The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

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.     

Tissue-specific defects are caused by loss of the Drosophila MAN1 LEM domain protein

The nuclear lamina represents a protein network required for nuclear structure and function. One family of lamina proteins is defined by an approximately 40-aa LAP2, Emerin, and MAN1 (LEM) domain (LEM-D) that binds the nonspecific DNA-binding protein, barrier-to-autointegration factor (BAF). Through interactions with BAF, LEM-D proteins serve as a bridge between chromosomes and the nuclear envelope. Mutations in genes encoding LEM-D proteins cause human laminopathies that are associated with tissue-restricted pathologies. Drosophila has five genes that encode proteins with LEM homology. Using yeast two-hybrid analyses, we demonstrate that four encode proteins that bind Drosophila (d)BAF. In addition to dBAF, dMAN1 associates with lamins, the LEM-D protein Bocksbeutel, and the receptor-regulated Smads, demonstrating parallel protein interactions with vertebrate homologs. P-element mobilization was used to generate null dMAN1 alleles. These mutants showed decreased viability, with surviving adults displaying male sterility, decreased female fertility, wing patterning and positioning defects, flightlessness, and locomotion difficulties that became more severe with age. Increased phospho-Smad staining in dMAN1 mutant wing discs is consistent with a role in transforming growth factor (TGF)-beta/bone morphogenic protein (BMP) signaling. The tissue-specific, age-enhanced dMAN1 mutant phenotypes are reminiscent of human laminopathies, suggesting that studies in Drosophila will provide insights into lamina dysfunction associated with disease.     

Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.     

Interactions among Drosophila Nuclear Envelope Proteins Lamin, Otefin, and YA

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin’s rod domain interacts with the complete otefin protein, with otefin’s hydrophilic NH₂-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA’s COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.     

Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery

Antibodies directed against nuclear envelope lamin proteins have been used in conjunction with three-dimensional light and electron microscope methodologies to determine the spatial organization of lamins in diploid interphase nuclei and to relate this organization to the positions of chromatin in the nuclear periphery. Using Drosophila early embryos, Drosophila Kc cells, and human HeLa cells, it is qualitatively and quantitatively observed that lamins are organized as a highly discontinuous, apparently fibrillar network that leaves large voids in the nuclear periphery containing little or no lamin. Using fluorescence microscopy to compare and quantitate the relationship between chromatin and the lamin network, it is found that although there is a strong tendency for the most peripheral chromatin to be positioned directly underneath a lamin fiber, only a small fraction of the chromatin in the nuclear periphery is sufficiently close to a lamin fiber to possibly be in direct contact.     

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.     

Lamin A precursor induces barrier-to-autointegration factor nuclear localization

Lamin A, a protein component of the nuclear lamina, is synthesized as a precursor named prelamin A, whose multi-step maturation process involves different protein intermediates. As demonstrated in laminopathies such as familial partial lipodystrophy, mandibuloacral dysplasia, Werner syndrome, Hutchinson-Gilford progeria syndrome and restrictive dermopathy, failure of prelamin A processing results in the accumulation of lamin A protein precursors inside the nucleus which dominantly produces aberrant chromatin structure. To understand if nuclear lamina components may be involved in prelamin A chromatin remodeling effects, we investigated barrier-to-autointegration factor (BAF) localization and expression in prelamin A accumulating cells. BAF is a DNA-binding protein that interacts directly with histones, lamins and LEM-domain proteins and has roles in chromatin structure, mitosis and gene regulation.In this study, we show that the BAF heterogeneous localization between nucleus and cytoplasm observed in HEK293 cycling cells changes in response to prelamin A accumulation. In particular, we observed that the accumulation of lamin A, non-farnesylated prelamin A and farnesylated carboxymethylated lamin A precursors induce BAF nuclear translocation. Moreover, we show that the treatment of human fibroblasts with prelamin A interfering drugs results in similar changes. Finally, we report that the accumulation of progerin, a truncated form of farnesylated and carboxymethylated prelamin A identified in Hutchinson-Gilford progeria syndrome cells, induces BAF recruitment in the nucleus. These findings are supported by coimmunoprecipitation of prelamin A or progerin with BAF in vivo and suggest that BAF could mediate prelamin A-induced chromatin effects.     

Solution NMR structure of the barrier-to-autointegration factor-Emerin complex

The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF(2) dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF(2) and Em(LEM). Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM) bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/- 2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that the LEM and DNA binding sites on BAF(2) are non-overlapping and that both subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM) binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.     

The vaccinia-related kinases phosphorylate the N' terminus of BAF, regulating its interaction with DNA and its retention in the nucleus

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N' terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture.     

C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes.