Search

Large protein complexes assemble at the nuclear envelope to transmit mechanical signals between the cytoskeleton and nucleoskeleton. These protein complexes are known as the linkers of the nucleoskeleton and cytoskeleton complexes (LINC complexes) and are formed by the interaction of SUN and KASH domain proteins in the nuclear envelope. Ample evidence suggests that SUN–KASH complexes form higher-order assemblies to withstand and transfer forces across the nuclear envelope. Herein, we present a review of recent studies over the past few years that have shed light on the mechanisms of SUN–KASH interactions, their higher order assembly, and the molecular mechanisms of force transfer across these complexes.

Nuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as the nuclear lamina, nuclear matrix or nucleoli. Recently, nuclear architecture has emerged as a major regulator of gene expression in mammalian cells. However, studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or gene expression within a population. In this report we present a method for combining 3D DNA fluorescence in situ hybridization (FISH) with single-molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine our method with imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of the cyclin-A2 (CCNA2) gene, which has known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position - which usually lies close to nuclear lamina - nor on the expression from other copies of the gene.This article has an associated First Person interview with the first author of the paper.

The nuclear envelope is much more than a simple barrier between nucleoplasm and cytoplasm. Nuclear envelope bridging complexes are protein complexes spanning both the inner and outer nuclear envelope membranes, thus directly connecting the cytoplasm with the nucleoplasm. In metazoans, they are involved in connecting the cytoskeleton with the nucleoskeleton, and act as anchoring platforms at the nuclear envelope for the positioning and moving of both nuclei and chromosomes. Recently, nucleocytoplasmic bridging complexes have also been identified in more evolutionarily diverse organisms, including land plants. Here, I discuss similarities and differences among and between eukaryotic supergroups, specifically of the proteins forming the cytoplasmic surface of these complexes. I am proposing a structure and function for a hypothetical ancestral nucleocytoplasmic bridging complex in the last eukaryotic common ancestor, with the goal to stimulate research in more diverse emerging model organisms.

The nuclear mitotic apparatus (NuMA) protein is a component of the nuclear matrix in interphase cells and an essential protein for the formation of mitotic spindle poles. We used herpes simplex virus (HSV), an enveloped DNA virus that replicates in the nucleus, to study the intra-nuclear dynamics of NuMA in infected cells. This study shows that NuMA is extensively modified following HSV infection, including phosphorylation of an unidentified site(s), and that it depends to an extent on viral DNA synthesis. Although NuMA is insoluble in uninfected interphase cells, HSV infection induced solubilisation and dynamic relocalisation of NuMA, whereupon the protein became excluded from viral replication compartments – sites of virus transcription and replication. Live cell, confocal imaging showed that NuMA localisation dramatically changed from the early stages (diffusely nuclear, excluding nucleoli) to late stages of infection (central diminuition, but remaining near the inner nuclear peripheries). In addition, NuMA knockdown using siRNA suggested that NuMA is important for efficient viral growth. In summary, we suggest that NuMA is required for efficient HSV infection, and identify further areas of research that address how the virus challenges host cell barriers.

Spatial organisation of nuclear compartments is an important regulator of chromatin function, yet the molecular principles that maintain nuclear architecture remain ill-defined. We have used RNA interference to deplete key structural nuclear proteins, the nuclear lamins. In HeLa cells, we show that reduced expression of lamin B1, but not lamin A/C, severely inhibits RNA synthesis – first by RNA polymerase II and later by RNA polymerase I. Declining levels of transcription correlate with different morphological changes in major nuclear compartments, nucleoli and nuclear speckles. Ultimately, nuclear changes linked to the loss of synthetic activity result in expansion of the inter-chromatin domain and corresponding changes in the structure and spatial organisation of chromosome territories, which relocate towards the nuclear periphery. These results show that a lamin B1-containing nucleoskeleton is required to maintain RNA synthesis and that ongoing synthesis is a fundamental determinant of global nuclear architecture in mammalian cells.

The DNA glycosylase hOGG1 initiates base excision repair (BER) of oxidised purines in cellular DNA. Using confocal microscopy and biochemical cell fractionation experiments we show that, upon UVA irradiation of human cells, hOGG1 is recruited from a soluble nucleoplasmic localisation to the nuclear matrix. More specifically, after irradiation, hOGG1 forms foci colocalising with the nuclear speckles, organelles that are interspersed between chromatin domains and that have been associated with transcription and RNA-splicing processes. The use of mutant forms of hOGG1 unable to bind the substrate showed that relocalisation of hOGG1 does not depend on the recognition of the DNA lesion by the enzyme. The recruitment of hOGG1 to the nuclear speckles is prevented by the presence of antioxidant compounds during UVA irradiation, implicating reactive oxygen species as signals for the relocalisation of hOGG1. Furthermore, APE1, the second enzyme in the BER pathway, is also present in nuclear speckles in UVA-irradiated cells. The recruitment of DNA repair proteins to nuclear speckles after oxidative stress implicates these organelles in the cellular stress response.

Cancer cells exhibit modifications in nuclear architecture and transcriptional control. Tumor growth and metastasis are supported by RUNX-family transcriptional scaffolding proteins, which mediate assembly of nuclear matrix–associated gene regulatory hubs. We used proteomic analysis to identify RUNX2-dependent protein-protein interactions associated with the nuclear matrix in bone, breast and prostate tumor cell types and found that RUNX2 interacts with three distinct proteins that respond to DNA damage: RUVBL2, INTS3 and BAZ1B. Subnuclear foci containing these proteins change in intensity or number following UV irradiation. Furthermore, RUNX2, INTS3 and BAZ1B form UV-responsive complexes with the serine 139-phosphorylated isoform of H2AX (γH2AX). UV irradiation increases the interaction of BAZ1B with γH2AX and decreases histone H3, lysine 9 acetylation levels (H3K9-Ac), which mark accessible chromatin. RUNX2 depletion prevents the BAZ1B/γH2AX interaction and attenuates loss of H3K9 and H3K56 acetylation. Our data are consistent with a model in which RUNX2 forms functional complexes with BAZ1B, RUVBL2 and INTS3 to mount an integrated response to DNA damage. This proposed cytoprotective function for RUNX2 in cancer cells may clarify its expression in chemotherapy-resistant and/or metastatic tumors.

Phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P3] is a lipid second messenger, produced by Type I phosphoinositide 3-kinases (PI 3-kinases), which mediates intracellular responses to many growth factors. Although PI 3-kinases are implicated in events at both the plasma membrane and intracellular sites, including the nucleus, direct evidence for the occurrence of PtdIns(3,4,5)P3 at non-plasma membrane locations is limited. We made use of the pleckstrin homology (PH) domain of general receptor for phosphoinositides (Grp1) to detect PtdIns(3,4,5)P3 in an on-section labeling approach by quantitative immunogold electron microscopy. Swiss 3T3 cells contained low levels of PtdIns(3,4,5)P3 that increased up to 15-fold upon stimulation with platelet-derived growth factor (PDGF). The signal was sensitive to PI 3-kinase inhibitors and present mainly at plasma membranes, including lamellipodia, and in a surprisingly large pool within the nuclear matrix. Comparatively little labeling was observed in endomembranes. A similar distribution of PtdIns(3,4,5)P3 was observed in U87MG cells, which lack the PtdIns(3,4,5)P3 phosphatase, PTEN. Re-expression of PTEN into U87MG cells ablated plasma membrane PtdIns(3,4,5)P3, but not the nuclear pool of this lipid even when PTEN was targeted to nuclei. These data have important implications for the versatility of PI 3-kinase signaling and for the proposed functions of PTEN in the nucleus.

Although the mechanism of chromosomal segregation is well known, it is unclear how other nuclear compartments such as promyelocytic leukemia (PML) nuclear bodies partition during mitosis and re-form in G1. We demonstrate that PML nuclear bodies partition via mitotic accumulations of PML protein (MAPPs), which are distinct from PML nuclear bodies in their dynamics, biochemistry and structure. During mitosis PML nuclear bodies lose biochemical components such as SUMO-1 and Sp100. We demonstrate that MAPPs are also devoid of Daxx and these biochemical changes occur prior to chromatin condensation and coincide with the loss of nuclear membrane integrity. MAPPs are highly mobile, yet do not readily exchange PML protein as demonstrated by fluorescence recovery after photo-bleaching (FRAP). A subset of MAPPs remains associated with mitotic chromosomes, providing a possible nucleation site for PML nuclear body formation in G1. As the nuclear envelope reforms in late anaphase, these nascent PML nuclear bodies accumulate components sequentially, for example Sp100 and SUMO-1 before Daxx. After cytokinesis, MAPPs remain in the cytoplasm long after the reincorporation of splicing components and their disappearance coincides with new PML nuclear body formation even in the absence of new protein synthesis. The PML protein within MAPPs is not degraded during mitosis but is recycled to contribute to the formation of new PML nuclear bodies in daughter nuclei. The recycling of PML protein from one cell cycle to the next via mitotic accumulations may represent a common mechanism for the partitioning of other nuclear bodies during mitosis.

The neuronal nuclear matrix protein, NRP/B, contains a BTB domain and kelch repeats and is expressed in primary neurons but not in primary glial cells. To examine the function of NRP/B in neurons, we analyzed the structure/function of the NRP/B-BTB domain and its role in neurite outgrowth. Based on three-dimensional modeling of NRP/B, we generated an NRP/B-BTB mutant containing three mutations in the conserved amino acids D47A, H60A and R61D that was termed BTB mutant A. BTB mutant A significantly reduced the dimerization of NRP/B compared to wild-type NRP/B. The NRP/B-BTB domain was required for nuclear localization and mediated the association of NRP/B with p110RB through the TR subdomain within the B pocket of p110RB. Overexpression of wild-type NRP/B and NRP/B-BTB domain significantly induced neurite outgrowth in PC12 cells and enhanced the G0-G1 cell population by ∼23% compared to the control cells, whereas NRP/B-BTB mutant A reduced neurite outgrowth by 70-80%, and inhibited NRP/B-p110RB association. Single cell microinjection of NRP/B-specific antibodies also blocked the neurite outgrowth of PC12 cells upon NGF stimulation. Interference of NRP/B expression by small interfering RNA (NRP/B-siRNA) inhibited neurite outgrowth and suppressed the NGF-induced outgrowth of neurites in PC12 cells. Additionally, p110RB phosphorylation at serine residue 795 was significantly reduced in PC12 cells treated with NRP/B siRNA compared to those treated with control GFP-siRNA, indicating that p110RB is a downstream target of NRP/B. Thus, the BTB domain of NRP/B regulates neurite outgrowth through its interaction with the TR subdomain within the B pocket of p110RB, and the conserved amino acids D47A, H60A and R61D within this domain of NRP/B are crucial residues for neurite extension in neuronal cells. These findings support a role for the BTB-domain of NRP/B as an important regulator of neuronal differentiation.

Global changes in gene expression and exit from the cell cycle underlie differentiation. Therefore, understanding chromatin behavior in differentiating nuclei and late G1 is key to understanding this developmental event. A nuclear event that has been shown to specifically occur in late G1 is the association of two heterochromatic blocks in Drosophila. The brownDominant (bwD) chromosome of Drosophila melanogaster contains a large block of heterochromatin near the end of 2R. This distal block associates with centric heterochromatin (2Rh), but not until at least 5 hours into G1. We used the bwD allele as a model for nuclear organization to determine whether its association with the heterochromatic compartment of the second chromosomes (2Rh) strictly requires differentiation or if this change is a stochastic event, its occurrence being proportional to time spent in G1/G0 phase of the cell cycle. Fluorescence in situ hybridization on eye imaginal discs showed increased association between the bw locus and 2Rh in differentiated cells. Interestingly, an increase in the number of nuclei showing bwD-2Rh association in the brains of developmentally delayed larvae that were compromised for differentiation was also observed. Live fluorescence imaging showed that the kinetics of chromatin movement remains unchanged in the developmentally arrested nuclei. These observations suggest that nuclear reorganization is not directly controlled by specific inductive signals during differentiation and that this nuclear reorganization can happen in a cell, regardless of differentiation state, that is arrested in the appropriate cell cycle stage. However, we did see changes that appear to be more directly correlated with differentiation. Dynamic imaging in eye imaginal discs showed that the movement of chromatin is more constrained in differentiated cells, implying that confinement of loci to a smaller nuclear space may help to maintain the changed organization and the transcription profile that accompanies differentiation.

We purified a 68-kDa protein from the mouse nuclear matrix using ion exchange and affinity chromatography. Column fractions were tested for specific binding to mouse minor satellite DNA using a gel mobility shift assay. The protein was identified by mass spectrometry as RNA helicase P68. In fixed cells, P68 was found to shuttle in and out of SC35 domains, forming fibres and granules in a cell-cycle dependent manner. Analysis of the P68 sequence revealed a short potential coiled-coil domain that might be involved in the formation of P68 fibres. Contacts between centromeres and P68 granules were observed during all phases of the cycle but they were most prominent in mitosis. At this stage, P68 was found in both the centromeric regions and the connections between chromosomes. Direct interaction of P68/DEAD box RNA helicase with satellite DNAs in vitro has not been demonstrated for any other members of the RNA helicase family.

Regulatory machinery for replication and gene expression is punctately organized in supramolecular complexes that are compartmentalized in nuclear microenvironments. Quantitative approaches are required to understand the assembly of regulatory machinery within the context of nuclear architecture and to provide a mechanistic link with biological control. We have developed `intranuclear informatics' to quantify functionally relevant parameters of spatially organized nuclear domains. Using this informatics strategy we have characterized post-mitotic reestablishment of focal subnuclear organization of Runx (AML/Cbfa) transcription factors in progeny cells. By analyzing point mutations that abrogate fidelity of Runx intranuclear targeting, we establish molecular determinants for the spatial order of Runx domains. Our novel approach provides evidence that architectural organization of Runx factors may be fundamental to their tissue-specific regulatory function.

The spatial organization in interphase nuclei of the breakpoint cluster regions (BCRs) of the AML-1 and ETO genes frequently participating in reciprocal t(8;21) translocations was studied using cytological and biochemical approaches. Both BCRs were found to be localized preferentially, but not exclusively, to the nuclear matrix, as shown by hybridization of specific probes with nuclear halos. This association was not related to transcription, because the transcribed regions of both genes located far from BCRs were located preferentially in loop DNA, as shown by in situ hybridization. The sites of association with the nuclear matrix of the intensely transcribed AML-1 gene were mapped also using the biochemical PCR-based approach. Only the BCR was found to be associated with the nuclear matrix, whereas the other transcribed regions of this gene turned out to be positioned randomly in respect to the nuclear matrix. The data are discussed in the framework of the hypothesis postulating that the nuclear matrix plays an important role in determining the positions of recombination-prone areas.

Genotoxic agents deform DNA structure thus eliciting a complex genetic response allowing recovery and cell survival. The Kin17 gene is up-regulated during this response. This gene encodes a conserved nuclear protein that shares a DNA-binding domain with the bacterial RecA protein. The KIN17 protein binds DNA and displays enhanced expression levels in proliferating cultured cells, suggesting a role in nuclear metabolism. We investigated this by studying the expression profile of KIN17 protein during mouse spermatogenesis. As expected, the expression level of Kin17 is higher in proliferating than in differentiated cells. KIN17 is selectively extracted from this tissue by detergents and a fraction was tightly associated with the nuclear matrix. Germinal cells ubiquitously express Kin17 and the protein is located mainly in the nucleus except in elongated spermatids where cytoplasmic staining is also observed. Sertoli and germ cells that are no longer mitotically active express KIN17, suggesting a general role in all testicular cell types. In adult testis a significant proportion of KIN17 co-purifies with polyadenylated RNA. KIN17 directly binds RNA, preferentially poly(G) and poly(U) homopolymers. These results together with the identification of KIN17 as a component of the human spliceosome indicate that this protein may participate in RNA processing.

We imaged the interiors of relatively intact Xenopus oocyte nuclei by field emission scanning electron microscopy (feSEM) and visualized a network of filaments that attach to nuclear pore complexes and extend throughout the nucleus. Within the nucleus, these 'pore-linked filaments' (PLFs) were embedded into spherical structures 100 nm to approximately 5 microm in diameter. A subset of spheres was identified as Cajal bodies by immuno-gold labeling; the rest were inferred to be nucleoli and snurposomes both of which are abundant in Xenopus oocyte nuclei. Most PLFs were independent of chromatin. The thickness of a typical PLF was 40 nm (range, approximately 12-100 nm), including the 4 nm chromium coat. PLFs located inside the nucleus merged, bundled and forked, suggesting architectural adaptability. The PLF network collapsed upon treatment with latrunculin A, which depolymerizes actin filaments. Jasplakinolide, which stabilizes actin filaments, produced PLFs with more open substructure including individual filaments with evenly-spaced rows of radially projecting short filaments. Immuno-gold labeling of untreated oocyte nuclei showed that actin and protein 4.1 each localized on PLFs. Protein 4.1-gold epitopes were spaced at approximately 120 nm intervals along filaments, and were often paired ( approximately 70 nm apart) at filament junctions. We suggest that protein 4.1 and actin contribute to the structure of a network of heterogeneous filaments that link nuclear pore complexes to subnuclear organelles, and discuss possible functions for PLFs in nuclear assembly and intranuclear traffic.

The biological significance of nuclear scaffold/matrix-attachment regions (S/MARs) remains a topic of long-standing interest. The key to understanding S/MAR behavior relies on determining the physical attributes of in vivo S/MARs and whether they serve as rigid or flexible chromatin loop anchors. To analyze S/MAR behavior, single and multiple copies of the S/MAR-containing constructs were introduced into various host genomes of transgenic mice and transfected cell lines. These in vivo integration events provided a system to study the association and integration patterns of each introduced S/MAR. By utilizing FISH to visualize directly the localization of S/MARs on the nuclear matrix or chromatin loop, we were able to assign specific attributes to the S/MAR. Surprisingly, when multiple-copy S/MARs were introduced they were selected and used as nuclear matrix anchors in a discriminatory manner, even though they all contained identical primary sequences. This selection process was probably mediated by S/MAR availability including binding strength and copy number, as reflected by the expression profiles and association of multi-copy tandem inserted constructs. Whereas S/MARs functioned as the mediators of loop attachment, they were used in a selective and dynamic fashion. Consequently, S/MAR anchors were necessary but not sufficient for chromatin loops to form. These observations reconcile many seemingly contradictory attributes previously associated with S/MARs.

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are composed of a major vault protein (MVP), two minor vault proteins [telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase (VPARP)] and small untranslated RNA molecules. Not all expressed TEP1 and VPARP in cells is bound to vaults. TEP1 is known to associate with the telomerase complex, whereas VPARP is also present in the nuclear matrix and in cytoplasmic clusters (VPARP-rods). We examined the subcellular localization and the dynamics of the vault complex in a non-small cell lung cancer cell line expressing MVP tagged with green fluorescent protein. Using quantitative fluorescence recovery after photobleaching (FRAP) it was shown that vaults move temperature independently by diffusion. However, incubation at room temperature (21°C) resulted in the formation of distinct tube-like structures in the cytoplasm. Raising the temperature could reverse this process. When the vault-tubes were formed, there were fewer or no VPARP-rods present in the cytoplasm, suggesting an incorporation of the VPARP into the vault-tubes. MVP molecules have to interact with each other via their coiled-coil domain in order to form vault-tubes. Furthermore, the stability of microtubules influenced the efficiency of vault-tube formation at 21°C. The dynamics and structure of the tubes were examined using confocal microscopy. Our data indicate a direct and dynamic relationship between vaults and VPARP, providing further clues to unravel the function of vaults.

Eukaryotic gene expression can be viewed within a conceptual framework in which regulatory mechanisms are integrated at three hierarchical levels. The first is the sequence level, i.e. the linear organization of transcription units and regulatory sequences. Here, developmentally co-regulated genes seem to be organized in clusters in the genome, which constitute individual functional units. The second is the chromatin level, which allows switching between different functional states. Switching between a state that suppresses transcription and one that is permissive for gene activity probably occurs at the level of the gene cluster, involving changes in chromatin structure that are controlled by the interplay between histone modification, DNA methylation, and a variety of repressive and activating mechanisms. This regulatory level is combined with control mechanisms that switch individual genes in the cluster on and off, depending on the properties of the promoter. The third level is the nuclear level, which includes the dynamic 3D spatial organization of the genome inside the cell nucleus. The nucleus is structurally and functionally compartmentalized and epigenetic regulation of gene expression may involve repositioning of loci in the nucleus through changes in large-scale chromatin structure.

NuMA is a nuclear matrix protein that has an essential function in the organization of the mitotic spindle. Here we have studied the fate of NuMA in Fas-treated apoptotic Jurkat T and HeLa cells. We show that in both cell lines NuMA is an early target protein for caspases and that NuMA is cleaved coincidently with poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear lamin B. NuMA is cleaved differently in Jurkat T and HeLa cells, suggesting that different sets of caspases are activated in these cell lines. The normal diffuse intranuclear distribution of NuMA changed during apoptosis: first NuMA condensed, then concentrated in the center of the nucleus and finally encircled the nuclear fragments within the apoptotic bodies. NuMA seems to be preferentially cleaved by caspase-3 in vivo since it was not cleaved in staurosporine-treated caspase-3-null MCF-7 breast cancer cells. The cleavage of NuMA, lamin B and PARP-1 was inhibited in the presence of three different caspase inhibitors: z-DEVD-FMK, z-VEID-FMK and z-IETD-FMK. Furthermore, in the presence of caspase inhibitors approximately 5-10% of the cells showed atypical apoptotic morphology. These cells had convoluted nuclei, altered chromatin structure and additionally, they were negative for NuMA and lamins. Since caspase-8, -3 and -7 were not activated and PARP was not cleaved in these cells as judged by western blotting and immunofluorescence studies, it is likely that this is an atypical form of programmed cell death owing to a proteinase(s) independent of caspases. These results characterize the role of NuMA in programmed cell death and suggest that cleavage of NuMA plays a role in apoptotic nuclear breakdown.

The runt-related transcription factors (RUNX/Cbfa/AML) are essential for cellular differentiation and fetal development. C-terminal truncations of RUNX factors that eliminate the targeting of these factors to subnuclear foci result in lethal hematopoietic and skeletal phenotypes. Here we demonstrate that in living cells the RUNX C-terminus is necessary for the dynamic association of RUNX into stable subnuclear domains. Time-lapse fluorescence microscopy shows that RUNX1 and RUNX2 localize to punctate foci that remain stationary in the nuclear space. By fluorescence recovery after photobleaching assays, both proteins are shown to dynamically associate at these subnuclear foci, with a 10 second half-time of recovery. A truncation of RUNX2, removing its intranuclear targeting signal (NMTS), increases its mobility by an order of magnitude, resulting in a half-time of recovery equivalent to that of EGFP alone. We propose that the dynamic shuttling of RUNX factors in living cells to positionally stabilized foci, which is dependent on the C-terminus, is a component of the mechanism for gene regulation in vivo.

Emerin is the nuclear membrane protein defective in X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). The majority of X-EDMD patients have no detectable emerin. However, there are cases that produce mutant forms of emerin, which can be used to study its function. Our previous studies have shown that the emerin mutants S54F, P183T, P183H, Del95-99, Del236-241 (identified in X-EDMD patients) are targeted to the nuclear membrane but to a lesser extent than wild-type emerin. In this paper, we have studied how the mislocalisation of these mutant emerins may affect nuclear functions associated with the cell cycle using flow cytometry and immunofluorescence microscopy. We have established that cells expressing the emerin mutant Del236-241 (a deletion in the transmembrane domain), which was mainly localised in the cytoplasm, exhibited an aberrant cell cycle length. Thereafter, by examining the intracellular localisation of endogenously expressed lamin A/C and exogenously expressed wild-type and mutant forms of emerin after a number of cell divisions, we determined that the mutant forms of emerin redistributed endogenous lamin A/C. The extent of lamin A/C redistribution correlated with the amount of EGFP-emerin that was mislocalised. The amount of EGFP-emerin mislocalized, in turn, was associated with alterations in the nuclear envelope morphology. The nuclear morphology and redistribution of lamin A/C was most severely affected in the cells expressing the emerin mutant Del236-241.It is believed that emerin is part of a novel nuclear protein complex consisting of the barrier-to-autointegration factor (BAF), the nuclear lamina, nuclear actin and other associated proteins. The data presented here show that lamin A/C localisation is dominantly directed by its interaction with certain emerin mutants and perhaps wild-type emerin as well. These results suggest that emerin links A-type lamins to the nuclear envelope and that the correct localisation of these nuclear proteins is important for maintaining cell cycle timing.

To examine the intracellular localization of neutral sphingomyelinase 1 (nSMase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E. coli. It has been reported that, in rat liver or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amount in nucleus and cytoplasm. The biochemical properties, dithiothreitol requirement and high salt concentration dependency, of cloned and expressed nSMase 1 resemble those of previously described nuclear neutral SMase of AH7974. The present study was therefore focused on the nuclear localization of this enzyme. Western blotting of subcellular fractions using anti-rat nSMase 1 antibody revealed most nSMase 1 to be associated with the nuclei and some with microsomes, but not with plasma membranes. Consistently, neutral SMase activity in nuclear extract was immunoprecipitated by the antibody, while that of plasma membranes was not. The results indicate that nSMase 1 mainly resides in the nucleus and may thus differ from neutral SMase in plasma membrane. On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive protein bands on western blotting, suggesting that a large part of nuclear neutral SMase may be nSMase 1. Removal of the nuclear envelope by treatment with Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e. nuclear envelope, chromatin, and nuclear matrix) revealed nSMase 1 signal exclusively in the nuclear matrix. Immunocytochemistry with AH7974, as well as rat fibroblast cell line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, with some in the cytoplasm. Moreover, immuno-electron microscopy clearly showed the signal of nSMase 1 to be more dense in the nucleus than in the cytoplasm of AH7974.

LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, we have cloned a novel LAP2beta-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein. In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope. Here, we show that, in mammalian cells, GCL is co-localized with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. Recently, mGCL was found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. We demonstrate here that LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein.

The Polycomb-group (PcG) is a diverse set of proteins required for maintenance of gene silencing during development. In a screen for conserved partners of the PcG protein Polycomblike (Pcl), we have identified a new protein, human CHMP1 (CHromatin Modifying Protein; CHarged Multivesicular body Protein), which is encoded by an alternative open reading frame in the PRSM1 gene and is conserved in both complex and simple eukaryotes. CHMP1 contains a predicted bipartite nuclear localization signal and distributes as distinct forms to the cytoplasm and the nuclear matrix in all cell lines tested. We have constructed a stable HEK293 cell line that inducibly overexpresses CHMP1 under ecdysone control. Overexpressed CHMP1 localizes to a punctate subnuclear pattern, encapsulating regions of nuclease-resistant, condensed chromatin. These novel structures are also frequently surrounded by increased histone H3 phosphorylation and acetylation. CHMP1 can recruit a PcG protein, BMI1, to these regions of condensed chromatin and can cooperate with co-expressed vertebrate Pcl in a Xenopus embryo PcG assay; this is consistent with a role in PcG function. In combination, these observations suggest that CHMP1 plays a role in stable gene silencing within the nucleus.

Hair follicle cycling is an exquisitely regulated and dynamic process consisting of phases of growth, regression and quiescence. The transitions between the phases are governed by a growing number of regulatory proteins, including transcription factors. The hairless (hr) gene encodes a putative transcription factor that is highly expressed in the skin, where it appears to be an essential regulator during the regression of the catagen hair follicle. In hairless mice, as well as humans with congenital atrichia, the absence of hr gene function initiates a premature and abnormal catagen due to a dysregulation of apoptosis and cell adhesion, and defects in the signaling required for hair follicle remodeling. Here, we report structure-function studies of the hairless gene product, in which we identify a novel bipartite nuclear localization signal (NLS) of the form KRA(X13) PKR. Deletion analysis of the mouse hr gene mapped the NLS to amino acid residues 409–427. Indirect immunofluorescence microscopy of cells transiently transfected with hairless-green fluorescent fusion proteins demonstrated that these amino acid residues are necessary and sufficient for nuclear localization. Furthermore, nuclear fractionation analysis revealed that the hr protein is associated with components of the nuclear matrix.

A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunoreactivity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.