Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the ammount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from ‘lamin depleted nuclei’ contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.

The major residual structure that remains associated with the nuclear envelope following extraction of isolated nuclei or oocyte germinal vesicles with non-ionic detergents, nucleases and high salt is the lamina (Dwyer and Blobel, 1976). The nuclear lamina is mainly composed of protein sub-units called lamins (Gerace and Blobel, 1980) which are now classified as type V intermediate filament proteins (McKeon et al., 1986). Consistent with this classification, ultrastructural analyses of isolated oocyte GV envelopes indicate that the lamina comprises a lattice of 10 nm diameter fibrils which covers the entire inner surface of the nuclear envelope and which interlinks nuclear pores (Aebi et al., 1986; Goldberg and Allen, 1992).

Lamins fall into two sub-groups, the A-type and B-type lamins. One or more B-type lamins are thought to be expressed constitutively in all embryonic and somatic tissues (Lehner et al., 1987; Wolin et al., 1987; Vorburger et al., 1989a) although different tissues may contain different B-type lamins (Stick and Hausen, 1985; Benevente et al., 1985; Höger et al., 1988). In contrast, expression of A-type lamins is restricted to differentiated tissues (Lehner et al., 1987; Rober et al., 1989). Both types of lamin contain a sequence motif CaaX (C, cysteine; a, aliphatic amino acid, X, any amino acid) at the carboxyl terminus. This sequence serves as a site for modification by isoprenylation (Beck et al., 1990; Wolda and Glomset, 1988; Vorburger et al., 1989b; Firmbach-Kraft and Stick, 1993) and methylation (Chelsky et al., 1987). However, A-type and B-type lamins differ in that the modified cysteine residue in the CaaX motif can be removed by proteolytic cleavage of the final 18 amino acids in lamin A (Beck et al., 1990; Vorburger et al., 1989b; Weber et al., 1989). In addition, A-type lamins can be synthesised from an alternatively spliced mRNA species that lacks codons for the final 82 amino acids and therefore lacks the CaaX motif (this shortened lamin is termed lamin C; Fisher et al., 1986). Isoprenylation at the CaaX motif is thought to function in positioning the lamins at the nuclear envelope since the farnesyl residues (the isoprene added to the lamins) incorporated at this site can associate with the inner nuclear membrane probably via an isoprene receptor (Firmbach-Kraft and Stick, 1993; Meier and Georgatos, 1994). Consequently, removal of farensylated and methylated cysteine residues from A-type lamins during interphase, may result in these species dissociating from the nuclear envelope at mitosis. In contrast, B-type lamins, which retain their modified cysteine residues, segregate with nuclear envelope vesicles on nuclear envelope breakdown (Gerace and Blobel, 1980; Stick et al., 1988; Meier and Georgatos, 1994).

The other major modification process which regulates lamin behaviour is phosphorylation/dephosphorylation. At mitosis, in vertebrate cells, the nuclear envelope is disassembled at prometaphase and must therefore be reassembled at telophase (reviewed by Cox and Hutchison, 1994). Disassembly of the lamina is regulated through phosphorylation (Ottaviano and Gerace, 1985; Miake-Lye and Kirschner, 1985) by cdc2/cyclin B (Peter et al., 1991). Phosphorylation occurs at two sites situated at the amino- and carboxyl ends of the rod domains (Peter et al., 1990). If these sites are mutated by serine to arginine substitutions, lamina disassembly at mitosis is blocked (Heald and McKeon, 1990). Lamina reassembly is regulated by both phosphorylation and dephosphorylation processes. Drugs which inhibit the activity of protein phosphatase 2a also prevent lamina assembly in Xenopus eggs (Murphy et al., 1995). In addition, phosphorylation of lamin B2 by protein kinase C (PKC), at a site adjacent to the nuclear localisation signal sequence, prevents translocation of this lamin across the nuclear envelope. Lamins which are transported across the nuclear envelope during interphase are incorporated into the lamina (Goldman et al., 1992). Therefore it seems likely that PKC phosphorylation also regulates lamina assembly processes.

In addition to the lamina, a second filamentous network has been identified in vertebrate nuclei. This network has been termed the nuclear matrix (Fey et al., 1986) or the nucle-oskeleton (Jackson and Cook, 1988). Because of the high concentration of chromatin within nuclei the ultrastructure of supporting filamentous networks is often obscure. Nevertheless extraction of nuclei with hypotonic buffers, detergents, nucleases and salt (Fey et al., 1986) or extraction of whole cells with detergents, nucleases and electroelution following encapsulation in agarose (Jackson and Cook, 1988) removes 95% of chromatin but preserves a proteinaceous filamentous network. This network has been viewed using resinless section EM (Fey et al., 1986; He et al., 1990; Jackson and Cook, 1988) and has the appearance of a network of branched filaments with diameters ranging between 9 and 13 nm. While these filaments interact with the lamina, they also extend throughout the nucleoplasm and are not restricted to the inner aspect of the nuclear envelope. For simplicity, this structure is referred to below as the nuclear matrix.

Using biochemical and ultrastructural approaches, several groups have demonstrated a close association between the nuclear matrix and sites of DNA replication. Both replicative DNA polymerases (Smith and Berezney, 1983; Jackson and Cook, 1986a) and nascent DNA (Jackson and Cook, 1986b; Berezney and Buchold, 1981) remain associated with the nuclear matrix at the end of extraction procedures. Moreover, centres of DNA replication have been visualised as residual components of nuclear matrix preparations using light microscopy (Nakayasu and Berezney, 1989) and electron microscopy (Hozak et al., 1993). Thus it seems likely that the nuclear matrix provides a structural framework which interacts with and supports the sites of DNA replication.

Cell-free extracts of Xenopus eggs support the assembly of replication competent nuclei in vitro (Blow and Laskey, 1986). DNA replication in these nuclei is entrained to normal embryonic cell cycle events, initiation occuring once per cell cycle (Hutchison et al., 1988) and being dependent upon the breakdown and reformation of the nuclear envelope (Blow and Watson, 1987; Blow and Laskey, 1988). Indirect immunofluorescence analysis indicates a close temporal and spatial correlation between lamina assembly and the initiation of DNA replication (Hutchison et al., 1988). Furthermore, removal of lamin B3 (the major lamin sub-type in Xenopus eggs) from the extracts, using monoclonal or polyclonal antibodies, permits the assembly of nuclei which lack a lamina and which do not support DNA replication (Newport et al., 1990; Meier et al., 1991; Jenkins et al., 1993). These nuclei have complete nuclear envelopes and well formed nuclear pores (Jenkins et al., 1995; Goldberg et al., 1995) and support active transport of karyophilic proteins such as the proliferating cell nuclear antigen (PCNA) (Jenkins et al., 1993). However, the assembly of replication centres containing PCNA is prevented (Jenkins et al., 1993). Replication centres are distributed throughout the nucleoplasm (Mills et al., 1989) and are only associated with the lamina indirectly (Hutchison, 1995). Thus the absence of replication centres in nuclei assembled in lamin B3 depleted extracts probably arises because these nuclei lack a properly organised nuclear matrix. The corollary of this is that the assembly of a nuclear matrix is dependent upon the prior assembly of a nuclear lamina (Hutchison et al., 1994).

It has recently been suggested that lamins are structural components of both nuclear matrix filaments (Hozak et al., 1995) and replication centres (Moir et al., 1994). Therefore a second interpretation consistent with the results of lamin depletion of egg extracts is that lamin B3 is directly involved in the assembly of nuclear matrix filaments. To test this hypothesis we prepared nuclear matrices from sperm pronuclei assembled in vitro. Nuclear matrices were viewed as DGD sections and as whole mounts by transmission electron microscopy (TEM) and as whole mounts using field emission in lens scanning electron microscopy (FEISEM). Typical preparations consisted of a continuous network of filaments which extended throughout the nucleus. The filaments at the surface of nuclear matrices interconnected nuclear pores. These filaments were labelled with anti-lamin B3 mAbs. Other filaments extended into the nuclear matrix. Many of these filaments were not labelled with anti-lamin B3 mAbs. When nuclear matrices were prepared from nuclei assembled in lamin B3 depleted extracts filamentous structures were still observed. The density of these filaments was greatly reduced and the filaments were generally poorly organised. Moreover, these filaments were not labelled with anti-lamin B3 antibodies. This result supports the hypothesis that in Xenopus sperm pronuclei, the lamin B3 lamina is required for the correct assembly of a nuclear matrix but is not itself part of the nuclear matrix.

Preparation and use of egg extracts

Extracts were prepared from unfertilised Xenopus eggs according to the method described by Hutchison (1994). Extracts stored in liquid nitrogen were thawed rapidly and supplemented with demembranated sperm heads, an energy regenerating system and cycloheximide. Sperm pronuclear assembly occurred after incubating the reaction mixture for 90 minutes at 21°C. To recover sperm pronuclei for nuclear matrix preparation, the reaction mixture was diluted in 1/3 strength extraction buffer (100 nm KCl, 20 mM Hepes-KOH, pH 7.5, 5 mM MgCl2, 1 mM DTT) and layered over 30% glycerol. The nuclei were then recovered onto polylysine treated glass coverslips or Formvar and carbon-coated nickel grids by centrifugation at 1,500 g for 15 minutes.

Immunodepletion of Xenopus egg extracts

Egg extracts were specifically depleted of lamin B3 using monoclonal antibody L6 5D5 covalently linked to paramagnetic Dynabeads (Jenkins et al., 1993); 200 µl of Dynabead were mixed with 250 µl of the anti-lamin B3 mAb L6 5D5 (Stick, 1988) tissue culture supernatant by rotation at 2 rpm overnight at 4°C. The antibody was removed and the beads washed three times in PBS containing 1% BSA before being suspended in SUNaSp (Gurdon, 1976). The beads were recovered using a magnet and divided into 2 equal aliquots. The first aliquot was mixed with 125 µl of egg extract for 30 minutes at RT by rotation at 2 rpm. The beads were then removed from the extract using a magnet. The second aliquot of beads was mixed with the partially depleted extract for a further 30 minutes ar RT by rotation at 2 rpm. The beads were again removed using a magnet and the fully depleted extract stored in liquid nitrogen. As controls for specificity, parallel ‘mock depletions’ were performed with irrelevent monoclonal antibodies linked to Dynabeads. In addition, purified lamin B3 was readded to a sub-fraction of the depleted extract (Goldberg et al., 1995). To confirm that all lamin B3 was removed from depleted extracts, samples of the extract at each stage of the depletion were suspended in SDS sample buffer and analysed by western blotting. In addition, the Dynabeads recovered at each step were also suspended in SDS sample buffer and analysed.

Nuclear matrix preparation

Nuclear matrix preparations were performed using a modification of the method described by He et al. (1990), essentially as described by Zhang et al. (1993). Briefly, nuclei were incubated, either in situ or in suspension, in CSK buffer (10 mM Pipes-KOH, pH 6.8, 10 mM KCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1.2 mM PMSF). Next, nuclei were incubated for 10 minutes in CSK buffer containing 0.5% Triton X-100. The nuclei were then rinsed in RSB (42.5 mM Tris-HCl, pH 8.3, 8.5 mM NaCl, 6 mM MgCl2, 1.2 mM PMSF) before incubation in RSB-Magik (RSB supplemented with 1% Tween-20 and 0.5 mM sodium deoxycholate) for 10 minutes. Next the nuclei were rinsed in digestion buffer (DB: 10 mM Pipes, pH 6.8, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1.2 mM PMSF) and then incubated with DB supplemented with 100 U/ml RNase-free DNase I and 5 U/ml RNasin for 30 minutes at RT. Finally digested material was eluted by adding 1 M (NH4)2SO4 to a final concentration of 0.25 M and incubating for a further 5 minutes. Except where mentioned, all the steps were carried out at 4°C. At each step of the procedure samples were removed for immunoblotting, indirect immunofluorescence or EM.

Indirect immunofluorescence

Indirect immunofluorescence was performed according to the method described by Hutchison (1994). mAb L6 5D5 was used to detect antilamin immunofluorescence. Secondary antibodies were FITC goat anti-mouse Ig supplied by DAKOPATTS.

Immunblotting

Samples prepared for SDS-PAGE were resolved on 10% polyacrylamide gels. Immunoblotting was performed as described by Jenkins et al. (1993) using mAb L6 5D5 to detect lamin B3 polypeptides. Revelation was achieved using HRP-conjugated rabbit anti-mouse Ig followed by ECL (see Goldberg et al., 1995).

DGD sections

DGD (diethylene glycol distearate) embedded sections were prepared according to the method of Zhang et al. (1993). Briefly, isolated nuclear matrices were fixed using 2% gluteraldehyde and 1% OsO4 and embedded in DGD and then sectioned. The DGD was then melted and removed with n-butanol (three changes at RT). n-butanonl was then replaced by acetone. Finally, the samples were CO2 critical point dried and viewed directly using a Joel 1200 transmission electron microscope.

Field emmision in lens scanning electron microscopy (FEISEM) and immunogold labelling

Nuclear matrices prepared in situ on 7 mM diameter glass coverslips were washed in Sørensen’s phosphate buffer (PB) and fixed with 2% glutaraldehyde followed by 1% osmium tetroxide (both in PB). After washing in PB, the nuclear matrices were dehydrated through 30%, 50%, 70%, 85%, 95% and 100% alcohol. After washing in 100% alcohol the samples were transferred to Arklone for critical point drying using the highest purity CO2. The samples were then sputter coated with 4 nm Tantalum in an Edwards Auto 306 cryopump vacuum system and finally viewed in a Topcon DS130F scanning electron microscope at 30 kV accelerating voltage.

For immunogold labelling, nuclear matrices were fixed with 4% paraformaldehyde, 0.2% glutaraldehyde for 20 minutes at 4°C. The samples were rinsed 3 times with PBS and blocked by incubation with 1% NCS in PBS (1 hour at RT). The samples were then incubated with mAb L6 5D5 (diluted 1:50 in PBS containing 1% NCS), or an irrelevant mAb (human specific Ki67 antibody diluted 1/25 in PBS/NCS-DAKO), overnight at 4°C. The following morning the samples were washed thoroughly in PBS and then incubated with biotinylated goat anti-mouse IgG (DAKO, diluted 1:100 in PBS containing 1% NCS) for 1 hour at RT. The samples were again washed thoroughly with PBS before final reaction with 10 nm gold-conjugated avidin (Sigma; diluted 1:20 in PBS containing 1% NCS) for 30 minutes at RT. The samples were then processed as described above for FEISEM except that coating was performed with 4 nm chromium.

Transmission electron microscopy

Nuclear matrices collected on Formvar and carbon-coated nickel grids were processed as described for FEISEM and viewed using a Joel 1200 transmission electron microscope at 80 kV accelerating voltage.

Preparation of nuclear matrices from in vitro assembled nuclei

Nuclear matrices were prepared from sperm pronuclei using a modification of the procedure originally described by Fey et al. (1986). Pronuclei were assembled in Xenopus egg extracts and then recovered on polylysine coated glass coverslips. The nuclei were then extracted sequentially by first washing in CSK buffer, then incubating with CSK buffer containing 0.5% Triton X-100 followed by RSB-Magik solution, before DNase digestion and salt elution of digested material. At each step of the procedure samples were fixed and prepared for indirect immunofluorescence to monitor both extraction of chromatin and retention of the nuclear lamina. Fig. 1 shows representative micrographs of nuclei at the start and finish of the extraction procedure. Judged by the distribution and intensity of lamin fluorescence the integrity of nuclear lamina was maintained throughout the extraction procedure. Judged by DAPI fluorescence most of the chromatin was removed. In contrast to other reports (Hozak et al., 1995) we did not observe an increase in lamin immunofluorescence, consistent with the presence of an internal lamin-nuclear matrix, after removal of the chromatin (see Fig. 1e and g).

Fig. 1.

Lamin and DNA distribution of extracted nuclei. Sperm pronuclei were assembled in vitro and extracted to prepare nuclear matrices. Whole nuclei (start) and nuclear matrices (finish) were examined by indirect immunofluorescence microscopy using the lamin B3 specific mAb L6 5D5 (a,c,e,g) and the DNA fluorescent dye DAPI (b,d,f,h). Low power (a-d) and high power micrographs (e-h) are shown. In typical extracts, the size of a nucleus will vary from 12-30 nm diameter (Hutchison, 1995). The difference in size between the nuclei in e and g reflects this variation. Bar, 15 µm.

Fig. 1.

Lamin and DNA distribution of extracted nuclei. Sperm pronuclei were assembled in vitro and extracted to prepare nuclear matrices. Whole nuclei (start) and nuclear matrices (finish) were examined by indirect immunofluorescence microscopy using the lamin B3 specific mAb L6 5D5 (a,c,e,g) and the DNA fluorescent dye DAPI (b,d,f,h). Low power (a-d) and high power micrographs (e-h) are shown. In typical extracts, the size of a nucleus will vary from 12-30 nm diameter (Hutchison, 1995). The difference in size between the nuclei in e and g reflects this variation. Bar, 15 µm.

To confirm that the lamina was resistant to extraction the same procedure was performed on nuclei in suspension. At each step 2 ×105 nuclei were recovered by centrifugation and prepared for SDS-PAGE. Fig. 2 shows Coomassie stained gels and immunoblots which compare total protein and lamin B3 at the start and finish of the extraction procedure. While several protein species are lost during extraction (compare lanes 1 and 3), the amount of lamin B3 in the final nuclear matrix fraction (lane 4) is comparable to the amount in whole nuclei (lane 2). Thus, while chromatin associated proteins were efficiently solubilised during the extraction procedure, lamin B3 remained insoluble.

Fig. 2.

Lamin B3 is retained in extracted nuclei. Sperm pronuclei assembled in vitro were subjected to sequential extraction as described in Materials and Methods. Samples of whole nuclei (start) and fully extracted nuclei (finish) equivalent to 2×105 nuclei were prepared for SDS-PAGE and immunoblotting. Lane 1 reveals the protein profile of whole nuclei, resolved by 10% SDS-PAGE and stained with Coomassie brilliant blue. Lane 2 shows an immunoblot of the same material probed with the anti-lamin B3 antibody L6 5D5. Lane 3 reveals the protein profile of nuclear matrices, resolved by 10% SDS-PAGE and stained with Coomassie brilliant blue. Lane 4 shows an immunoblot of the same material probed with the antilamin B? antibody L6 5D5. Molecular mass markers are 80 kDa, 49.5 kDa and 32.5 kDa.

Fig. 2.

Lamin B3 is retained in extracted nuclei. Sperm pronuclei assembled in vitro were subjected to sequential extraction as described in Materials and Methods. Samples of whole nuclei (start) and fully extracted nuclei (finish) equivalent to 2×105 nuclei were prepared for SDS-PAGE and immunoblotting. Lane 1 reveals the protein profile of whole nuclei, resolved by 10% SDS-PAGE and stained with Coomassie brilliant blue. Lane 2 shows an immunoblot of the same material probed with the anti-lamin B3 antibody L6 5D5. Lane 3 reveals the protein profile of nuclear matrices, resolved by 10% SDS-PAGE and stained with Coomassie brilliant blue. Lane 4 shows an immunoblot of the same material probed with the antilamin B? antibody L6 5D5. Molecular mass markers are 80 kDa, 49.5 kDa and 32.5 kDa.

Nuclear matrix organisation in sperm pronuclei is similar to that observed in somatic cells

To date the morphology of nuclear matrix/nucleoskeletons has only been investigated in a variety of higher eukaryotes including plants. However, such preparations have never been described in amphibian embryos. Since the size, lamin content and distribution of chromatin is distinctly different in embryonic nuclei compared to somatic nuclei, we wished to determine the general morphology of nuclear matrices prepared from sperm pronuclei assembled in vitro. Nuclear matrices prepared from in vitro assembled nuclei were embedded in DGD resin, sectioned and viewed by TEM. A typical section is illustrated in Fig. 3. The boundary of the nucleus is formed by a dense array of filaments which correspond to the position of the lamina (L). Fine branched filaments having an average diameter of 13 nm were also observed within the body of the nucleus. These filaments are referred to below as internal nuclear matrix filaments (Nm). The similar-ity between the morphology of nuclear matrix filaments shown and those reported elsewhere (Jackson and Cook, 1988; He et al., 1990) is striking and indicates that despite several general differences in the organisation of embryonic and somatic nuclei, the organisation of the nuclear matrix is identical.

Fig. 3.

DGD section of nuclear matrix. Sperm pronuclei were extracted through five sequential steps, fixed and embedded in DGD resin. After sectioning, the resin was disolved and the sample viewed directly by TEM using a Joel 1200 transmission electron microscope. L shows the position of the nuclear lamina, Nm shows the nuclear matrix. Bar, 500 nm.

Fig. 3.

DGD section of nuclear matrix. Sperm pronuclei were extracted through five sequential steps, fixed and embedded in DGD resin. After sectioning, the resin was disolved and the sample viewed directly by TEM using a Joel 1200 transmission electron microscope. L shows the position of the nuclear lamina, Nm shows the nuclear matrix. Bar, 500 nm.

Insoluble lamin B3 is part of a filamentous network in nuclear matrix preparations

To investigate the composition and organisation of different filaments, whole mount nuclear matrices were viewed by field emission in lens scanning electron microscopy (FEISEM). Whole mounts were preferred because some structures are not easily observed in DGD sections. In particular nuclear pores are not easily identified. Since we wished to distinguish between pore-connecting filaments (the lamina) and internal nuclear matrix filaments, FEISEM seemed to offer greater potential. In addition, FEISEM can also be used to collect images at different levels in a whole mount preparation. Fig. 4a shows the surface of a typical nuclear matrix preparation.

Fig. 4.

Whole-mount FEISEM reveals the structures of the nuclear lamina and pore complexes. The surface of an extracted sperm pronucleus is illustrated at low power (a) and at higher power (b and c). The arrows show structures resembling nuclear pores which appear at high density within this area. The nuclear pores are interconnected by 1013 nm filaments which were readily labelled with anti-lamin B? mAb L6 5D5. Gold labelling of 10-13 nm filaments is visualised in backscattered images (e). As a control similar preparations were also labelled with human Ki67 antibody (d). Bars, 200 nm.

Fig. 4.

Whole-mount FEISEM reveals the structures of the nuclear lamina and pore complexes. The surface of an extracted sperm pronucleus is illustrated at low power (a) and at higher power (b and c). The arrows show structures resembling nuclear pores which appear at high density within this area. The nuclear pores are interconnected by 1013 nm filaments which were readily labelled with anti-lamin B? mAb L6 5D5. Gold labelling of 10-13 nm filaments is visualised in backscattered images (e). As a control similar preparations were also labelled with human Ki67 antibody (d). Bars, 200 nm.

A high density of structures resembling nuclear pores (arrows) was retained in these preparations. Each pore was connected by a minimum of two and a maximum of eight filaments of diameter 10-13 nm. The association of nuclear pores with 1013 nm diameter filaments is illustrated in detail in Fig. 4b and c). The appearance of parallel filaments extending from the pores is a common feature of these preparations. Nuclear matrix preparations which had been gold labelled using mAb L6 5D5 were also viewed with FIESEM by using a backscat-tered image. This technique revealed densely labelled filaments, again at the surface of the preparation (Fig. 4e) indicating that lamin B3 is a major component of the pore connecting filaments. As a control for specificity, nuclear matrix preparations were also immunolabelled with an irrelevent monoclonal antibody (Human Ki67) (Fig. 4d). In contrast to the ‘blank’ negative images used in immunolabelling controls in immunofluorescence microscopy, the use of backscattered electron (BSE) images in SEM still show the underlying features of interest, albeit at the reduced levels of signal inherent in this method. A strong BSE signal is generated by elements of high atomic number, and colloidal gold will clearly give a much stronger BSE signal than the underlying biological material. Thus in Fig. 4e gold is imaged as bright dots, overlying ‘reduced’ imaging from the filaments. In the absence of gold the BSE signal in Fig. 4d can still be optimised to show the filament, but they clearly lack any colloidal gold over their surface.

While a network of pore-connecting lamin B3 filaments was observed in our preparations, these filaments did not appear to form a clearly defined basket weave pattern. To investigate the effects of extraction on lamina morphology, we also viewed Xenopus oocyte germinal vesicle (GV) laminae with FEISEM. Oocyte GVs were manually dissected, spread on a silicon chip and extracted with Triton X-100 as done previously (Goldberg and Allen, 1992). The resulting pore complex-lamina preparations were then gold labelled using mAb L6 5D5. Typical structures viewed under FIESEM are illustrated in Fig. 5. In these preparations a much higher density of nuclear pores was observed (Fig. 5a). The nuclear pores were interconnected by 13 nm fibres which formed a branched filamentous network. These filaments were gold labelled in preparations which were incubated with mAb L6 5D5 (Fig. 5b) but not with irrelevant antibodies (Fig. 5a). While the filaments in these preparations were highly branched, a tightly organised square lattice was not observed. Instead the network was similar to the one observed in nuclear matrix preparations. It is quite likely that Triton solubilisation of the nuclear envelope stretches the lamin filaments thus altering their morphology or that extraction of the membrane exposes hydrophobic regions of lamina filaments which aggregate. However, the additional steps required to prepare nuclear matrices do not appear to cause greater disruption than Triton solubilisation of oocyte germinal vesicle envelopes.

Fig. 5.

Whole-mount FEISEM reveals the nuclear lamina and pore structures of germinal vesicles manually isolated from Xenopus oocytes. Isolated germinal vesicle envelopes were spread on silicon chips, treated with Triton X-100, and then processed for immuno-gold labelling using mAb L6 5D5 (b) or an irrelevant antibody (a). The nucleoplasmic face of the pore complex-lamina is shown. Arrows indicate the pore baskets while arrowheads indicate the antibody-gold particles which are situated on the lamina filaments. Bar, 100 nm.

Fig. 5.

Whole-mount FEISEM reveals the nuclear lamina and pore structures of germinal vesicles manually isolated from Xenopus oocytes. Isolated germinal vesicle envelopes were spread on silicon chips, treated with Triton X-100, and then processed for immuno-gold labelling using mAb L6 5D5 (b) or an irrelevant antibody (a). The nucleoplasmic face of the pore complex-lamina is shown. Arrows indicate the pore baskets while arrowheads indicate the antibody-gold particles which are situated on the lamina filaments. Bar, 100 nm.

Other filaments within nuclear matrix preparations

While filaments at the surface of nuclear matrix preparations were extensively labelled with mAb L6 5D5, other filaments, lying in the centre of typical preparations, were unlabelled. These filaments were easily distinguished from filaments lying at the surface of nuclear matrix preparations because structures resembling nuclear pores were absent. The morphology of filaments found in the centre of the nuclear matrix is illustrated in Fig. 6a. These filaments vary in diameter and in some areas the filaments appear to be aggregated (arrow). Nevertheless smooth branched filaments having a diameter of 10-13 nm are clearly visible. Moreover, the filaments extended throughout the nucleoplasm. Based upon the absence of nuclear pores and their proliferation through the nucleoplasm, we concluded that the filaments illustrated in Fig. 6 corresponded to filaments of the internal nuclear matrix (see Fig. 3). Consistent with this hypothesis, immunostaining with anti-lamin B3 mAbs (Fig. 3c) was indistiguishable from immunostaining with irrelevent antibodies (Fig. 3c with Fig. 4d) indicating that the filaments proliferating through the nucleoplasm were not lamina filaments.

Fig. 6.

Whole-mount FEISEM reveals the structure of the nuclear matrix. (a) Filaments not associated with nuclear pores and proliferating throughout extracted sperm pronuclei. (b) The organisation of filaments in extracted sperm pronuclei assembled in lamin B3 depleted extracts. Arrows (a) and arrowheads (b) show aggregated filaments. (c) A backscattered image of internal filaments after antibody labelling with mAb L6 5D5 followed by 10 nm gold conjugated secondary antibodies. Bars, 500 nm.

Fig. 6.

Whole-mount FEISEM reveals the structure of the nuclear matrix. (a) Filaments not associated with nuclear pores and proliferating throughout extracted sperm pronuclei. (b) The organisation of filaments in extracted sperm pronuclei assembled in lamin B3 depleted extracts. Arrows (a) and arrowheads (b) show aggregated filaments. (c) A backscattered image of internal filaments after antibody labelling with mAb L6 5D5 followed by 10 nm gold conjugated secondary antibodies. Bars, 500 nm.

Sperm pronuclei assembled in lamin B3 depleted extracts have a disrupted nuclear matrix

Nuclei assembled in lamin B3 depleted extracts do not possess a lamina, are unable to accumulate PCNA in replication centres and fail to initiate semi-conservative DNA replication (Jenkins et al., 1993). We have recently proposed that the lamina provides a surface upon which nuclear matrix filaments are assembled (Hutchison et al., 1994). The corollary of this hypothesis is that nuclei which do not possess a lamina cannot assemble a well organised nuclear matrix and thus do not initiate DNA replication. Since the investigations described above indicate the presence of lamin B3 and non-lamin B3 containing filaments within nuclear matrix preparations, we wished to investigate the morphology of the nuclear matrix filaments in nuclei assembled in lamin B3 depleted extracts. Lamin B3 depletion of egg extracts was performed as described previously (Jenkins et al., 1993; Goldberg et al., 1995). To confirm that the depletion was successful, western blotting was performed on depleted and mock depleted extracts. Mock depleted extracts contained lamin B3 which was detected as an 80 kDa band by 8% SDS-PAGE (Fig. 7, lane 6). In contrast no lamin B3 was detected in depleted extracts (Fig. 7 lane 5) but instead was recovered in each Dynabead fraction (Fig. 7, lanes 1 and 2). As expected sperm pronuclei assembled in lamin B3 depleted extracts were small (<10 nm diameter) lacked a lamina and failed to initiate DNA replication (Fig. 7d-f). Sperm pronuclei assembled in mock-depleted extracts (Fig. 7a-c) or depleted extracts which were supplemented with purified lamin B3 (Fig. 7g-i) were on the other hand large (>20 nm diameter) possessed a well formed lamina and supported DNA replication.

Fig. 7.

Sperm pronuclear assembly in lamin B? depleted extracts. Extracts were depleted of lamin B? as described in Materials and Methods. Samples (1 µl) of depleted (lane 6) and mock depleted (lane 5) extract were resolved by 8% SDS-PAGE along with 10 µl samples of each Dynabead fraction. Fully resolved proteins were transferred to nitrocellulose and probed with mAb L6 5D5 followed by alkaline phosphatase conjugated secondary antibodies. Lanes 1 and 2 contain L6 5D5 Dynabeads, lanes 3 and 4 contain control Dynabeads. Molecular mass markers are 109 kDa, 80 kDa and 49.5 kDa. Sperm pronuclei were assembled in mock depleted extracts (a-c), lamin B3 depleted extracts (d-f) and lamin B3 depleted extracts to which purified lamin B3 was readded (g-i). The extracts were supplemented with biotin-16-dUTP to reveal DNA replication. After incubation for 3 hours at 21°C sperm pronuclei were recovered on glass coverslips and immunolabelled with mAb L6 5D5 followed by FITC-conjugated secondary antibody to reveal the distribution of lamins. The samples were counter-stained with DAPI to reveal the distribution of DNA and Texas red streptavidin to reveal biotin incorporation. Images were acquired using a Hamamatsu CCD camera using Fluovision software (Improvision). Montages were assembled in Adobe Photoshop 3.0. Bars, 20 nm.

Fig. 7.

Sperm pronuclear assembly in lamin B? depleted extracts. Extracts were depleted of lamin B? as described in Materials and Methods. Samples (1 µl) of depleted (lane 6) and mock depleted (lane 5) extract were resolved by 8% SDS-PAGE along with 10 µl samples of each Dynabead fraction. Fully resolved proteins were transferred to nitrocellulose and probed with mAb L6 5D5 followed by alkaline phosphatase conjugated secondary antibodies. Lanes 1 and 2 contain L6 5D5 Dynabeads, lanes 3 and 4 contain control Dynabeads. Molecular mass markers are 109 kDa, 80 kDa and 49.5 kDa. Sperm pronuclei were assembled in mock depleted extracts (a-c), lamin B3 depleted extracts (d-f) and lamin B3 depleted extracts to which purified lamin B3 was readded (g-i). The extracts were supplemented with biotin-16-dUTP to reveal DNA replication. After incubation for 3 hours at 21°C sperm pronuclei were recovered on glass coverslips and immunolabelled with mAb L6 5D5 followed by FITC-conjugated secondary antibody to reveal the distribution of lamins. The samples were counter-stained with DAPI to reveal the distribution of DNA and Texas red streptavidin to reveal biotin incorporation. Images were acquired using a Hamamatsu CCD camera using Fluovision software (Improvision). Montages were assembled in Adobe Photoshop 3.0. Bars, 20 nm.

Sperm pronuclei assembled in lamin B3 depleted extracts were recovered on glass coverslips, extracted to prepare nuclear matrices and viewed with FEISEM. A typical example is illustrated in Fig. 6b. In nuclear matrices prepared from nuclei which ‘lack a lamina’, structures resembling nuclear pores were hard to identify. In addition, while branched filamentous structures were observed throughout the preparations, these were not smooth and often appeared to aggregate (arrowheads). Moreover, in many areas large gaps were seen between filaments. Nevertheless, the diameter of the finest filaments was still in the range 10-13 nm. In contrast to the appearance of nuclear matrix filaments in pronuclei assembled in lamin B3 depleted extracts, nuclear matrix filaments in sperm pronuclei assembled in mock depleted extracts were indistinguishable from the networks shown in Fig. 6a (not shown).

Investigation of nuclear matrices using whole mount TEM

The results of investigations using FEISEM indicated the presence of two types of filament in nuclear matrices prepared from sperm pronuclei. Pore-connecting filaments containing lamin B3 and internal nuclear matrix filaments. Consistent with this finding, nuclear matrices prepared from ‘nuclei which lack a lamina’ possess poorly organised filaments and lack easily identifiable pore structures and pore-connecting filaments. Using FEISEM, immunogold labelling is best seen using a back-scattered image. Therefore, in micrographs such as those seen in Fig. 6, in the absence of labelling the underlying filament network is always visible albeit without any associated gold. We wished to confirm the absence of lamin B3 from the filaments observed in ‘nuclei which lack a lamina’ using a second technique. We therefore performed immunogold labelling on whole mount nuclear matrices prepared for TEM. Positively stained nuclear matrices, prepapared from control nuclei, were spread on nickel grids and viewed at 80 kV accelerating voltage. Using this procedure nuclear pores could not be identified with confidence. However, highly branched continuous networks of filaments were readily observed (Fig. 8a). The diameter of the finest filaments was within the range 1013 nm. However, other filaments were much thicker, probably due to aggregation of smaller filaments. Many (but not all) of the filaments were labelled with 10 nm gold particles following incubation with anti-lamin B3 antibodies. The gold particles often appeared in clusters, with up to 10 particles in a cluster (arrows -Fig. 8a and shown in detail in Fig. 8b). In addition some filaments were lined with gold particles (Fig. 8b and c). Similar preparations stained with human Ki67 antibody had only a low concentration of gold particles (Fig. 8e). The density of gold labelling in L6 5D5 stained material was between 5 and 15 times higher than in Ki67 stained material (compare Fig. 8b and c with e). The absence of gold particles from certain areas of the network in L6 5D5 stained preparations (Fig. 8a) is consistent with the hypothesis that two types of filaments are present in the nuclear matrix. Namely, lamina filaments and internal nuclear matrix filaments.

Fig. 8.

Whole-mount TEM reveals the structure of the nuclear lamina and the nuclear matrix. (a) Extracted sperm pronuclei were gold labelled using mAb L6 5D5 and viewed as whole mounts by TEM. (b and c) Detailed views of selected areas of a. (d) Extracted sperm pronuclei assembled in lamin B3 depleted egg extract and immuno-gold labelled with L6 5D5. (e) Extracted sperm pronuclei which had been gold labelled using Human Ki67 mAb. Arrows indicate the highest density of gold labelling in each of micrographs a,d and e. Bars, 500 nm.

Fig. 8.

Whole-mount TEM reveals the structure of the nuclear lamina and the nuclear matrix. (a) Extracted sperm pronuclei were gold labelled using mAb L6 5D5 and viewed as whole mounts by TEM. (b and c) Detailed views of selected areas of a. (d) Extracted sperm pronuclei assembled in lamin B3 depleted egg extract and immuno-gold labelled with L6 5D5. (e) Extracted sperm pronuclei which had been gold labelled using Human Ki67 mAb. Arrows indicate the highest density of gold labelling in each of micrographs a,d and e. Bars, 500 nm.

In contrast, continuous networks of filaments were absent from nuclear matrices prepared from ‘nuclei which lack a lamina’. Instead, the filaments appeared to have collapsed into dense aggregates (Fig. 8d). Following incubation with mAb L6 5D5, gold particles were observed in these preparations. However, the density of labelling was greatly reduced (in the preparation shown in Fig. 8c, which is typical, control nuclear matrices contained 5-10 times as many gold particles compared to a similar area in nuclear matrices prepared from ‘nuclei which lack a lamina’) and clusters of gold particles were never seen. Moreover, the density of gold labelling in nuclear matrices stained with irrelevant antibodies was similar to that observed in ‘nuclei which lack a lamina’(Fig. 8e).

Nuclear matrix organisation

Complex branched filamentous networks were first described within the nucleus by Berezney and Coffey (1974, 1977). Since these pioneering studies other groups using completely different extraction procedures have also observed similar structures in a range of mammalian cells (Fey et al., 1986; Nickerson et al., 1989; Jackson and Cook, 1988). The general conclusion derived from these studies is that the nuclear matrix consists of 10 nm filaments which are morphologically indistinguishable from intermediate filaments (He et al., 1990; Jackson and Cook, 1988). However, the protein components of these filaments is disputed. The nuclear lamina forms a fibrillar meshwork lying subjacent to the nuclear envelope (Aebi et al., 1986). The major protein components of the lamina are lamins (Gerace et al., 1978) which have now been characterised as type V intermediate filament proteins (Fisher et al., 1986; McKeon et al., 1986). In most EM preparations the lamina interconnects nuclear pores (e.g. Goldberg and Allen, 1992) and directly interacts with filaments which extend throughout the nucleoplasmic space (He et al., 1990; Jackson and Cook, 1988). The filaments which extend into the nucleo-plasmic space are variously termed core filaments of the nuclear matrix (He et al., 1990), the diffuse nucleoskeleton (Hozak et al., 1995) or the internal matrix (Fey et al., 1986). It has recently been reported that these filaments are labelled with anti-lamin antibodies and therefore are partially or totally assembled from lamins (Hozak et al., 1995). However, other candidate nuclear matrix proteins which have the potential to form filaments have been characterized. In particular NuMa is a nuclear matrix protein (Lydersen and Pettijohn, 1980) which possesses an extended alpha-helical domain (Yang et al., 1992; Compton et al., 1992) and is capable of forming in-register double-stranded coiled-coil dimers in vitro (Harborth et al., 1995).

In the study described above we have adapted the procedure of Fey et al. (1986) to investigate nuclear matrix organisation in sperm pronuclei assembled in vitro. Using TEM and FEISEM we observed an extensive array of nuclear filaments in whole mount preparations and DGD sections. At the surface of the nuclear matrices, 10-13 nm filaments which interconnected nuclear pores were clearly visible. These filaments were similar in morphology to lamina filaments observed in Triton X-100 treated isolated oocyte germinal vesicles. Moreover, filaments from both preparations were extensively labelled with 10 nm gold particles following incubation with the antilamin B3 mAb L6 5D5. Curiously, this is the first direct evidence that lamin polypeptides are components of lamina filaments.

Other filaments extended throughout the nucleoplasmic space. Although we have used a different preparative technique and different nuclei, the internal nuclear matrix filaments we observed were very similar to the core filaments of the nuclear matrix described in MCF-7 cells (He et al., 1990). Unlike the filaments which interconnected nuclear pores, the internal nuclear matrix filaments were not labelled with 10 nm gold particles following incubation with mAb L6 5D5. Since we obtain extensive labelling of some (but not all) filaments using mAb L6 5D5 we conclude that in sperm pronuclei the nuclear matrix consists of at least two distinct types of filament. The first type of filament is assembled from lamin B3 and is situated at the nuclear periphery. The second type of filament we term internal nuclear matrix filaments and is equivalent to core filaments of the nuclear matrix (He et al., 1990). Consistent with this view, nuclear matrix filaments are still detected in sperm pronuclei assembled in extracts depleted of lamin B3. These filaments are not organised into an integrated network. Moreover, no significant labelling of these filaments is obtained using mAb L6 5D5. In a previous investigation we reported that sperm pronuclei assembled in lamin depleted extracts still possess a nuclear matrix which was characterised by 2-dimensional gel electrophoresis (Jenkins et al., 1993). Our new data confirm and extend this finding. Moreover, these data imply that while lamins are necessary for the correct organisation of a nuclear matrix, the lamina is distinct from internal nuclear matrix filaments. Our observations that poorly organised filaments and characteristic insoluble protein subsets (Jenkins et al., 1993) are still observed in nuclei which ‘lack a lamina’ implies that ‘internal nuclear matrix filaments’ form from proteins which have self-assembly properties that are independent of lamin B3.

Interdependence between the lamina and core filaments of the nuclear matrix in the assembly of a replication competent nucleus

The major lamin isoform in Xenopus eggs has been classified as lamin B3 (Döring and Stick, 1990). Like most B-type lamins, lamin B3 is stably isoprenylated and methylated at its carboxyl terminal cysteine residue (Firmbach-Kraft and Stick, 1993). However, >95% of the protein becomes soluble at mitosis (Firmbach-Kraft and Stick, 1993). Because of this lamin B3 can be readily immunodepleted from Xenopus egg extracts (Newport et al., 1990). Surprisingly, nuclei are still assembled in extracts depleted of lamin B3 but these nuclei are unable to support semi-conservative DNA replication (Newport et al., 1990; Meier et al., 1991; Jenkins et al., 1993). While nuclei assembled in lamin B3 depleted extracts lack a lamina (Jenkins et al., 1993) they possess well formed nuclear pores (Jenkins et al., 1995; Goldberg et al., 1995) and are able to support active nuclear transport (Jenkins et al., 1993). Moreover, a range of proteins which have been characterised as part of the nuclear matrix are still resistent to DNase/salt extraction in nuclei which ‘lack a lamina’. Despite this, proteins such as PCNA are unable to form stable associations with replication centres (Meier et al., 1991; Jenkins et al., 1993). Based upon these observations we proposed that the correct assembly of an internal nuclear matrix was dependent upon the prior assembly of a lamina (Hutchison et al., 1994).

The present investigation confirms the finding that a nuclear matrix is still assembled in nuclei which ‘lack a lamina’. However, this nuclear matrix has poorly organised filaments which appear to collapse onto each other. Thus the hypothesis that nuclear matrix assembly is dependant upon nuclear lamina assembly is only partially correct. Nuclear proteins which assemble into filaments must remain in Xenopus egg extracts depleted of lamin B3. Thus the assembly properties of these filaments are not dependent upon the presence of lamin B3. However, the formation of a stable and integrated network of filaments is apparently prevented. Therefore it is the stability of this network which must be required for the initiation of semi-conservative DNA replication.

Why should a stable and integrated network of nuclear filaments permit DNA replication? One possible explanation is that the nuclear matrix stabilizes nuclear form by acting as a tensile element within a tensegrity array (Ingber, 1993). The tensegrity model predicts that the load resisting properties of the nucleus, and hence its mechanical stability would be dependent upon two types of interconnected support elements: compression-resistant struts and a continuous series of tensile filaments. It has been proposed that in the nucleus, individual chromatin loops represent local compression-resistant elements that are pulled up and open through interconnection with the continuous tensile nuclear matrix network (Ingber, 1993). One possible consequence of removal of tensional continuity (in this instance lamina integrity) would be collapse and aggregation of the normally separated compression elements (the chromatin loops). Interestingly, indirect experimental evidence suggests that it is necessary to maintain chromatin in a relatively extended form to initiate DNA synthesis. For example, nuclear expansion has been shown to precede entrance into S-phase in many cell types (Yen and Pardee, 1979; Nicolini et al., 1986; Ingber et al., 1987). Also, when cells growing in culture are forced to round up by treatment with trypsin/EDTA, synthetic RDG peptides or exposure to high concentrations of ATP, both nuclear retraction (Sims et al., 1992) and inhibition of DNA replication result (Ingber, 1990). Intriguingly, nuclei which are re-swollen following injection of heparin can be re-induced to synthesise DNA (Pienta and Coffey, 1992). The most characteristic features of self-assembled nuclei which ‘lack a lamina’ in our study also were their small size and their inability to initiate semi-conservative DNA replication (Jenkins et al., 1993; Jenkins et al., 1995; Goldberg et al., 1995).

The investigations described above indicate that the nuclear matrix consists of two distinct elements. A lamin B3 containing lamina which interconnects nuclear pores and an internal nuclear matrix which has assembly properties which are independant of the lamina. We propose that both filamentous networks are required to provide tensional integrity within the nucleus and therefore to support DNA replication.

The authors are indebted to Prof. Birgitte Lane and Prof. Donald Ingber for critical reading of the manuscript. This work was supported by a Wellcome Trust Travelling Research Fellowship to C.Z. and by the Cancer Research Campaign.

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