The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells

Recent evidence shows that the COOH-terminal CaaX motif of lamins is necessary to target newly synthesized proteins to the nuclear envelope membranes. Isoprenylation at the CaaX-cysteine has been taken to explain the different fates of A- and B-type lamins during cell division. A-type lamins, which loose their isoprenylation shortly after incorporation into the lamina structure, become freely soluble upon mitotic nuclear envelope breakdown. Somatic B-type lamins, in contrast, are permanently isoprenylated and, although depolymerized during mitosis, remain associated with remnants of nuclear envelope membranes. However, Xenopus lamin B3, the major B-type lamin of amphibian oocytes and eggs, becomes soluble after nuclear envelope breakdown in meiotic metaphase. Here we show that Xenopus lamin B3 is permanently isoprenylated and carboxyl methylated in oocytes (interphase) and eggs (meiotic metaphase). When transfected into mouse L cells Xenopus lamin B3 is integrated into the host lamina and responds to cell cycle signals in a normal fashion. Notably, the ectopically expressed Xenopus lamin does not form heterooligomers with the endogenous lamins as revealed by a coprecipitation experiment with mitotic lamins. In contrast to the situation in amphibian eggs, a significant portion of lamin B3 remains associated with membranes during mitosis. We conclude from these data that the CaaX motif-mediated modifications, although necessary, are not sufficient for a stable association of lamins with membranes and that additional factors are involved in lamin-membrane binding.     

Type B lamins remain associated with the integral nuclear envelope protein p58 during mitosis: implications for nuclear reassembly.

p58 (also referred to as the lamin B receptor) is an integral membrane protein of the nuclear envelope known to form a multimeric complex with the lamins and other nuclear proteins during interphase. To examine the fate of this complex during mitosis, we have investigated the partitioning and the molecular interactions of p58 in dividing chicken hepatoma (DU249) cells. Using confocal microscopy and double immunolabelling, we show here that lamins B1 and B2 co-localize with p58 during all phases of mitosis and co-assemble around reforming nuclei. A close juxtaposition of p58/lamin B-containing vesicles and chromosomes is already detectable in metaphase; however, p58 and lamin reassembly proceeds slowly and is completed in late telophase--G1. Flotation of mitotic membranes in sucrose density gradients and analysis of mitotic vesicles by immunoelectron microscopy confirms that p58 and most of the type B lamins reside in the same compartment. Co-immunoprecipitation of both proteins by affinity-purified anti-p58 antibodies shows that they are physically associated in the context of a mitotic p58 'sub-complex'. This sub-assembly does not include the type A lamins which are fully solubilized during mitosis. Our data provide direct, in vivo and in vitro evidence that the majority of type B lamins remain connected to nuclear membrane 'receptors' during mitosis. The implications of these findings in nuclear envelope reassembly are discussed below.     

p34cdc2 acts as a lamin kinase in fission yeast

The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.     

Regulated docking of nuclear membrane vesicles to vimentin filaments during mitosis

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.     

Differential expression of nuclear lamin proteins during chicken development

By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B1, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B1 and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B1 decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell- or tissue-specific differences in nuclear architecture.     

The nuclear lamin protein family in higher vertebrates. Identification of quantitatively minor lamin proteins by monoclonal antibodies

The nuclear lamina, a structure closely apposed to the inner nuclear membrane, is believed to provide a framework important for nuclear envelope integrity and interphase chromatin organization. So far, in mammalian and avian species three major constituents of the lamina, lamins A, B, and C, have been identified. These proteins migrate to characteristic positions on two-dimensional gels, lamin B being more acidic than lamins A and C. Here, we show that the composition of the nuclear lamina in avian and mammalian cells is more complex than previously assumed. When analyzed on two-dimensional gels, the major 66-kDa chicken "lamin B" protein can readily be identified. However, an additional 68-kDa protein migrates to a similarly acidic position. Based on the following evidence, both proteins can be considered as two distinct members of the lamin protein family. First, peptide mapping experiments and immunological criteria demonstrate that these two proteins are not related to each other or to lamin A via postsynthetic modifications or precursor-product relationships. Second, as determined by immunocytochemical techniques, both proteins are located exclusively at the nuclear periphery. Third, both proteins display the biochemical properties characteristic of lamin proteins, i.e. they are resistant to extraction of nuclei with nonionic detergents, nucleases, and high salt. Fourth, both proteins are immunologically related to previously characterized lamin proteins: the major 66-kDa chicken "lamin B" protein shares at least two epitopes with lamin A. However, contrary to what current nomenclature might suggest, this 66-kDa chicken "lamin B" protein is not related to rat liver lamin B, but to a minor component of rat liver pore-complex lamina preparations that had not previously been recognized as a lamin protein. Conversely, the minor 68-kDa component of chicken lamina preparations that had not previously been considered to be a lamin protein is immunologically related to rat liver lamin B. Thus, in addition to demonstrating the existence of quantitatively minor lamin proteins in higher vertebrates, our results caution against assigning structural homologies between lamin proteins from different species on the basis of gel electrophoresis analyses.     

Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.     

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.     

The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2

Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.     

Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies

The nuclear lamins are major components of a proteinaceous polymer that is located at the interface of the nuclear membrane and chromatin; these lamins are solubilized and dispersed throughout the cytoplasm during mitosis. It has been postulated that these proteins, assembled into the lamina, provide an architectural framework for the organization of the cell nucleus. To test this hypothesis we microinjected lamin antibodies into cultured PtK2 cells during mitosis, thereby decreasing the soluble pool of lamins. The antibody injected was identified, together with the lamins, in cytoplasmic aggregates by immunoelectron microscopy. We show that microinjected cells are not able to form normal daughter nuclei, in contrast to cells injected with other immunoglobulins. Although cells injected with lamin antibodies are able to complete cytokinesis, the chromatin of their daughter nuclei remains arrested in a telophase-like configuration, and the telophase-like chromatin remains inactive as judged from its condensed state and by the absence of nucleoli. These results indicate that lamins and the nuclear lamina structure are involved in the functional organization of the interphase chromatin.     

Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis

The nuclear envelope is a dynamic structure that completely disassembles in response to MPF/cdc2 activity in mitosis. A key feature of this process is the hyperphosphorylation of the major structural proteins of the envelope, the nuclear lamins A, B, and C. Two highly conserved serine residues of the lamin protein (Ser-22 and Ser-392 of lamins A and C) are symmetrically positioned 5 amino acids from the ends of the large alpha-helical domain and are shown in the accompanying paper by Ward and Kirschner to be among four sites phosphorylated during nuclear envelope breakdown. Mutations in Ser-22 and Ser-392 that prevent phosphorylation at these sites block the disassembly of the nuclear lamina during mitosis. We propose a model for the regulation of lamin assembly in which phosphorylation just outside the ends of the alpha-helical domain controls the assembly dynamics of the lamin coiled-coil dimers.     

The myristoylation site of meiotic lamin C2 promotes local nuclear membrane growth and the formation of intranuclear membranes in somatic cultured cells

Lamin C2 is a splice product of the mammalian lamin A gene and expressed in primary spermatocytes where it is distributed in the form of discontinuous plaques at the nuclear envelope. We have previously shown that the aminoterminal hexapetide GNAEGR of lamin C2 following the start methionine is essential for its association with the nuclear envelope and that the aminoterminal glycine of the hexapeptide is myristoylated. Here we have analyzed the ultrastructural changes induced in COS-7 and Xenopus A6 cells by overexpressing rat lamin C2 or a human lamin C mutant possessing the lamin C2-specific hexapeptide at its aminoterminus. Both lamins were targeted to the nuclear envelope of mammalian and amphibian cells and induced the formation of intranuclear membranes, whereas wild-type human lamin C and a lamin C2 mutant, that both lack this lipid moiety, did not. Our data indicate that the myristoyl group of lamin C2 has besides its demonstrated role in nuclear envelope association additional functions during spermatogenesis. Our present study complements previously published results where we have shown that the CxxM motif of lamins promotes nuclear membrane growth (Prüfert et al., 2004. J. Cell Sci. 117, 6105-6116).     

Nuclear envelope proteins: Identification of lamin B subtypes

The lamins are a group of nuclear envelope proteins thought to form a structural layer at the nuclear periphery. Lamins A, B and C occur in many cell types although lamin C, a subtype of lamin A, is relatively decreased in chicken cells. A subtype of lamin B has been found in chicken cells. This subtype, called lamin B1, is slightly larger and more acidic than the quantitatively major subtype now called lamin B2. The lamin B subtypes have very similar primary sequences and share a distinctive topology. Two lamin B subtypes have not been observed in mammalian tissues but have been found in three avian tissues, erythrocytes of mature chickens and liver and erythrocytes of 11- to 13-day-old embryos. As these tissues differ widely in metabolic activity, both subtypes appear to be constitutive nuclear proteins.     

Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection

During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.     

Disassembly of in vitro formed lamin head-to-tail polymers by CDC2 kinase.

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. At the onset of mitosis it depolymerizes, presumably in response to phosphorylation of the lamin proteins. Recently, cdc2 kinase, a major regulator of the eukaryotic cell cycle, was shown to induce lamina depolymerization when incubated with isolated nuclei. Here, we have analysed the structural consequences of lamin phosphorylation by cdc2 kinase using lamin head-to-tail polymers reconstituted in vitro from bacterially expressed chicken lamin B2 protein as a substrate. The effects of phosphorylation were monitored by both a pelleting assay and electron microscopy. We show that lamin B2 head-to-tail polymers disassemble in response to phosphorylation of specific sites that are phosphorylated also during mitosis in vivo. These sites are located within SP/TP motifs N- and C-terminal to the central alpha-helical rod domain of lamin proteins. Subsequent dephosphorylation of these sites by purified phosphatase 1 allows reformation of lamin head-to-tail polymers. The relative importance of N- and C-terminal phosphorylation sites for controlling the assembly state of nuclear lamins was assessed by mutational analysis. Polymers formed of lamin proteins carrying mutations in the C-terminal phosphoacceptor motif could still be disassembled by cdc2 kinase. In contrast, a single point mutation in the N-terminal site (Ser16----Ala) rendered head-to-tail polymers resistant to disassembly. These results emphasize the importance of the N-terminal end domain for lamin head-to-tail polymerization in vitro, and they demonstrate that phosphorylation-dephosphorylation is sufficient to control the longitudinal assembly of lamin B2 dimers.     

The inner nuclear membrane protein LAP1 forms a native complex with B-type lamins and partitions with spindle-associated mitotic vesicles.

We have examined the in situ organization and nearest neighbours of the 'lamina-associated polypeptide-1' (LAP1), a type II membrane protein and a major constituent of the mammalian nuclear envelope. We show here that, during interphase, LAP1 forms multimeric assemblies which are suspended in the inner nuclear membrane and are specifically associated with B-type lamins. The LAP1-lamin B complex is distinct from analogous complexes formed by the 'lamina-associated polypeptide-2' (LAP2), another inner nuclear membrane protein, and includes a protein kinase. Upon nuclear envelope breakdown, LAP1 partitions with mitotic vesicles which carry nuclear lamin B. The LAP1 vesicles can be distinguished from fragments of the nuclear envelope containing LAP2 and exhibit a striking co-alignment with spindle microtubules. These observations suggest that the inner nuclear membrane comprises discrete territories which accommodate specific integral membrane proteins and are differentially disassembled during mitosis.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. Phosphorylation of lamin proteins is believed to cause lamina disassembly during meiotic and mitotic M phase, but the M phase-specific lamin kinase has not been identified. Here we show that the cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type lamins in vitro on sites that are specifically phosphorylated during M phase in vivo. Concomitantly, cdc2 kinase is capable of inducing lamina depolymerization upon incubation with isolated nuclei. One of the target sites of cdc2 kinase is identified as a motif (SPTR) conserved in the N-terminal domain of all lamin proteins. These results lead us to propose that mitotic disassembly of the nuclear lamina results from direct phosphorylation of lamins by cdc2 kinase.     

The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase.

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.