1 Department of Medical Biochemistry, Max F. Perutz Laboratories, Vienna Biocenter, Medical University of Vienna, Dr Bohrgasse 9/3, A-1030 Vienna, Austria
2 Department of Molecular Cell Biology, University of Vienna, Max F. Perutz Laboratories, Vienna Biocenter, Dr Bohrgasse 9/3, A-1030 Vienna, Austria
* Authors for correspondence (e-mail: roland.foisner{at}meduniwien.ac.at; josef.gotzmann{at}meduniwien.ac.at)
Accepted 20 September 2005
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Summary |
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Key words: Chromatin organization, Lamina-associated proteins, Lamins, LEM domain, Nuclear architecture, Nuclear envelope
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Introduction |
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A group of the lamin-binding proteins share a common structural motif of 40 amino acids (aa) at their N-termini, called the lamina-associated polypeptideemerinMAN1 (LEM) domain (Cai et al., 2001; Laguri et al., 2001
; Lin et al., 2000
), which mediates the interaction with a highly conserved DNA-crosslinking protein, barrier-to-autointegration factor (BAF) (Cai et al., 2001
; Furukawa, 1999
; Lee et al., 2001
; Mansharamani and Wilson, 2005
; Segura-Totten and Wilson, 2004
; Shumaker et al., 2001
). The founding members of the LEM-domain protein family include several alternatively spliced LAP2 isoforms (Berger et al., 1996
; Dechat et al., 2000b
; Furukawa et al., 1995
; Harris et al., 1994
), emerin, and MAN1 (Lin et al., 2000
). The LAP2 isoforms contain an additional LEM-like motif at the extreme N-terminus, which interacts with DNA (Cai et al., 2001
). Other members of the LEM-domain protein family include the Drosophila-specific proteins otefin (Ashery-Padan et al., 1997
) and Bocksbeutel (Wagner et al., 2004
), and the uncharacterized Caenorhabditis elegans LEM3 (Lee et al., 2000
). A comprehensive database screen identified a mammalian orthologue of Ce-LEM3 and three novel LEM-domain proteins in higher eukaryotes, which were termed LEM2, LEM4 and LEM5 based on and extending the C. elegans nomenclature (Lee and Wilson, 2004
; Mansharamani and Wilson, 2005
).
On the basis of their interaction with BAF and DNA, the LEM-domain proteins have been implicated in chromatin organization (Dechat et al., 2004; Haraguchi et al., 2001
; Liu et al., 2003
; Segura-Totten et al., 2002
; Segura-Totten and Wilson, 2004
; Shimi et al., 2004
). Emerin and LEM2 might have overlapping and redundant functions, as RNA interference-mediated knockdown of both proteins in C. elegans was embryonic lethal at the 100-cell stage, whereas single knockouts showed no or less-severe phenotypes (Liu et al., 2003
). Furthermore, emerin and MAN1 were found to bind to each other in vitro (Mansharamani and Wilson, 2005
). Numerous novel recently identified binding partners of LEM-domain proteins indicate many important functions of these proteins in diverse cellular processes (Bengtsson and Wilson, 2004
; Gruenbaum et al., 2005
). LAP2ß and emerin bind the transcriptional repressor germ-cell less (Holaska et al., 2003
; Nili et al., 2001
), and LAP2
interacts with the tumor suppressor retinoblastoma protein (Markiewicz et al., 2002
), implicating functions in transcriptional regulation. Other potential functions of LEM-domain proteins can be envisaged based on the observation that emerin can act as a capping protein for nuclear actin (Holaska et al., 2004
). Interestingly, XMAN1, the Xenopus orthologue of human MAN1, has been shown to serve as antagonist of the bone morphogenetic protein (BMP) signalling pathway by binding to downstream regulatory Smads 1, 5 and 8 (Osada et al., 2003
). Similarly, human MAN1 can augment BMP signalling as well as signalling through transforming growth factor ß (TGF-ß) and activin by binding to Smads 1, 2 and 3, but not Smad 4 (Hellemans et al., 2004
; Lin et al., 2005
; Pan et al., 2005
).
Mutations in genes encoding A-type lamins and some of their binding partners give rise to a diverse group of diseases termed `laminopathies' (Burke and Stewart, 2002; Gotzmann, 2004; Gruenbaum et al., 2005
; Hutchison and Worman, 2004
; Mounkes and Stewart, 2004
). The disease phenotypes are diverse, affecting skeletal muscles, heart, adipose, bone, skin and neuronal tissues, and also include premature aging; the underlying molecular mechanisms for the laminopathies are still unclear. Apart from disease-causing mutations in the LMNA gene, mutations in genes encoding the LEM-domain proteins emerin (Bione et al., 1994
; Emery, 1987
; Emery and Dreifuss, 1966
; Manilal et al., 1996
) and MAN1 (Hellemans et al., 2004
) give rise to muscular dystrophy and bone-related diseases, respectively. Since some disease-linked mutations in LMNA or lack of lamin A cause mislocalization of emerin to the endoplasmic reticulum (ER) (Holt et al., 2003
; Muchir et al., 2004
; Ostlund et al., 2001
; Raharjo et al., 2001
; Sullivan et al., 1999
; Vaughan et al., 2001
), it is likely that disruption of lamin A complexes caused by mutations in either lamin A/C or in associated proteins are the molecular cause of these diseases. Therefore, in order to gain more insights into the functions of lamin A/C complexes and their involvement in disease, it is extremely important to identify and characterize novel proteins of these complexes.
Here we describe and characterize a novel human MAN1-related LEM-domain protein, LEM2. Importantly, we demonstrate that LEM2 binds to lamin C and requires association with A-type lamins for proper NE localization. Furthermore, highly overexpressed LEM2 formed clusters and membranous invaginations at the NE as well as tubular structures interlinking nuclei of adjacent cells, to which it recruits lamin A/C and associated proteins emerin and BAF. This suggests a role of LEM2 in the organization of lamin A/C complexes.
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Materials and Methods |
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Antibodies
Mouse and rabbit anti-V5 antibodies were purchased from Invitrogen and Sigma, respectively. Rabbit anti-phospho-histone-H3 antibody was obtained from Upstate Biotechnologies; mouse anti--tubulin antibody and a polyclonal serum against actin were from Sigma; mouse monoclonal anti-lamin A (clone#133A2) was from Abcam; and mouse anti-NUP62 was from BD Pharmingen. The rabbit antiserum to LAP2
has been described previously (Vlcek et al., 2002
). The antiserum to BAF was a generous gift of K. Furukawa [Niigata University, Japan (Furukawa, 1999
)]. The antiserum to ER-marker
-calnexin was provided by E. Ivessa [Medical University Vienna, Austria (de Virgilio et al., 1998
)]. The guinea pig anti-LBR antibody was provided by H. Hermann [DKFZ, Heidelberg, Germany (Dreger et al., 2002
)]. The mouse anti-lamin B1 monoclonal antibody 8D1 was provided by D. Vaux [Sir William Dunn School of Pathology, Oxford, UK (Maske and Vaux, 2004
)].
Plasmids and cloning strategy
A cDNA clone (#T5554) containing full-length human LEM2 was obtained from Genecopoeia (Germantown, USA) and used as template to amplify cDNA of full-length human LEM2 or the N-terminus (LEM2-NT) using the following primers: full-length, 5'-CACCATGGCCGGCCTGTCGGACCTGGAACTGCGGC-3' and 5'-GCATCGCTCTGAGTCAGAGAAGGAAGAGG-3'; LEM2-NT, 5'-CACCATGGCCGGCCTGTCGGACCTGGAACTGCGGC-3' and 5'-AAGCTTCGCGCCAGCAGGGCCCGCTCGAGT-3'. cDNAs were cloned into pENTR/D-TOPO (Invitrogen). For eukaryotic expression, hLEM2 and LEM2-NT were shuttled using the LR-recombination reaction (Invitrogen) into pTRACER-B, made Gateway®-compatible by insertion of the `conversion cassette' (Invitrogen) into the EcoRV site. Full-length human MAN1 was amplified by PCR (5'-CACCATGGCGGCGGCAGCAGCTTC-3'; 5'-GCAGGAACTTCCTTGAGAAT-3') and cloned into pTRACER-B as above. Vectors expressing deletion mutants of hLEM2 were constructed by restriction digest and re-ligation using pTRACER with full-length hLEM2. For LEM2CT (
aa 378-486) we used MslI, for LEM2
NT (
aa 130-203) SfoI, for LEM2-LEM (
aa 74-503) BssHII, and for LEM2
LEM+NT (
aa 28-203) SmaI and SfoI. All expression constructs were tagged with a C-terminal V5 epitope. For the LEM2-GFP construct, a SpeI-XbaI fragment from the pTRACER vector expressing full-length LEM2 was subcloned into the NheI site of peGFP-C1 (Clontech). A construct expressing GFP-tagged pre-lamin A (Sullivan et al., 1999
) was a generous gift of C. Stewart; the plasmid GFP-xLaminB1
2+ was kindly provided by C. Hutchison [University of Durham, UK (Izumi et al., 2000
)].
Subcellular fractionation, gel electrophoresis and immunoblotting
Subcellular fractionation was done essentially as described (Dechat et al., 1998; Gotzmann et al., 2000
). In brief, cells were broken in hypotonic buffer using a dounce homogenizer with a tight-fitting pestle. After addition of 8% sucrose, the soluble cytoplasmic and insoluble nuclear fractions were separated by centrifugation at 2000 g for 15 minutes at 4°C. The nuclei-containing pellet fractions were extracted in the same buffer supplemented with 1% Triton X-100, or 200 mM NaCl, or combinations of both, or 7 M urea followed by centrifugation at 15,000 g for 10 minutes; analyses of supernatant and pellet fractions were carried out by western blotting. SDS-PAGE and immunoblotting was performed essentially as described previously (Gotzmann et al., 2002
; Gotzmann et al., 2000
).
PCR analysis
Total RNA was purified by standard techniques from whole mouse embryos (dpc 12, 14, 16, 18) or purchased as total RNA collections of human and mouse tissues (Clontech). Poly(A)+ mRNA was extracted from total RNA with an mRNA Isolation Kit and reverse transcribed using the first-strand cDNA Synthesis Kit (both from Roche). Aliquots of the resulting products were employed as templates for specific PCR amplifications using Ready-To-Go PCR beads (Amersham Pharmacia Biotech). The conditions for the PCR reactions were optimized for each primer pair. All cDNAs were normalized according to actin expression levels. Real-time PCR analysis of LEM2 expression at developmental stages was performed using an iCycler real-time PCR detection system (Bio-Rad) and the DNA-binding dye SYBR Green I. The following pairs of forward and reverse primers (all in 5'-3' direction) were used for amplifications: human and mouse actin, ATCTGGCACCACACCTTCTAC and CAGCCAGGTCCAGACGCAGG; human LEM2, GCCGGCCTGTCGGACCTGGAACT and GGCGGCGCAGCTTGTTGCGGTAG; mouse LEM2, AAGCGAGTATGGGACCGTGCTGTG and AGGTGAGACCCGGCTGAAGAGTTG.
Blot overlay assay
Full-length hLEM2 and an N-terminal fragment (LEM2-NT; aa 46-195) of hLEM2 were subcloned into a pET102 vector backbone using TOPO technology (Invitrogen). Expression plasmids for lamin C fragments (head, aa 1-171; rod, aa 171-319; tail, aa 319-572) and full-length LAP2 are described elsewhere (Dechat et al., 2000a
). Coupled transcription and translation in the presence of [35S]-methionine was performed using the TNT® Quick Coupled Transcription/Translation according to the manufacturer's instructions (Promega). Recombinant lamin C fragments and LAP2
were separated by SDS-PAGE and transferred onto nitrocellulose. Proteins were stained with PonceauS, and blots were incubated in overlay buffer (10 mM Hepes, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 2 mM EGTA, 0.1% Triton X-100, 1 mM DTT) for 3 hours, blocked with 5% skim milk in overlay buffer, and probed with whole reticulocyte lysate containing 35S-labelled proteins diluted 1:50 in overlay buffer with 1% skim milk and 1 mM phenylmethylsulphonylfluoride (PMSF) overnight at 4°C. After extensive washing in phosphate-buffered saline/Tween (PBST), bound proteins were detected by autoradiography.
Immunofluorescence microscopy
Cells were grown on poly-L-lysine-coated glass coverslips, fixed in methanol at 20°C for 3 minutes, or fixed in 4% paraformaldehyde for 10 minutes and permeabilized with 0.5% Triton X-100 in PBS for 5 minutes. Digitonin extraction of fixed cells was done with 40 µg/ml digitonin (Calbiochem) in PBS for 3 minutes on ice. Cells were blocked in 0.5% gelatin in PBS for 15 minutes, incubated with primary antibodies for 45 minutes, washed and probed with the appropriate secondary antibodies conjugated to either Texas Red (Jackson Immuno Research) or Alexa Fluor 488 (Molecular Probes) for 45 minutes. DNA was stained with Hoechst for 5 minutes, and samples were mounted in Mowiol (Fluka). Confocal images of the samples were taken with a confocal laser scanning microscope (LSM510 and Axiovert 100; Zeiss). Digital images were analysed and adjusted for brightness and contrast using the software LSM-Image-Browser (Zeiss) and Adobe Photoshop.
Electron microscopy
Cells grown on glass coverslips were fixed in 3% glutaraldehyde in 0.15 M Sorensen's buffer, pH 7.4, for 1 hour, incubated in 1% OsO4 in Sorensen's for 1 hour and dehydrated with increasing concentrations of ethanol. Samples were subsequently `flat' embedded in epoxy resin (Agar 100). Thin sections of 60-80 nm were cut with a LEICA Ultracut S ultramicrotome, mounted on copper grids, contrasted with uranyl acetate and lead citrate, and examined in a JEOL JEM-1210 electron microscope at 60 kV. Images were acquired using a Mega View III digital camera and analySIS FIVE software (SIS, Praha, Czech Republic).
Computer-assisted analysis
Alignments of cDNA sequences, genomic contig sequence alignments and database searches were performed by NCBI-BLAST (http://www.ncbi.nlm.nih.gov/blast/) and ClustalW (http://www.ebi.ac.uk/clustalw/) (Thompson et al., 1994). Genomic analysis was done using the ENSEMBL Genome Browser (http://www.ensembl.org/; Sanger Institute). The sequence of the murine orthologue of hLEM2 was calculated using the GENSCAN gene-prediction software [http://genes.mit.edu/GENSCAN.html (Burge and Karlin, 1997
)]. Protein motifs and pattern searches were performed using SMART [http://smart.embl-heidelberg.de/ (Letunic et al., 2004
; Schultz et al., 1998
)], CDD (http://www.ncbi.nlm.nih.gov/; NCBI) and PSORT II (http://psort.nibb.ac.jp/form2.html). Transmembrane domains were calculated using the membrane protein topology database [http://blanco.biomol.uci.edu/mptopo/ (Jayasinghe et al., 2001
)], the TMHMM 2.0 prediction software [http://www.cbs.dtu.dk/services/TMHMM/ (Krogh et al., 2001
)], the SOSUI system (http://sosui.proteome.bio.tuat.ac.jp/) and the DAS-TM filter algorithm (Cserzo et al., 2004
). Phylogenetic tree predictions were done using eSHADOW [http://eshadow.dcode.org/ (Ovcharenko et al., 2004
)] and visualized using the Phylodendron software (http://iubio.bio.indiana.edu/treeapp/).
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Results |
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We detected LEM2 orthologues in C. elegans (Fig. 1A), rat, chicken and dog (data not shown). The overall domain organization of hLEM2 is identical to that of hMAN1 and other MAN1 orthologues (e.g. Xenopus XMAN1), except that LEM2 lacked an RNA-recognition motif found in the C-termini of MAN1-type proteins (Fig. 1, RRM domain, yellow). Except for the LEM domain, hLEM2 and hMAN1 are less well conserved in their N-termini (upstream of the first predicted transmembrane domain) and in the region between the transmembrane domains (11 and 27% identity, respectively), whereas a high degree of identity (87%) was detected in the C-terminal MSC domains of these proteins (Fig. 1A). Thus, we suggest that MAN1 and LEM2 comprise a subfamily within the LEM-domain proteins characterized by the presence of the MSC domain. Interestingly, Ce-LEM2, which has originally been termed Ce-MAN1 (Lee et al., 2000), is more homologous in the N-terminus and in the region between the transmembrane domains to hLEM2 than to hMAN1, and also lacks the C-terminal RNA recognition motif of MAN1. Therefore, we concur that the protein encoded by Ce-LEM2 is orthologous to human LEM2 (Mansharamani and Wilson, 2005
), not MAN1, as originally thought (Lee et al., 2000
). By contrast, a highly related protein from Drosophila (dmCG3167, Fig. 1A) seems to be a MAN1 rather than a LEM2 orthologue.
Phylogenetic analysis led us to speculate that LEM2 and MAN1 have a common ancestor in evolution (Fig. 1B). Intriguingly, a domain organization related to that of the predicted ancestor was also detected in yeast Src1p-related proteins. Src1p contains an N-terminal SAP domain, which by structural criteria might be similar to the LEM domain, two transmembrane domains and the conserved C-terminal MSC domain (Fig. 1B) (Mans et al., 2004). Thus, the LEM motif might have been derived from the SAP motif, and proteins containing the SAP/LEM and MSC domains might represent an evolutionarily conserved LEM2 protein family. In addition, a group of MAN1-type proteins, characterized by the presence of an additional C-terminal RRM domain (yellow), might have evolved from LEM2 ancestors.
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In order to determine the subcellular localization of LEM2 protein, we stably expressed V5-tagged hLEM2 in various cell types. Confocal immunofluorescence microscopy of stable HeLa clones showed that the expressed LEM2 localized exclusively to the NE, giving rise to a typical rim-like staining of the nuclear periphery (Fig. 2A). Identical data were obtained in COS7 and MDCK cells, and in human and mouse primary skin fibroblasts (data not shown). In order to distinguish localization of LEM2 in the INM versus ONM, we permeabilized cells with digitonin, which selectively disrupts the plasma membrane leaving the NE membranes intact (Adam et al., 1990). Antibodies to the C-terminal V5 tag of hLEM2 and to the nucleoplasmic protein LAP2
did not detect the proteins in digitonin-treated interphase cells in the nucleus; by contrast, in mitotic cells, in which the nuclear membrane was disassembled, LEM2-V5 and LAP2
were visible in the cytoplasm (arrows, Fig. 2B). A weak LEM2 staining in digitonin-treated interphase cells in the cytoplasm might represent a small fraction of LEM2-V5 in the ER. Since LEM2 in the NE was not accessible for antibodies in digitonin-treated interphase cells, we concluded that the LEM2 C-terminus is located in the nucleoplasm, implying that hLEM2 is a constituent of the INM.
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During mitosis, when the nucleus is disintegrated and reassembled after sister chromatid separation, LEM2 behaved similarly to other LEM-domain-containing membrane proteins. From prophase to prometaphase, identified by the presence of phosphorylated histone H3 (Juan et al., 1998), the nuclear membrane was progressively disintegrated and LEM2 diffused into the cytoplasm, where it remained until telophase (Fig. 3A). Similar to emerin and LAP2ß (Dechat et al., 2004
; Haraguchi et al., 2001
), LEM2 re-localized to the NE only at later stages of assembly, clearly after LAP2
was accumulated in chromatin-associated core regions and after LBR was detectable at peripheral regions of decondensing chromosomes (Fig. 3B). Altogether, based on its subcellular localization in the interphase nucleus, its dynamic behaviour during mitosis and its biochemical properties, LEM2 is indistinguishable from other integral nuclear membrane proteins of the LEM-domain family.
NE localization of hLEM2 requires A-type lamins at the NE
Extraction studies indicated that hLEM2 is associated with the lamina-nucleoskeleton scaffold (see above). Furthermore, in C. elegans, Ce-LEM2 localized to the NE in a lamin-dependent manner (Liu et al., 2003), and in mammals the LEM-domain protein emerin requires A-type lamins for NE targeting (Sullivan et al., 1999
; Vaughan et al., 2001
). To explore whether NE localization of hLEM2 also depends on A-type lamins, we analysed the localization of hLEM2 in MEFs. In wild-type MEFs, LEM2-V5 preferentially localized to the NE (Fig. 4A); however, in Lmna/ MEFs, LEM2-V5 was distributed throughout the ER (Fig. 4B). Expression of GFP-lamin A in Lmna/ MEFs, which localized to the NE, restored the predominantly rim-like localization of hLEM2-V5 at the NE (Fig. 4C), suggesting that lamin A is required for retention of LEM2 at the INM. To test this model further, we disrupted the distribution of endogenous A-type lamins in LEM2-V5-expressing HeLa cells by introducing a headless Xenopus lamin mutant, GFP-xLaminB1
2+, which accumulated in nucleoplasmic aggregates and caused the redistribution of endogenous A-type lamins from the peripheral lamina to these aggregates (Dechat et al., 2000a
; Izumi et al., 2000
). In cells containing nuclear aggregates of mutated lamins, a significant fraction of hLEM2 localized to the presumed ER (Fig. 4D, arrows) whereas, in untransfected cells, hLEM2 was localized predominantly at the NE. As the mutated lamins clearly caused a mislocalization of endogenous lamin A/C in these cells (Fig. 4E), this finding is consistent with the hypothesis that loss of lamin A/C at the NE destabilizes the anchorage of LEM2 at the INM and causes its diffusion into the ER.
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To test whether hLEM2 associates with A-type lamins, we performed blot overlay assays. 35S-labelled full-length hLEM2 (FL) or an N-terminal hLEM2 fragment (NT) or LAP2 were overlaid onto immobilized GST-tagged lamin C fragments comprising the lamin C head, rod or tail domains, and onto immobilized LAP2
(Dechat et al., 2000a
). Similar to the known lamin C-binding protein LAP2
, hLEM2 strongly bound to the C-terminus of lamin C (Fig. 4F, lane 3). The lamin C fragments containing the lamin C head (lane 1) or rod (lane 2) or LAP2
(lane 4) or GST alone (lane 5) did not bind hLEM2 above background level. The N-terminus of hLEM2 also bound to the lamin C C-terminus, although the interaction was weaker than those of full-length LEM2 (Fig. 4F). Altogether, our data strongly support a role of A-type lamins in targeting and stabilizing hLEM2 at the NE by a direct interaction.
The N-terminus of hLEM2 is required for retention at the INM
To determine the domain(s) of hLEM2 responsible for NE targeting and retention, we investigated the localization of hLEM2 fragments with a C-terminal V5 tag in HeLa cells. Whereas fragments lacking the C-terminus, including the MSC and the second transmembrane region (Fig. 5, LEM2CT), still localized to the NE in a continuous rim, fragments missing nearly the complete N-terminus were no longer retained at the NE and localized mostly to the ER (LEM2
LEM+NT). This suggested that the N-terminus is essential for NE retention of LEM2. As LEM2 fragments missing a
70aa region (aa 130-203) within the N-terminus close to the first transmembrane domain (LEM2
NT) behaved like wild-type protein, we concluded that the potential N-terminal nuclear retention signal is located upstream of position 130. To test whether the N-terminal retention signal is sufficient for LEM2 targeting, we expressed the N-terminus without the transmembrane domains and the C-terminus (LEM2-NT). This fragment localized to the nucleus but did not accumulate at the NE. Thus, the N-terminus is sufficient for nuclear targeting but not for targeting to the INM. The latter process requires an additional transmembrane region. This observation is consistent with previous studies showing that the localization of INM proteins to the nuclear periphery requires both a transmembrane domain and a retention signal mediating binding to a nuclear component (Holmer and Worman, 2001
). In order to test whether the retention signal might reside within the LEM domain, which could retain LEM2 in the INM by binding to the chromatin protein BAF, we expressed the N-terminal 74 aa fragment of LEM2 containing the entire LEM motif (LEM2-LEM). However LEM2-LEM localized throughout the cell, indicating that the LEM domain of LEM2 is not sufficient for nuclear retention. On the basis of our observation that NE targeting of LEM2 is dependent on the presence of A-type lamins, we reasoned that the retention domain within the N-terminus of LEM2 might mediate the association with lamin A/C complexes. To test this possibility, we expressed the LEM2 N-terminus in Lmna/ MEFs. In contrast to wild-type HeLa cells, LEM2-NT did not accumulate in the nucleus in lamin A-deficient cells (Fig. 5, lower right panel). Taking all data together, targeting of LEM2 to the NE requires both a transmembrane domain and an N-terminal retention signal located between residues 74-130, which mediates association with lamin A/C complexes.
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To test whether formation of these LEM2 clusters can affect the distribution of other INM or lamina proteins, we analysed the localization of various NE proteins in transfected versus untransfected cells by double immunofluorescence microscopy. Lamin B1 (data not shown) and the lamin B-binding partner LBR (Fig. 7) did not significantly accumulate in LEM2 clusters. By contrast, A-type lamins were partially enriched in LEM2 patches, although they still localized throughout the entire NE (Fig. 7, arrowheads). Furthermore, emerin, which interacts with lamin A (Clements et al., 2000; Holaska et al., 2003
), and BAF, which might be associated with the LEM domains of LEM2 and/or emerin, showed the most dramatic reorganizations to LEM2 clusters at the NE (Fig. 7). Therefore, we concluded that highly overexpressed LEM2 accumulated in patches in the INM and recruited A-type lamins and A-type lamin-binding proteins, whereas B-type lamins and their interaction partners (LBR) were not affected. These results indicate that LEM2 is associated with lamin A/C structures and support the model of a lamin A/C-dependent stabilization and retention of LEM2 at the NE shown above. Interestingly, overexpressed hMAN1-V5 did not accumulate in patches, but coexpression of MAN1-V5 and LEM2-GFP caused accumulation of both proteins in clusters, indicating that LEM2 can also recruit MAN1.
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LEM2 overexpression induced formation of tubular structures between nuclei
Unlike untransfected control cells, a significant number (up to 10%) of LEM2-overexpressing cells contained tubular structures between nuclei of adjacent cells. These structures, which also contained LEM2, were up to 30 µm long and originated frequently from LEM2 patches at the NE (Fig. 8B, arrows). Double staining of these cells for actin (Fig. 8A) revealed that the tubular connections occur between individual cells that have completed cytokinesis. Furthermore, the tubular structures seem to be stable and can be maintained for a long time, as we detected up to four interconnected cells (Fig. 8F). Regarding the biogenesis of these tubular structures, it seems very likely that they are formed during NE assembly in telophase and G1. LEM2-containing tubular structures extend from LEM2 patches on chromatin in telophase and increase in length during progression to G1 phase (Fig. 8E, arrows). The formation of the tubular connections between adjacent cells apparently did not affect cell-cycle progression and subsequent cell divisions. Connected interphase cells contain phospho-histone 3, a marker for dividing cells (Fig. 8C), and cell pairs in late telophase/G1 phase can be linked to large interphase cells (Fig. 8D), indicating that cells with tubular connections can divide normally.
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Discussion |
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LEM2 is an INM protein linked to A-type lamin complexes
Our studies demonstrate that LEM2 is localized to the INM and is a genuine lamina protein, characterized by its co-fractionation with insoluble lamin structures upon treatment of nuclei with detergent and high salt (Foisner and Gerace, 1993). Unfortunately, our efforts to produce antibodies against N-terminal peptides remained unsuccessful and the characterization of mammalian LEM2 thus relies on ectopic expression data. Interestingly, we found that LEM2 required A-type lamins at the nuclear periphery for NE targeting. Lack of A-type lamins or disruption of endogenous lamin A structures caused a mislocalization of LEM2 to the ER. Thus, LEM2 can be considered as an A-type lamin-associated protein. Our in vitro binding data revealing an interaction of in-vitro-translated LEM2 with lamin C suggest direct interaction of these proteins, which is in accordance with various studies on Ce-LEM2 and human MAN1. First, Ce-LEM2, which is the only LEM2-type protein to date that has been analysed by biochemical and genetic means, also required lamins for its NE localization and has been shown to interact with Ce-lamin through the N-terminus (Ce-LEM2; aa 1-333) in blot overlay assays (Liu et al., 2003
). Second, the N-terminus of MAN1 was found to interact in vitro with the globular tail domains of both A- and B-type lamins (Mansharamani and Wilson, 2005
). In line with these findings, we also found that the domain mediating lamin A-dependent retention of LEM2 in the NE is localized in a region
60 residues in length within the N-terminus of LEM2. A similar behaviour has been described for emerin, which binds to A-type lamins in vitro (Clements et al., 2000
; Holaska et al., 2003
) and requires A-type lamins for NE localization (Sullivan et al., 1999
; Vaughan et al., 2001
).
Potential functions of LEM2
High-level overexpression of LEM2 caused accumulation of the protein in patches at the NE and formation of NE invaginations, effects that were not observed with overexpressed human MAN1. Intriguingly, lamin A, emerin and BAF were, unlike lamin B1 and LBR, also reorganized to these structures, suggesting a role of LEM2 in the structural organization of a subset of NE components. As this subset of NE proteins included mostly proteins that have previously been detected in A-type lamin complexes, we propose that LEM2 is involved in the organization of A-type lamins within the NE. Given the fact that emerin and BAF seem to be recruited into these patch-like structures more efficiently than A-type lamins, it is tempting to speculate that these proteins might bind directly to LEM2 with high affinity. Whereas binding of BAF to LEM2 might be mediated by the LEM domain and a C-terminal region (Liu et al., 2003), binding of emerin might be mediated by a region in LEM2 related to MAN1, as an interaction between emerin and MAN1 has recently been demonstrated (Mansharamani et al., 2005
). Interestingly, the LEM2 structures seem to be distinct from lamin B-containing protein complexes. Previous studies showed that overexpressed GFP-tagged LBR (Ellenberg et al., 1997
), as well as endogenous LBR, accumulated in `microdomains' within the NE (Makatsori et al., 2004
). These observations suggest that the NE might be organized in different subdomains, each of which contains a specific subset of INM and lamina proteins.
In addition to LEM2 patches, we also observed tubular structures interconnecting nuclei of adjacent cells, which contained LEM2, lamin A, emerin, MAN1 and BAF, but rarely lamin B and LBR. Currently, the molecular details of how these membranous internuclear connections are formed are unknown. Analyses of the appearance of these structures in living cells suggested that they are formed at telophase emanating from LEM2 patches at chromatin, but we did not see anaphase bridges described in Ce-LEM2Ce-emerin double-deficient or in Ce-BAF-deficient worms (Liu et al., 2003). Although we detected little chromatin in about 20% of these structures, we never found phosphorylated histone H3 as described in C. elegans. Excess LEM2 might disturb the assembly of A-type lamins and/or associated proteins, such as emerin and BAF, during post-metaphase NE reassembly. One could speculate that the internuclear bridges in LEM2-overexpressing cells are also caused by a defect in nuclear membrane assembly after sister chromatid separation, although the insertion of functional NPCs into these bridges was not observed. Interestingly, connected cells remained replication competent and re-entered mitosis normally. Altogether, the observed phenotypes upon overexpression of LEM2 imply functions of the protein in membrane assembly and in the dynamic NE organization during the cell cycle.
The properties of LEM2 presented here also make the protein an interesting candidate for involvement in laminopathy-type diseases (Burke and Stewart, 2002; Gotzmann, 2004; Gruenbaum et al., 2005
; Hutchison and Worman, 2004
; Mounkes and Stewart, 2004
). First, LEM2 was found to associate with A-type lamin complexes. Disease-linked mutations in the LMNA gene could disrupt the potential function of this complex in nuclear and chromatin organization. Second, upon loss of lamin A, LEM2 behaved exactly like emerin, implying overlapping functions of these proteins also in vertebrates. As mutations in emerin were found to cause Emery-Dreifuss muscular dystrophy (EDMD) (Bione et al., 1994
; Emery, 1987
; Emery and Dreifuss, 1966
; Manilal et al., 1996
), LEM2 might be linked to similar diseases. In this context, it is important to note that only 40% of clinically diagnosed EDMD cases are linked to mutations in either lamin A or emerin, whereas 60% of cases are probably caused by mutations in other NE components with similar functions as lamin A/C and emerin. Third, mutations in MAN1 have recently been linked to osteopoikilosis, Buschke-Ollendorf syndrome and melorheostosis characterized by increased bone density (Hellemans et al., 2004
). However, it is likely that at least some of the clinical phenotypes detected in these diseases are related to the recently identified role of MAN1 as an antagonist of the pathways mediated by BMP, TGF-ß or activin (Lin et al., 2004; Lin et al., 2005
; Osada et al., 2003
; Pan et al., 2005
). So far, we have not been able to detect a similar antagonistic signalling activity for LEM2 (our unpublished data), most likely because LEM2 lacks the C-terminal RRM motif, known to mediate binding to the R-Smads (Osada et al., 2003
; Pan et al., 2005
). Nevertheless, signalling-independent functions of MAN1 might also contribute to the disease phenotype, in which case one would also expect clinical symptoms for loss of LEM2.
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