hour at 21 °C. The Staph.a was then pelleted by centrifugation at 15 000 g for 5 min and the pellet and supernatant fractions were analysed by immunoblotting following SDS–PAGE.

A 100 μl sample of L6 8A7 ascites was precipitated with 40% saturated ammonium sulphate and resuspended in 0.25 M (pH 9.0) carbonate-bicarbonate buffer (Hudson and Hay, 1980). Following dialysis in this buffer, fluorescein isothyocyanate (FITC, 0.05 mg mg⁻¹ of total protein) was added and mixed overnight at 4°C. Conjugated protein was separated from free fluorochrome by gel filtration using a Sephadex G-25 column equilibrated in phosphate-buffered saline. Direct immunofluorescence was performed with this FITC-conjugated antibody as follows: nuclei were fixed and spun onto glass coverslips as previously described (Hutchison et al. 1988). Each coverslip was then incubated with 30 pl of FITC-conjugated anti-lamin antibody (diluted 1/25 in PBS containing 1% foetal calf serum) and incubated overnight at 4 °C. The coverslips were then washed in PBS and mounted in 50% PBS/glycerol containing l μg ml⁻¹ DAPI.

A 10 μl sample of egg extract containing 10⁴ nuclei was dropped into 1ml of 0.2 M sodium cacodylate (pH 7.4) containing 2.5% glutaraldehyde. After incubation for 2h on ice, the drop of cytoplasm had fixed into a loose ball. This was washed twice in lml of 0.2 M sodium cacodylate (pH 7.4) before post-fixation in OsO₄ for 1 h at 4 °C. The sample was then dehydrated in a graded ethanol series (30% to 100%) and incubated overnight in 50% LR Whites resin in 50% ethanol at 4 °C. The following day the sample was transferred to 100% LR Whites and incubated overnight at 4°C. After a further change of resin and incubation for a further 48h at 4°C the sample was polymerized at 60°C for 24h. Thin sections were cut using a Reichert OM U3 microtome and poststained on copper grids with uranyl acetate/lead citrate. Sections were viewed using a JOEL JEM 1200EX transmission electron microscope.

Nuclei were isolated from egg extracts by the following procedure: 200 pl of extract containing 2×10⁵ nuclei were diluted in 2 ml of nuclear isolation buffer (NIB: 60 mM KC1, 15 mM Tris-HCl, pH7.4, 15mM NaCl, ImM 2-mercaptoethanol, 0.5mM spermidine, 0.15 mM spermine). The diluted suspension of nuclei was layered over a 60% Percol cushion (1ml) and subjected to centrifugation at 3000 g for 10 min at 4 °C. Nuclei were recovered from the interface, diluted in 1 ml of NIB and pelleted by centrifugation at 6000 g for 10 min. The nuclear pellet was either resuspended in SDS sample buffer or in NIB containing 0.5% Triton X-100 and 2 M NaCl. In the case of resuspension in modified NIB, nuclei were incubated for 10 min on ice before centrifugation for a second time at 6000 g for 10 min. Both the pellet and supernatant were prepared for SDS–PAGE by the addition of sample buffer.

Protein samples resolved on 10% SDS–PAGE, were transfered to nitrocellulose (Schleicher and Schuell) by electrophoresis in transfer 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, pH 7.4, 1 mM EDTA, 75mM NaCl, 0.1% TWEEN 20) and then incubated with a 1/1000 dilution of L6 8A7 (in blot rinse buffer containing 1% FCS) overnight at 4°C. The filter was washed thoroughly in blot rinse buffer before incubation for 4 h at 4 °C with peroxidase-conju gated rabbit anti-mouse IgG (1/400 dilution DAKOPATTS). After a final wash the filter was developed by incubation in 24 MM citric acid, 51mM Na₃HPO₄ (pH5.0) 1 mg ml⁻¹ DAB and 0.3% H₂O₂.

In order to establish that antibodies inoculated into egg extracts were absorbing all free lamins, the following experiment was performed. Extracts were incubated with monoclonal 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 a further 60 min at 21°C. The resulting Staph.a–IgG complex was pelleted by centrifugation at 6000 g for 10 min and the distribution of lamins between the pellet and the supernatant fractions was determined by SDS–PAGE followed by immunoblotting, using a second monoclonal anti-lamin antibody L6 8A7. Lamins were only detected in the pellet fraction following incubations with L6 5D5 and Staph.a. This was in contrast to incubations of extracts with Staph.a only or with Staph.a plus an irrelevant antibody, where only small amounts of lamins co-precipitated with the pellet fraction while the bulk remained in the supernatant (results not shown). These data imply that essentially all lamins were specifically bound to IgG upon inoculation of L6 5D5 into egg extracts.

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

To confirm that the structures observed under the light microscope were indeed nuclei, samples were prepared for transmission electron microscopy. In Fig. 2 typical structures isolated from both lamin-depleted (Fig. 2A) and undepleted (Fig. 2B) extracts are compared. Both nuclei are large (>8 μm) and are surrounded by double unit membranes interupted by pore complexes (arrows). We did observe more variation in the size of nuclei isolated from lamin-depleted extracts (of 50 such nuclei counted the mean diameter was 5 gm with variations between 2 μm and 9 urn) but all of these nuclei were otherwise identical to controls.

The results described above contrast with previous studies in which lamins had been depleted from somatic cells or somatic cell extracts during mitosis. The former studies demonstrated that the re-formation of interphase nuclei required lamins both for chromosome decondensation and membrane accumulation around decondensed chromosomes (Burke and Gerace, 1986). While previous data have been obtained using polyclonal antisera, in which the antibody–antigen complexes where removed from the extracts, in our experiments a monoclonal antibody was used and the antibody-antigen complex remained in the extract. Thus the contrast between the results reported here and previous studies might be explained if absorbtion of lamins to monoclonal antibodies failed to prevent either the accumulation of lamins within nuclei or their subsequent polymerization. To test this, we isolated nuclei formed in extracts that had been inoculated with L6 5D5 and fixed them in preparation for direct immunofluorescence. Fixed nuclei were probed with TRITC-conjugated rabbit anti-mouse IgG to reveal the distribution of L6 5D5 antibodies, and then with FITC-conjugated monoclonal anti-lamin antibodies (L6 8A7) to reveal independently the distribution of the bulk of the lamins.

TRITC-conjugated rabbit anti-mouse IgG stained nuclei that had been isolated from L6 5D5-treated extracts, with an intense punctate pattern (Fig. 3B). This fluorescence pattern indicated that L6 5D5 IgG was distributed throughout the nucleoplasm with no particular association with the nuclear envelope. In contrast, TRITC-labelled rabbit anti-mouse IgG did not stain nuclei that were formed in extracts containing an irrelevant mouse IgG (Fig. 3e). These results imply that while IgG is normally excluded from nuclei, L6 5D5 anti-lamin antibodies were able to penetrate the nuclear envelope. Co-staining of these nuclei with FITC-coiyugated L6 8A7 (anti-lamin) monoclonal antibodies revealed that the distribution of lamins in nuclei isolated from L6 5D5-treated extracts was identical to the distribution of L6 5D5 IgG (compare Fig. 3B and 3C) and did not resemble the halo-like pattern obtained with nuclei isolated from control extracts (Fig. 3F).

One interpretation of these data is that small amounts of free lamins in L6 5D5-treated extracts are able to enter nuclei without assembling into a continuous lamina and that, subsequently, during the fixation period free antibody penetrates the nuclei and associates with such lamins. To exclude this possibility we inoculated extracts containing nuclei with L6 5D5 antibodies just before fixation and stained them with TRITC-labelled rabbit anti-mouse IgG followed by FITC-labelled L6 8A7 antibodies. Only FITC fluorescence was detectable in such nuclei, implying that L6 5D5 antibodies had not penetrated the nuclei during the fixation period (Fig. 3H and I). Thus we infer that the presence of L6 5D5 in nuclei isolated from lamin-depleted extracts results from cotransport of antibody-lamin complex into those nuclei. Co-transport of antibody-nuclear antigen complexes has been reported previously, and presumably occurs when the antibody does not cover the transport sequence on the antigen (Einck and Bustin, 1984; Borer et al. 1989).

While the immunofluorescence data imply that L6 5D5 anti-lamin antibodies prevent the formation of a continuous lamina, the residual lamin structures that were formed may be incorporated into a Triton-insoluble nucleoskeleton, which could provide sites for the organization of interphase chromatin. To investigate this possibility, nuclei assembled in antibody-treated extracts were isolated, by centrifugation onto Percol cushions, and extracted in 0.5% Triton X-100 and 2 M NaCL The distribution of lamins between Triton-soluble and Tritoninsoluble fractions was determined by immunoblotting following gel electrophoresis on SDS-PAGE.

The results, illustrated in Fig. 4, show that monoclonal anti-lamin antibodies L6 8A7 detected three polypeptides of M_r 58× 10³, 60× 10³ and 62× 10³ in nuclei isolated from egg extracts (lane 1). Only one of these polypeptides (60× 10³) was pelleted with bulk chromatin following Triton/high-salt extraction (lane 2), while the other two polypeptides were Triton/high-salt-soluble (lane 3). Previous studies have demonstrated the presence of only a single lamin species (lamin LUI) in lamina fractions prepared from nuclei formed in Xenopus extracts and from early embryos as revealed by immunoblotting with either polyclonal anti-lamin serum or monoclonal antibody L6–8A7 (Stick and Hausen, 1985). The results illustrated in Fig. 4 lanes 2 and 8 are in agreement with these observations. A single band of 60×10³Af_r (LIII) was pelleted after Triton/high-salt extraction of nuclei isolated from extracts preincubated with either buffer alone (Fig. 4, lane 2) or an irrelevant antibody (Fig. 4, lane 8). In contrast, no lamin proteins were detected in the Triton/high-salt-resistent fraction of nuclei isolated from extracts preincubated with anti-lamin monoclonal antibody L6–5D5 (Fig. 4, lane 5). In the latter case lamin proteins are present in the Triton/high-salt-soluble fraction (Fig. 4, lane 6) while the corresponding fractions in the control experiments have been largely depleted of LIU (Fig. 4, lanes 3 and 9). The monoclonal antibody L6–8A7 recognizes, in addition to the lamin LIII, two other polypeptides in whole nuclei isolated from egg extracts. We do not know what these proteins are. However, they are always found in the soluble fraction after extraction and thus do not contribute to the formation of the lamina structure. In conclusion, these results imply that inoculation of L6–5D5 monoclonal anti-lamin antibodies into egg extracts functionally depletes the lamins; thus preventing the formation of a lamina within pronuclei.

DNA replication is usually extremely efficient in nuclei incubated in Xenopus egg extracts. Since sperm heads that had been inoculated into lamin-depleted extracts were assembled into nuclei that lacked a lamina, we investigated whether such nuclei were able to replicate DNA. Extracts containing nuclei were either pulse labelled with biotin-ll-dUTP or labelled continuously with [³²P]dCTP. Following biotin labelling nuclei were fixed and prepared for fluorescence microscopy. Fig. 5A–C shows typical nuclei isolated from lamin-depleted extracts; FITC-labelled anti-lamin antibody L6 8A7 detected only islands of lamins with no particular association with the nuclear envelope (Fig. 5B). Such nuclei did not incorporate biotin-11-dUTP even after prolonged (2 h) incubations (Fig. 5C). In contrast, nuclei isolated from extracts that had been inoculated with an irrelevant IgG had a continuous lamina (Fig. 5E) and after only 30 min showed extensive incorporation of biotin-II-dUTP (Fig. 5F). Fig. 6 illustrates a typical time course of biotin-11-dUTP incorporation in antibody-treated and control extracts. Up to 80% of sperm heads had been assembled into nuclei that were synthesizing 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 still assembled into nuclei in lamin-depleted extracts, less than 10% of these nuclei incorporated biotin-ll-dUTP. Furthermore, biotin incorporation in these nuclei was extremely weak.

To estimate the amount of DNA replication in antibody-treated extracts, [³²P]dCTP labelling was performed as previously described (Hutchison et al. 1988). Table 1 shows that, whereas inoculation of an irrelevant antibody into extracts resulted in a small (9%) reduction in the total amount of DNA replication occurring over a 2h period (compared to extracts without antibody), inoculation of L6 5D5 antibodies resulted in a 94% reduction in the amount of DNA synthesized.

Several authors have suggested that intermediate filaments within the nucleus provide sites for the organization of the replication machinery (Jackson and Cook, 1988; Nakayasu and Berezney, 1989). Furthermore, it now appears that this structural organization leads to clustering of replicons (Mills et al. 1989) and that the sites of such replicon clusters can be detected with antibodies recognising proliferating cell nuclear antigen (PCNA) (Bravo and MacDonald-Bravo, 1987; Hutchison and Kill, 1989). If lamins participate in the organization of replicon clusters, then lamin depletion may give rise to changes in the distribution of PCNA. To test this we have stained nuclei that had been assembled in lamin-depleted extracts, with anti-PCNA antibodies. Fig. 7F illustrates a typical distribution of PCNA in S-phase nuclei, in which anti-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-depleted extracts was clearly different, being diffuse and restricted to areas of lamin fluorescence (Fig. 7B and C). Thus it appears that nuclei that are unable to form a lamina are also unable to target replication enzymes correctly.

We have used monoclonal antibodies to deplete lamins functionally from cell-free extracts of Xenopus eggs. Extracts depleted in this way are still able to decondense sperm DNA and to assemble double membranes and pore structures around that DNA. Furthermore, as nuclear proteins accumulate within such nuclei it seems likely that the pore complexes are functional. These resulta contrast with previous studies in which lamins were depleted from extracts of CHO cells, using polyclonal antisera (Burke and Gerace, 1986). Burke and Gerace reported that lamin depletion in their extracts inhibited both chromatin decondensation at telophase and membrane assembly around telophase chromosomes. Similar studies in which polyclonal anti-lamin antisera were microinjected into mitotic cells, demonstrated that functional depletion of the lamins at mitosis prevented chromosome decondensation at telophase but did not completely inhibit the formation of membrane structures around telophase chromosomes (Benevante and Krohne, 1986). Thus in somatic cells, it seems clear that lamins are required for chromosome decondensation, and may be required for the assembly of the nuclear membrane.

Nuclear assembly in Xenopus egg extracts may differ from nuclear assembly in somatic cell extracts. It has already been demonstrated that decondensation of sperm DNA can be achieved independantly of nuclear envelope assembly (Lohka and Masui, 1984; Sheehan et al. 1988). Other studies have showm that chromosome decondensation and membrane assembly occur before lamin polymerization, as extracts progress from mitosis to interphase (Hutchison et al. 1988, 1989). Furthermore, the association of structures resembling nuclear pores with the chromatin also occurs independantly of envelope assembly, implying that a lamina is not required for the organization of pore complexes (Sheehan et al. 1988). Thus there are considerable data that suggest that the key processes of nuclear assembly in Xenopus egg extracts do not require lamins.

There are several reasons why Xenopus eggs and early embryos may differ from somatic cells of other species in their requirement for lamins. Both Xenopus oocytes and eggs contain only a single lamin species LUI (Stick and Hausen, 1985), which is structurally dissimilar to any of the two subtypes of lamins found in somatic cells (Stick, 1988). Although lamin LUI seems to be more closely related to B-type rather than A-type lamins, unlike the B-type lamins of somatic cells, lamin LUI is freely soluble in the egg cytoplasm and does not segregate with membrane vesicles during metaphase in early cleavage stages. The segregation of B-type lamins in ER-like membranes (Burke and Gerace, 1986; Stick et al. 1988) has led to the suggestion that B-type lamins target the association of membranes with telophase chromosomes (Gerace et al. 1984). In early Xenopus embyros and oocytes, other membrane proteins may fulfil this role (Wilson and Newport, 1988). Furthermore, the existence of nuclei without a lamina structure is not without precedence. Nuclei at specific meiotic phases have well-defined double membranes and pore complexes despite the lack of any lamin structures (Stick and Schwarz, 1982, 1983). The finding that lamins are not required for chromosome decondensation in Xenopus eggs could be explained by the unusually large stockpiles of enzymes required for DNA replication and nuclear assembly in these cells. In particular, topoisomerase II has been implicated as being required for chromatin decondensation in Xenopus egg extracts (Newport, 1987). In somatic cells, topoisomerase II has been shown to be associated with the pore-complex lamina during interphase and with chromatin scaffolds during mitosis (Rottman et al. 1987; Earnshaw et al. 1985). Perhaps these structural associations are needed in order to ensure local concentrations of the enzyme. In Xenopus eggs the higher levels of topoisomerase II may negate this requirement, thus allowing chromatin decondensation in the absence of a lamina in Xenopus eggs but not in somatic cells.

Structural organization appears to be an essential feature of DNA replication in eukaryotes. Analyses of the distribution of the sites of DNA replication in S-phase nuclei, by fluorescence microscopy, has indicated that replicons are clustered in groups of 100–300 (Nakamura et al. 1986; Mills et al. 1989). Furthermore, these replicon clusters are retained at the nuclear matrix following extraction of biotin-ll-dUTP-labelled nuclei with Triton and high-salt (Nakayasu and Berezney, 1989). When lamins are depleted from cell-free extracts of Xenopus eggs using monoclonal antibodies, DNA replication is inhibited and PCNA is prevented from being distributed in the punctate pattern that corresponds to replicon clusters. Inhibition of DNA replication, using aphidicolin, does not prevent the normal distribution of PONA and DNA polymerase (Hutchison and Kill, 1989). Therefore, do lamins provide the structures at which replicon clusters are organized?

As it has already been shown that the assembly of a nuclear envelope is essential for the initiation of DNA replication (Blow and Laskey, 1986; Blow and Watson, 1987; Sheehan et al. 1988), one trivial explanation of our results is that lamin-depleted extracts fail, to complete nuclear membrane assembly around decondensed chromatin, thus inhibiting DNA replication. If this was so then perhaps these nuclei could not maintain essential differences in the concentration of replication factors between the nucleoplasm and cytosol (Blow and Watson, 1987). However, electron-microscopy (EM) data indicate no difference between the membrane structures surrounding nuclei in depleted and undepleted extracts. Furthermore, nuclear antigens accumulate within the nuclei assembled in depleted extracts. Thus it seems unlikely that DNA replication is prevented as a result of the nuclear membrane failing to act as a barrier.

In Xenopus egg extracts, lamin polymerization and the re-initiation of DNA replication both appear to occur at the same time and in the same place in nuclei that are reforming during telophase (Hutchison et al. 1988). However, in nuclei assembled from sperm heads in similar extracts, replicons are distributed throughout the nucleus that have no particular association with the nuclear envelope (Mills et al. 1989). Since studies using both immunofluorescence and EM indicate that the lamina is restricted to the inner surface of the nuclear membrane (Gerace et al. 1978; Stick and Krohne, 1980; Brakenhoff et al. 1985; Aebi et al. 1986), it would appear that replicons are not directly associated with the lamina. Moreover, it is possible, by adding maturation promoting factor (MPF), to disrupt both the lamina and nuclear membrane of replicating Xenopus nuclei residing in egg extracts, without disrupting replicon clusters (Hutchison and Kill, 1989). Thus, while the assembly of a lamina appears to be a pre-requisite for the initiation of DNA replication, it does not appear to be the structure at which replicons are organized.

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 et al. 1984, 1986; Pouchelet et al. 1986). These matrices appear to consist of two clearly defined 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 intermediate filaments that form a continuous lattice of fibres, communicating with cytoplasmic intermediate filaments via the pore complex-lamina (Jackson and Cook, 1988). Thus the lamina may function as a supporting substructure for the internal nuclear matrix and may also provide continuity between the internal nuclear matrix and cytoskeletal structures. It has been suggested that this structural hierarchy provides a convenient highway for the transport of protein and RNA into and out of the nucleus (Jackson and Cook, 1988). Thus the abnormal distribution of PCNA in lamin-depleted nuclei may result from loss of structural integrity. Furthermore, without such structures to target replication proteins it may be impossible for the nucleus to assemble all of the components of the replication complex at the same time and in the same place.

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