LEM4/ANKLE-2 deficiency impairs post-mitotic re-localization of BAF,LAP2α and LaminA to the nucleus,causes nuclear envelope instability in telophase and leads to hyperploidy in HeLa cells

The human LEM-domain protein family is involved in fundamental aspects of nuclear biology. The LEM-domain interacts with the barrier-to-autointegration factor (BAF), which itself binds DNA. LEM-domain proteins LAP2, emerin and MAN1 are proteins of the inner nuclear membrane; they have important functions: maintaining the integrity of the nuclear lamina and regulating gene expression at the nuclear periphery.LEM4/ANKLE-2 has been proposed to participate in nuclear envelope reassembly after mitosis and to mediate dephosphorylation of BAF through binding to phosphatase PP2A. Here, we used CRISPR/Cas9 to create several cell lines deficient in LEM4/ANKLE-2. By using time-lapse video microscopy, we show that absence of this protein severely compromises the post mitotic re-association of the nuclear proteins BAF, LAP2α and LaminA to chromosomes. These defects give rise to a strong mechanical instability of the nuclear envelope in telophase and to a chromosomal instability leading to increased number of hyperploid cells. Reintroducing LEM4/ANKLE-2 in the cells by transfection could efficiently restore the telophase association of BAF and LAP2α to the chromosomes. This rescue phenotype was abolished for N- or C-terminally truncated mutants that had lost the capacity to bind PP2A. We demonstrate also that, in addition to binding to PP2A, LEM4/ANKLE-2 binds BAF through its LEM-domain, providing further evidence for a generic function of this domain as a principal interactor of BAF.     

Barrier-to-autointegration factor: major roles in chromatin decondensation and nuclear assembly

Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain approximately 12 microM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.     

Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics

The lamina-associated polypeptide (LAP) 2 family comprises up to six alternatively spliced proteins in mammalian cells and three isoforms in Xenopus. LAP2beta is a type II integral protein of the inner nuclear membrane, which binds to lamin B and the chromosomal protein BAF, and may link the nuclear membrane to the underlying lamina and provide docking sites for chromatin. LAP2alpha shares only the N-terminus with the other isoforms and contains a unique C-terminus. It is a nonmembrane protein associated with the nucleoskeleton and may help to organize higher order chromatin structure by interacting with A-lamins and chromosomes. Recent studies using mutant proteins have just begun to unravel functions of LAP2 isoforms during postmitotic nuclear reassembly. LAP2alpha associates with chromosomes via an alpha-specific domain at early stages of assembly, possibly providing a structural framework for chromosome reorganization. The subsequent interaction of both LAP2alpha and LAP2beta with the chromosomal BAF may stabilize chromatin structure and target membranes to the chromosomes. At later stages LAP2 may regulate the assembly of lamins. LAP2 isoforms have been found to share a homologous approximately 40 amino acid long region, the LEM domain, with nuclear membrane proteins MAN1 and emerin, which has been implicated in Emery-Dreifuss muscular dystrophy.     

Dependence of diffusional mobility of integral inner nuclear membrane proteins on A-type lamins

Integral proteins of the nuclear envelope inner membrane have been proposed to reach their sites by diffusion after their co-translational insertion in the rough endoplasmic reticulum. They are then retained in the inner nuclear membrane by binding to nuclear structures. One such structure is the nuclear lamina, an intermediate filament meshwork composed of A-type and B-type lamin proteins. Emerin, MAN1, and LBR are three integral inner nuclear membrane proteins. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts from wild-type mice and Lmna -/- mice, which lack A-type lamins. We then studied the diffusional mobilities of emerin, MAN1, and LBR using fluorescence recovery after photobleaching. We show that emerin and MAN1, but not LBR, are more mobile in the inner nuclear membrane of cells from Lmna -/- mice than in cells from wild-type mice. In cells from Lmna -/- mice expressing exogenous lamin A, the protein mobilities were similar to those in cells from wild-type mice. This supports a model where emerin and MAN1 are at least partly retained in the inner nuclear membrane by binding to A-type lamins, while LBR depends on other binding partners for its retention.     

Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly

Barrier-to-autointegration factor (BAF) is an essential, highly conserved, metazoan protein. BAF interacts with LEM (LAP2, emerin, MAN1) domain-carrying proteins of the inner nuclear membrane. We analyzed the in vivo function of BAF in Caenorhabditis elegans embryos using both RNA interference and a temperature-sensitive baf-1 gene mutation and found that BAF is directly involved in nuclear envelope (NE) formation. NE defects were observed independent of and before the chromatin organization phenotype previously reported in BAF-depleted worms and flies. We identified vaccinia-related kinase (VRK) as a regulator of BAF phosphorylation and localization. VRK localizes both to the NE and chromatin in a cell-cycle-dependent manner. Depletion of VRK results in several mitotic defects, including impaired NE formation and BAF delocalization. We propose that phosphorylation of BAF by VRK plays an essential regulatory role in the association of BAF with chromatin and nuclear membrane proteins during NE formation.     

CHMPions of repair: Emerging perspectives on sensing and repairing the nuclear envelope barrier

Understanding how the integrity of the nuclear membranes is protected against internal and external stresses is an emergent challenge. Work reviewed here investigated the mechanisms by which losses of nuclear–cytoplasmic compartmentalization are sensed and ameliorated. Fundamental to these is spatial control over interactions between the endosomal sorting complexes required for transport machinery and LAP2–emerin–MAN1 family inner nuclear membrane proteins, which together promote nuclear envelope sealing in interphase and at the end of mitosis. We suggest that the size of the nuclear envelope hole dictates the mechanism of its repair, with larger holes requiring barrier-to-autointegration factor and the potential triggering of a postmitotic nuclear envelope reassembly pathway in interphase. We also consider why these mechanisms fail at ruptured micronuclei. Together, this work re-emphasizes the need to understand how membrane flow and local lipid metabolism help ensure that the nuclear envelope is refractory to mechanical rupture yet fluid enough to allow its essential dynamics.     

Distribution of emerin during the cell cycle

Human emerin is a nuclear membrane protein that is lost or altered in patients with Emery-Dreifuss muscular dystrophy (EMD). While the protein is expressed in the majority of human tissues analyzed, the pathology predominates in cardiac and skeletal muscles of patients with EMD. Our results show that emerin can be detected by immunocytochemistry and immunoblotting in the nuclear envelope of all vertebrates studied from man to Xenopus. Immunolocalizations and nuclear envelope extraction experiments confirm that emerin possesses properties characteristic for integral membrane proteins of the inner nuclear membrane. Some nuclear envelope proteins are localized also in annulate lamellae (AL), i.e. cytoplasmic flattened membrane cisternae penetrated by pore complexes. To verify whether emerin is contained in these membrane stacks, we have induced the formation of AL by exposure of rat cells (line RV-SMC) to sublethal doses of the antimitotic drug vinblastine sulfate and found that emerin is present in the nuclear envelope, but is absent from AL. In contrast to the homogeneous distribution of emerin in the nuclear envelope of interphase cells, this protein shows a focal accumulation in the nuclear membranes of late telophase cells. During early reassembly of the nuclear envelope at this mitotic stage emerin colocalizes with lamin A/C but not with lamin B and LAP2 proteins. Confocal laser scanning microscopy after double-labeling experiments with emerin and tubulin shows that emerin is concentrated in areas of the mitotic spindle and in the midbody of mitotic cells suggesting a close interaction of these proteins. Our data suggest that emerin participates in the reorganisation of the nuclear envelope at the end of mitosis.     

SINC,a type III secreted protein of Chlamydia psittaci,targets the inner nuclear membrane of infected cells and uninfected neighbors

SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci–infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP–transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear “lamina” structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci.     

Differential dynamics of splicing factor SC35 during the cell cycle

Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear speckles are disassembled by metaphase and reassembled in telophase in structures termed mitotic interchromatin granules (MIGs). We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in MIGs. Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of cells with inhibitors of cyclin-dependent kinases (cdks) caused changes in the organization of nuclear compartments such as nuclear speckles and nucleoli, with corresponding changes in the mobility of GFP-SC35 and GFP-fibrillarin. Our results suggest that the dynamics of SC35 are significantly influenced by the organization of the compartment in which it is localized during the cell cycle.     

All in the family: evidence for four new LEM-domain proteins Lem2 (NET-25), Lem3, Lem4 and Lem5 in the human genome

LEM-domain proteins share a folded structure, the 'LEM-domain', which binds a conserved chromatin protein named BAF. Most LEM-domain proteins are found at the nuclear membrane, but some are nucleoplasmic. All characterized members of this family bind nuclear lamin filaments. We summarize the 'founding' LEM-domain proteins LAP2, emerin and MAN1 ('SANE' or 'XMAN' in Xenopus) and their emerging roles in gene regulation and nuclear assembly. These roles are placed in the context of human diseases ('laminopathies') caused by mutations in either emerin or A-type lamins. Other LEM-domain proteins might modify the phenotype or severity of human laminopathy, or cause new laminopathies. We summarize evidence that the human genome encodes at least four additional LEM-domain proteins, designated Lem2 (NET-25), Lem3, Lem4 and Lem5. Early adaptation of a consistent nomenclature, such as the "Lem" names proposed here, will facilitate rapid progress in this field. Further investigation of 'founder' and novel members of this family will be important to understand nuclear structure, and presents new opportunities to understand human disease.     

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel)

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.     

BAF-1 mobility is regulated by environmental stresses

Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones, and DNA. Under normal conditions, BAF protein is highly mobile when assayed by fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We report that Caenorhabditis elegans BAF-1 mobility is regulated by caloric restriction, food deprivation, and heat shock. This was not a general response of chromatin-associated proteins, as food deprivation did not affect the mobility of heterochromatin protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser-4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that, overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestinal cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.     

Distinct functions of the unique C terminus of LAP2alpha in cell proliferation and nuclear assembly

The non-membrane-bound lamina-associated polypeptide 2 isoform, LAP2alpha, forms nucleoskeletal structures with A-type lamins and interacts with chromosomes in a cell cycle-dependent manner. LAP2alpha contains a LEM (LAP2, emerin, and MAN1) domain in the constant N terminus that binds to chromosomal barrier-to-autointegration factor, and a C-terminal unique region that is essential for chromosome binding. Here we show that C-terminal LAP2alpha fragment efficiently bound to mitotic chromosomes and inhibited assembly of endogenous LAP2alpha, nuclear membranes, and lamins A/C in in vitro nuclear assembly assays. Full-length recombinant LAP2alpha, which bound to chromosomes, and N-terminal fragment, which did not bind, had no effect on assembly. This suggested an essential role for the LAP2alpha C terminus in chromosome association and for the N-terminal LEM domain in subsequent assembly stages. In vivo analysis upon transient expression of GFP-tagged LAP2alpha fragments confirmed that, unlike the N-terminal fragment, the C-terminal fragment was able to bind to chromosomes during mitosis, if expressed weakly. At higher expression levels, C-terminal LAP2alpha fragment and full-length protein led to cell cycle arrest in interphase and apoptosis, as shown by fluorescence-activated cell sorter analysis, time lapse microscopy, and BrdUrd incorporation assays. These data indicated distinct functions of LAP2alpha in cell cycle progression during interphase and in nuclear reassembly during mitosis.