B-type lamins in health and disease.

For over two decades, B-type lamins were thought to have roles in fundamental processes including correct assembly of nuclear envelopes, DNA replication, transcription and cell survival. Recent studies have questioned these roles and have instead emphasised the role of these proteins in tissue building and tissue integrity, particularly in tissues devoid of A-type lamins. Other studies have suggested that the expression of B-type lamins in somatic cells influences the rate of entry into states of cellular senescence. In humans duplication of the LMNB1 gene (encoding lamin B1) causes an adult onset neurodegenerative disorder, termed autosomal dominant leukodystrophy, whilst very recently, LMNB1 has been implicated as a susceptibility gene in neural tube defects. This is consistent with studies in mice that reveal a critical role for B-type lamins in neuronal migration and brain development. In this review, I will consider how different model systems have contributed to our understanding of the functions of B-type lamins and which of those functions are critical for human health and disease.

Nuclear lamins: laminopathies and their role in premature ageing.

It has been demonstrated that nuclear lamins are important proteins in maintaining cellular as well as nuclear integrity, and in maintaining chromatin organization in the nucleus. Moreover, there is growing evidence that lamins play a prominent role in transcriptional control. The family of laminopathies is a fast-growing group of diseases caused by abnormalities in the structure or processing of the lamin A/C (LMNA) gene. Mutations or incorrect processing cause more than a dozen different inherited diseases, ranging from striated muscular diseases, via fat- and peripheral nerve cell diseases, to progeria. This broad spectrum of diseases can only be explained if the responsible A-type lamin proteins perform multiple functions in normal cells. This review gives an overview of current knowledge on lamin structure and function and all known diseases associated with LMNA abnormalities. Based on the knowledge of the different functions of A-type lamins and associated proteins, explanations for the observed phenotypes are postulated. It is concluded that lamins seem to be key players in, among others, controlling the process of cellular ageing, since disturbance in lamin protein structure gives rise to several forms of premature ageing.

A-type lamins are essential for TGF-beta1 induced PP2A to dephosphorylate transcription factors.

Diseases caused by mutations in lamins A and C (laminopathies) suggest a crucial role for A-type lamins in different cellular processes. Laminopathies mostly affect tissues of mesenchymal origin. As transforming growth factor-beta1 (TGF-beta1) signalling impinges on the retinoblastoma protein (pRB) and SMADs, we tested the hypothesis that lamins modulate cellular responses to TGF-beta1 signalling, via the regulation of these transcription factors in mesenchymal cells. Here, we report that A-type lamins are essential for the inhibition of fibroblast proliferation by TGF-beta1. TGF-beta1 dephosphorylated pRB through PP2A, both of which, we show, are associated with lamin A/C. In addition, lamin A/C modulates the effect of TGF-beta1 on collagen production, a marker of mesenchymal differentiation. Our findings implicate lamin A/C in control of gene activity downstream of TGF-beta1, via nuclear phosphatases such as PP2A. This biological function provides a novel explanation for the observed mesenchymal dysfunction in laminopathies.

The lethal phenotype of a homozygous nonsense mutation in the lamin A/C gene.

The authors report the clinical and histologic phenotypes of a LGMD1B family including a newborn child with a homozygous LMNA nonsense mutation (Y259X). At the heterozygous state the nonsense mutation leads to a classic LGMD1B phenotype; the homozygous LMNA nonsense mutation causes a lethal phenotype.

Lamin expression in normal human skin, actinic keratosis, squamous cell carcinoma and basal cell carcinoma.

BACKGROUND: Aberrant expression patterns of nuclear lamins have been described in various types of cancer depending on the subtype of cancer, its aggressiveness, proliferative capacity and degree of differentiation. In general, the expression of A-type lamins (lamins A and C) has been correlated with a non-proliferating, differentiated state of cells and tissues. OBJECTIVES: To establish and compare the expression patterns of lamins in normal human skin, actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). METHODS: Expression patterns of the individual lamin subtypes were studied immunohistochemically. The proliferation capacity of the tumour cells was detected using a specific antibody to Ki-67, and was related to the A-type lamin expression patterns. RESULTS: In normal skin, lamin A was expressed in the suprabasal cell compartment of the epidermis, whereas the basal cells were mostly unstained. BCCs and SCCs stained positive in most cells, while the epidermis overlying BCC and SCC and the epidermis in AK stained homogeneously and strongly in the basal cells in addition to the suprabasal cells. Lamin C was expressed in some basal cells of normal epidermis while the suprabasal cells stained strongly positive. Both BCCs and SCCs stained strongly positive for lamin C, with the difference that in BCC the staining was predominantly present in nucleolar structures with occasional staining of the nuclear envelope. The epidermis overlying SCC showed strong positivity in the lamina of virtually all cells. The expression of lamin C in the basal cells of AK resembled the expression pattern seen in the epidermis overlying BCC, i.e. a nucleolar staining next to nuclear envelope staining. Lamin B1 and B2 were found in virtually all cells in normal epidermis, AK, BCC, SCC and the epidermis overlying cancer. The percentage of Ki-67-expressing cells was highest in BCC (45%), and gradually decreased via epidermis overlying BCC, AK, SCC, and epidermis overlying SCC, to normal skin (11%). Simultaneous expression of A-type lamins and Ki-67 occurred in approximately 50% of the proliferating (Ki-67 positive) cells in BCC and SCC. CONCLUSIONS: Significant changes occur in the expression patterns of A-type lamins in both premalignant and malignant lesions of the skin. The profound overlap of lamin A and Ki-67 staining patterns indicates that the proliferating tumour cells may obtain a certain degree of differentiation. Finally, lamin A expression in the basal cell layer of the apparently normal epidermis overlying BCC may suggest its involvement in the primary process.

Both emerin and lamin C depend on lamin A for localization at the nuclear envelope.

Physical interactions between lamins and emerin were investigated by co-immunoprecipitation of in vitro translated proteins. Emerin interacted with in vitro translated lamins A, B1 and C in co-immunprecipitation reactions. Competition reactions revealed a clear preference for interactions between emerin and lamin C. Structural associations between lamins and emerin were investigated in four human cell lines displaying abnormal expression and/or localisation of lamins A and C. In each cell line absence of lamins A and C from the nuclear envelope (NE) was correlated with mis-localisation of endogenous and exogenous emerin to the ER. In two cell lines that did not express lamin A but did express lamin C, lamin C as well as emerin was mis-localised. When GFP-lamin A was expressed in SW13 cells (which normally express only very low levels of endogenous lamin A and mis-localise endogenous emerin and lamin C), all three proteins became associated with the NE. When GFP-lamin C was expressed in SW13 cells neither the endogenous nor the exogenous lamin C was localised to the NE and emerin remained in the ER. Finally, lamins A and C were selectively eliminated from the NE of HeLa cells using a dominant negative mutant of lamin B1. Elimination of these lamins from the lamina led to the accumulation of emerin as aggregates within the ER. Our data suggest that lamin A is essential for anchorage of emerin to the inner nuclear membrane and of lamin C to the lamina.

Expression of individual lamins in basal cell carcinomas of the skin.

In this study we used a unique collection of type specific anti-lamin antibodies to study lamin expression patterns in normal human skin and in skin derived from patients with basal cell carcinomas (BCCs). Lamin expression in serial sections from frozen tissue samples was investigated by single and double indirect immunofluorescence. In normal skin, lamin A was expressed in dermal fibroblasts and in suprabasal epithelial cells but was absent from all basal epithelial cells. Lamin C was expressed in dermal fibroblasts, suprabasal epithelial cells and a majority of basal epithelial cells. However, lamin C was not expressed in quiescent basal epithelial cells. Lamin B1 was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2 was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2 was expressed in all cell types in normal skin. Lamin expression was also investigated in a collection of 16 BCCs taken from a variety of body sites. Based upon patterns of lamin expression the BCCs were classified into four groups: A-negative (10/16 tumours), C-negative (5/16 tumours), A/C-negative (1/16 tumours) and A/B2-negative (1/16 tumours). Lamin expression was also compared to cell proliferation index by staining serial sections with the proliferation marker Ki67. 9/10 of the lamin A negative tumours were highly proliferative, whereas 4/5 of the lamin C negative tumours were slow growing. Thus as a general rule absence of lamin A was correlated with rapid growth within the tumour, while absence of lamin C was correlated with slow growth within the tumour. Our data supports the hypothesis that lamin A has a negative influence on cell proliferation and its down regulation may be a requisite of tumour progression.

Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes?

The nuclear lamina is a filamentous structure composed of lamins that supports the inner nuclear membrane. Several integral membrane proteins including emerin, LBR, LAP1 and LAP2 bind to nuclear lamins in vitro and can influence lamin function and dynamics in vivo. Results from various studies suggest that lamins function in DNA replication and nuclear envelope assembly and determine the size and shape of the nuclear envelope. In addition, lamins also bind chromatin and certain DNA sequences, and might influence chromosome position. Recent evidence has revealed that mutations in A-type lamins give rise to a range of rare, but dominant, genetic disorders, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction-system disease and Dunnigan-type familial partial lipodystrophy. An examination of how lamins A/C, emerin and other integral membrane proteins interact at the INM provides the basis for a novel model for how mutations that promote disease phenotypes are likely to influence these interactions and therefore cause cellular pathology through a combination of weakness of the lamina or altered gene expression.

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells.

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.

Lamina-associated polypeptide 2alpha binds intranuclear A-type lamins.

The nucleoskeletal protein lamina-associated polypeptide 2(&agr;) (LAP2*) contains a large, unique C terminus and differs significantly from its alternatively spliced, mostly membrane-integrated isoforms, such as LAP2beta. Unlike lamin B-binding LAP2beta, LAP2alpha was found by confocal immunofluorescence microscopy to colocalize preferentially with A-type lamins in the newly formed nuclei assembled after mitosis. While only a subfraction of lamins A and C (lamin A/C) was associated with the predominantly nuclear LAP2alpha in telophase, the majority of lamin A/C colocalized with LAP2alpha in G(1)-phase nuclei. Furthermore, selective disruption of A-type lamin structures by overexpression of lamin mutants in HeLa cells caused a redistribution of LAP2alpha. Coimmunoprecipitation experiments revealed that a fraction of lamin A/C formed a stable, SDS-resistant complex with LAP2alpha in interphase cells and in postmetaphase cell extracts. Blot overlay binding studies revealed a direct binding of LAP2alpha to exclusively A-type lamins and located the interaction domains to the C-terminal 78 amino acids of LAP2alpha and to residues 319-566 in lamin A/C, which include the C terminus of the rod and the entire tail common to lamin A/C. These findings suggest that LAP2alpha and A-type lamins cooperate in the organization of internal nuclear structures.

Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs.

In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B(3) specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B(3) co-immunoprecipitate, and lamin B(3) interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB delta 2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.

Investigations of the pathway of incorporation and function of lamin A in the nuclear lamina.

Cell-free extracts of Xenopus eggs were used to investigate how differing lamin sub-types are incorporated into a lamina and how lamina composition influences DNA replication. Purified recombinant human lamin A (HlaminA) was inoculated into egg extracts, which support in vitro nuclear assembly. The route of incorporation of Hlamin A into the lamina was compared to Xenopus lamin B3 (XlaminB3), the major endogenous lamin sub-type in egg extracts. While Xlamin B3 was incorporated into the lamina directly, HlaminA first accumulated at nucleoplasmic foci, before entering the lamina. When HlaminA was inoculated into extracts depleted of XlaminB3 it entered nuclei efficiently and was incorporated into nucleoplasmic foci. However, in the absence of XlaminB3, HlaminA remained in the foci and did not enter the nuclear envelope. When HlaminA entered the nuclear envelope, it did not influence DNA replication. Nuclei containing HlaminA initiated DNA replication on queue and in addition the spatial distribution of replication centres in these nuclei was identical to controls. Taken together, these results suggest that the incorporation of A-type lamins into the nuclear envelope is dependent upon the presence of B-type lamins. However, the presence of A-type lamins alone is not sufficient to influence the spatial and temporal order of DNA replication.

Temporal differences in the appearance of NEP-B78 and an LBR-like protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types.

In this work, we have used novel mAbs against two proteins of the endoplasmic reticulum and outer nuclear membrane, termed NEP-B78 and p65, in addition to a polyclonal antibody against the inner nuclear membrane protein LBR (lamin B receptor), to study the order and dynamics of NE reassembly in the Xenopus cell-free system. Using these reagents, we demonstrate differences in the timing of recruitment of their cognate membrane proteins to the surface of decondensing chromatin in both the cell-free system and XLK-2 cells. We show unequivocally that, in the cell-free system, two functionally and biochemically distinct vesicle types are necessary for NE assembly. We find that the process of distinct vesicle recruitment to chromatin is an ordered one and that NEP-B78 defines a vesicle population involved in the earliest events of reassembly in this system. Finally, we present evidence that NEP-B78 may be required for the targeting of these vesicles to the surface of decondensing chromatin in this system. The results have important implications for the understanding of the mechanisms of nuclear envelope disassembly and reassembly during mitosis and for the development of systems to identify novel molecules that control these processes.

The role of the ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants.

The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. Here, we have investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin alpha and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin beta, Ran binding protein 1 and RCC1, an exchange factor for Ran. We show that Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system.

Cell cycle changes in A-type lamin associations detected in human dermal fibroblasts using monoclonal antibodies.

A new panel of anti-A-type lamin monoclonal antibodies was generated. Epitope mapping was performed by immunoblotting against GST-lamin fusion peptides. Epitopes were mapped to four different regions of human lamin A and three different regions of human lamin C. The distribution of A-type lamins was compared with the distribution of the proliferation marker Ki67 in proliferating and quiescent cultures of human dermal fibroblasts (HDFs) using a double indirect immunofluorescence assay. Antibodies that had been mapped to a region of the lamin C tail stained the nuclear envelope of proliferating and quiescent cells equally brightly. In contrast, antibodies recognizing epitopes in the head domain and rod domain of lamins A and C and the tail domain of lamin A stained the nuclear envelope of quiescent cells strongly but reacted poorly or not at all with the nuclear envelope of proliferating cells. Changes in the level of expression of lamins A and C were not detected in immunoblotting assays. However, epitope masking was revealed, and this occurred by two distinct mechanisms. Epitope masking in the head domain of lamins A and C occurred as a result of protein phosphorylation. Epitope masking in the rod domain of lamins A and C and in the tail domain of lamin A occurred through a physical association between the lamin and chromatin and/or other nuclear proteins. The cell cycle timing of epitope masking was investigated in HDFs that had been restimulated after serum starvation. Extensive epitope masking in restimulated cells only occurred after cells had passed through mitosis. These results are consistent with the hypothesis that rearrangement of A-type lamin filaments, as cells progress from a quiescent to a proliferating state, results in altered lamina associations.

GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication.

A cDNA encoding Xlamin B1 was cloned from a whole ovary mRNA by RT-PCR. GST-lamin fusion constructs were generated from this cDNA by first creating convenient restriction sites within the Xlamin B1 coding sequence, using PCR directed mutagenesis, and then sub-cloning relevant sequences into pGEX-4T-3. Two expression constructs were made, the first, termed delta 2+ lacked sequences encoding the amino-terminal 'head domain' of lamin B1 but included sequences encoding the nuclear localization signal sequence (NLS). The second expression construct, termed delta 2-, lacked sequences encoding the amino-terminal 'head domain' as well as sequences encoding the NLS. Purified fusion proteins expressed from these constructs, when added to egg extracts prior to sperm pronuclear assembly, formed hetero-oligomers with the endogenous lamin B3. The delta 2+ fusion protein prevented nuclear lamina assembly but not nuclear membrane assembly. The resulting nuclei were small (approximately 10 microns in diameter), did not assemble replication centers and failed to initiate DNA replication. When the delta 2- fusion protein was added to egg extracts prior to sperm pronuclear assembly, lamina assembly was delayed but not prevented. The resulting nuclei although small (approximately 12 microns), did form replication centers and initiated DNA replication. When added to egg extracts after sperm pronuclear assembly was completed delta 2+, but not delta 2-, entered the pre-formed nuclei causing lamina disassembly. However, the disassembly of the lamina by delta 2+ did not result in the disruption of replication centers and indeed these centres remained functional. These results are consistent with the hypothesis that lamina assembly precedes and is required for the formation of replication centers but does not support those centers directly.

Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract.

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 amount 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.

S-phase phosphorylation of lamin B2.

Lamin B2 modification in synchronously dividing populations of human diploid fibroblasts was determined by 2-dimensional gel electrophoresis and [32P]orthophosphate labelling. In quiescent (G0) and G1 cultures of HDF, lamin B2 migrated as 2 spots on 2-dimensional gels. In contrast, in S-phase populations of HDF lamin B2 migrated as a single basic species. The level of lamin B2 phosphorylation was determined after immunoisolation from [32P]orthophosphate labelled cells. The results of these experiments indicated a 2-3-fold increase in the steady state level of lamin B2 phosphorylation in S-phase HDF compared with G0 HDF. Consistent with this evidence, tryptic peptide maps revealed the presence of a phosphopeptide in S-phase lamin B2 which was absent from G0 lamin B2. Since all of the phosphate incorporated into S-phase and G0 lamin B2 was recovered in serine residues we conclude that the S-phase specific phosphopeptide did not represent either of the cdc2 sites associated with entry nuclear lamina breakdown.

Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts.

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of 'depleted' extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.