1 Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, 1030 Vienna, Austria
2 Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
3 CREST of JST Kansai Advanced Research Center, Kobe 651-2492, Japan
4 Department of Chemistry, Faculty of Sciences, Niigata University, Niigata 950-2181, Japan
* Author for correspondence (e-mail: roland.foisner{at}meduniwien.ac.at)
Accepted 10 September 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Chromosomes, Lamins, Mitosis, Nuclear assembly, Nuclear envelope
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to lamins, numerous lamin-binding proteins (Burke and Stewart, 2002; Foisner, 2001
; Simos and Georgatos, 1992
; Ye and Worman, 1994
) define the structure and function of lamin complexes. Lamin B receptor (LBR) is a protein of the inner membrane with eight transmembrane domains (Worman et al., 1990
) and binds B-type lamins (Simos and Georgatos, 1992
; Ye and Worman, 1994
). Lamina-associated polypeptide 2 (LAP2) is a family of six alternatively spliced proteins, of which four, LAP2ß, -
, -
and -
are type II membrane proteins (Berger et al., 1996
; Dechat et al., 2000b
; Harris et al., 1994
). LAP2
is structurally and functionally different, sharing only the N-terminus with the other isoforms. LAP2ß binds lamin B (Foisner and Gerace, 1993
; Furukawa et al., 1998
) at the NE whereas LAP2
interacts with A-type lamins in the nucleoplasm (Dechat et al., 1998
; Dechat et al., 2000a
). Emerin is an inner nuclear membrane protein (Manilal et al., 1996
) that binds both A- and B-type lamins in vitro (Clements et al., 2000
; Lee et al., 2001
; Sakaki et al., 2001
) and is retained in the NE by A-type lamins (Holt et al., 2003
; Sullivan et al., 1999
; Vaughan et al., 2001
). All LAP2 isoforms, emerin and the inner membrane protein, MAN1 (Lin et al., 2000
) share a
40 amino acid-long structural motif, the lamina-associated polypeptide emerin MAN1 or LEM domain (Cai et al., 2001
; Laguri et al., 2001
), which mediates binding to BAF (Furukawa, 1999
; Shumaker et al., 2001
). BAF is a small highly conserved protein in multicellular eukaryotes that binds double stranded DNA without sequence specificity (Zheng et al., 2000
).
In higher eukaryotes the nucleus is transiently disassembled during mitosis because of a phosphorylation-dependent disassembly of the lamina and pore complexes (Burke and Ellenberg, 2002; Foisner, 2003
), a process facilitated by a microtubule/dynein-mediated deformation and disruption of the nuclear envelope (Beaudouin et al., 2002
; Salina et al., 2002
). Mitosis-specific phosphorylation of lamins was found to be essential for lamina disassembly (Heald and McKeon, 1990
) and lamin-binding proteins are also phosphorylated in mitosis (Dechat et al., 1998
; Foisner and Gerace, 1993
; Nikolakaki et al., 1997
).
After sister chromatid separation the NE and nuclear structure reassemble in a tightly regulated manner, ensuring that the interphase chromatin organization can be re-established in daughter nuclei (Cohen et al., 2001). Nuclear reassembly requires phosphatase activity and, at least for B-type lamins, has been shown to involve the membrane-associated phosphatase PP1 (Steen and Collas, 2001
; Thompson et al., 1997
). The kinetics of the association of lamins and some lamin-binding proteins with the reforming nucleus have been studied in the past years. LBR and emerin are targeted to chromosomes about 5 minutes after metaphase-anaphase transition, followed by nuclear pore complex assembly (Haraguchi et al., 2000
). LAP2ß was also found to accumulate at around the same time as emerin (Bodoor et al., 1999
; Dabauvalle et al., 1999
; Foisner and Gerace, 1993
). B- and A-type lamins follow different pathways (Moir et al., 2000
). Stable B-type lamin structures are seen at the nuclear envelope after pore complex assembly (Chaudhary and Courvalin, 1993
; Dabauvalle et al., 1991
; Daigle et al., 2001
) followed by the bulk of lamin A that accumulates in the nuclear interior and at the NE later. Addition of protein mutants or antibodies to in vitro nuclear assembly assays or transient expression of mutants in cells has implicated lamins, LBR, LAP2ß and LAP2
in nuclear assembly (Gant et al., 1999
; Lopez-Soler et al., 2001
; Lourim and Krohne, 1994
; Pyrpasopoulou et al., 1996
; Vlcek et al., 2002
), but the molecular mechanisms remain unclear.
Here we perform detailed analyses of the dynamics of LAP2 during nuclear assembly in relation to other NE and chromatin proteins and show several major new findings. Following initial binding to telomeres, LAP2
is the first among a group of lamina proteins, including lamin C and emerin, detectable in the `core' structures on chromatin that have been described previously (Haraguchi et al., 2001
). In contrast, LBR, LAP2ß and lamin B initially bind to distinct, peripherally located regions of the chromatin bulk, and spread to core structures later. In support of previous studies implicating BAF in NE assembly (Haraguchi et al., 2001
; Segura-Totten et al., 2002
), we provide evidence that a subfraction of BAF relocalizes to core structures together with LAP2
. We propose that these structures may define chromatin organization in the reassembling nucleus.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture, transfection and synchronization
HeLa and NRK cells were routinely maintained in high glucose DME medium supplemented with 10% FCS, 10 mM HEPES, pH 7.0 and 50 µg/ml penicillin and streptomycin (Life Technologies, Paisley, UK) at 37°C and 5% CO2. For generation of stable HeLa cell clones expressing GFP- or YFP-LAP2, cells were transfected with plasmids using the standard calcium phosphate method and clones were selected in medium containing 700 µg/ml G418 (Life Technologies) and maintained in medium plus 100 µg/ml G418. Transient transfections were performed with Fugene 6 (Roche, Mannheim, Germany). For live cell microscopy, cells were cultured in two-well LabTek chambers with glass bottoms (Nunc, Rochester, NY) and cell growth was synchronized by arresting cells 24 hours post transfection in 0.5 µg/ml aphidicolin (Sigma-Aldrich, St Louis, MO) for 15 hours, followed by a 7-hour release in complete medium. For imaging, Phenol Red-free DME supplemented with 20% FCS and 0.5 mg/ml acetylsalicylic acid was used.
Immunofluorescence microscopy
Cells on plastic dishes were fixed with 3.7% formaldehyde or 2% paraformaldehyde in PBS for 20 minutes at room temperature, followed by incubation in 50 mM NH4Cl/PBS and 1% Triton X-100/0.1% SDS for 5 minutes each. Samples were incubated in 0.2% gelatin/PBS for 30 minutes prior to antibody incubation. Primary and secondary antibodies were applied in gelatin/PBS for 1 hour each at room temperature. Primary antibodies were goat antiserum N18 against lamins A/C (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit antiserum against LAP2 (Vlcek et al., 2002
), antiserum to BAF (Furukawa, 1999
), monoclonal antibody 4A794 to TRF2 (Upstate Biotechnology, Lake Placid, NY) and monoclonal antibodies 15-2 and 17 to LAP2
and LAP2ß, respectively (Dechat et al., 1998
); secondary antibodies used are donkey anti-goat IgG conjugated to Cy3 and goat anti-mouse IgG conjugated to Texas Red (Jackson ImmunoResearch, West Grove, PA) and goat anti-rabbit IgG conjugated to Alexa488 (Molecular Probes, Leiden, The Netherlands). DNA was stained with 1 µg/ml Hoechst dye 33258 (Calbiochem-Behring) for 10 minutes. Samples were mounted in Mowiol and viewed in a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany).
4D multicolor live cell imaging and image processing
Live cell imaging was performed at 37°C maintained by an air stream incubator (ASI 400; Nevtek, Burnsville, VA) in conjunction with an objective heater (Bioptechs, Butler, PA) on a customized LSM 510 confocal microscope fitted with a z-scanning stage, selected photomultiplier tubes (Carl Zeiss), a Kr 413 nm laser (Coherent, Dieburg, Germany) and custom dichroics and emission filters (Chroma, Brattleboro, VT) for fluorescent protein imaging as described elsewhere (Daigle et al., 2001; Gerlich et al., 2001
). Mitotic events were monitored with a PlanApochromat 63x N.A. 1.4 oil DIC objective (Carl Zeiss) every 10 seconds for 3 minutes. Image series from a single confocal z-plane representing selected time points were assembled and single confocal z-stacks of each time point were projected with the LSM 510 2.3 software (Carl Zeiss) and combined into movies using ImageJ software (http://rsb.info.nih.gov/ij/). False colors for optimal presentation were used: mainly green for YFP and red for CFP. Rendering of four-dimensional images was by graphical reconstruction. Noise levels were reduced using an anisotropic diffusion filter on individual image slices (Gerlich et al., 2001
). Isosurface reconstruction and rendering was carried out using the Amira 2.3 software package (TGS, San Diego, CA, USA). Photobleaching was done as described previously (Daigle et al., 2001
).
Chromosome spreads
Mitotic cells harvested from an unsynchronized culture were incubated in 0.075 mM KCl for 20 minutes at room temperature and lysed by adding 0.1% Tween 20. Samples were spun onto coverslips at 500 g for 3 minutes with a Cytospin 2 (Therme Shandon, Pittsburgh, USA), fixed either in methanol (-20°C for 5 minutes) or 2% formaldehyde (10 minutes at room temperature) and processed for immunofluorescence microscopy.
Immunoprecipitation
Mitotic NRK cells were lysed in KHM buffer and chromosomes spun out as described (Vlcek et al., 2002). The lysate was pre-cleared by addition of 50 µl 50% protein G-sepharose beads and centrifugation at 400 g for 5 minutes with a tabletop centrifuge. 10 µl protein G-sepharose beads, preincubated in 1 ml of hybridoma supernatant containing antibody to LAP2
or 10 µl untreated beads (control) were added to the lysates and harvested by centrifugation following a 2 hour incubation. Beads were washed in KHM buffer and proteins solubilized in 3x SDS-PAGE sample buffer.
LAP2-BAF in vitro binding
BAF was in vitro translated using the TNT® Quick Coupled Transcription/Translation System (Promega) from a pET15b vector (Novagene) containing full-length BAF (Lee and Craigie, 1998). The translation product was diluted sevenfold in binding buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.1% Triton, 1 mM DTT and 1 mM PMSF), precleared and used for coimmunoprecipitation. Recombinant LAP2
was expressed (Vlcek et al., 1999
; Vlcek et al., 2002
), renatured by dialyzing into binding buffer and centrifuged at 1500 g for 5 minutes. 100 µl BAF sample was mixed with 100 µl recombinant LAP2
or 100 µl binding buffer as a control and transferred to 50 µl protein G-sepharose beads, preincubated in 1 ml hybridoma supernatant containing antibody to LAP2
. Following a 30-minute incubation, beads were underlaid with 30% sucrose and harvested by centrifugation.
Polyacrylamide gel electrophoresis and immunoblotting
SDS-PAGE was performed according to Laemmli (Laemmli, 1970). For solubilizing BAF, 6 M urea was added to sample buffer. For immunoblotting, primary antibodies were hybridoma supernatants of LAP2 antibodies (Dechat et al., 1998
) and BAF antiserum 3273 (Haraguchi et al., 2001
); secondary antibodies, alkaline phosphatase or peroxidase-conjugated goat antibodies. Proteins were detected with the Protoblot Immunoscreening System (Promega) or the Super Signal ECL (Pierce, Rockford, CA, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
LAP2 associates with telomeric regions of chromosomes
The preferred association sites of LAP2 at the tips of lagging chromosomes observed by live cell imaging suggested localization at telomeric structures. To address this possibility we performed double immunofluorescence microscopy of fixed cells using antibodies to LAP2
and to the telomeric TTAGGG repeat binding factor 2 (TRF2) (Broccoli et al., 1997
; van Steensel et al., 1998
). Although in interphase cells TRF2 antibody stained dot-like telomeres throughout the nucleoplasm, the antibody had a complex dotted pattern on telophase chromosomes. Here, we observed partial overlap of TRF2 structures with LAP2
on chromatin (Fig. 2A), consistent with a targeting of LAP2
to telomeric regions in the early phases of nuclear reassembly.
|
To identify LAP2 on individual chromosomes we prepared mitotic chromosome spreads. Both a mouse monoclonal antibody (Fig. 2Ba) and a rabbit antiserum to LAP2
(Fig. 2Bb-d) detected LAP2
at the tips of chromosomes, whereas antibodies to LAP2ß (Fig. 2Be), or to lamins (data not shown) stained dots scattered throughout the sample without a preferred telomere association. The LAP2
structures on chromosomes differed significantly in size ranging from small dots (Fig. 2Bb) to larger clusters (Fig. 2Bc,d). Furthermore, most of the chromosomes in the preparations with LAP2
localized at the tips contained two chromatids, representing a metaphase stage, whereas in living cells LAP2
patches were first detected at chromosomes in anaphase after sister chromatid separation. From previous studies we knew that binding of LAP2
to chromosomes is controlled by phosphorylation (Dechat et al., 1998
; Vlcek et al., 2002
) and that incubation of mitotic cell extracts induces dephosphorylation-dependent association of LAP2
with chromosomes. Thus, we hypothesized that during chromosome spread preparation the cells proceeded partially to post-metaphase stages without chromatid separation owing to the lack of a functional spindle. This may induce dephosphorylation and chromosome association of LAP2
. In line with this model, spreads prepared in the presence of phosphatase inhibitors rarely showed LAP2
at telomeres (data not shown). Thus, the preparation of mitotic chromosome spreads in the absence of phosphatase inhibitors may represent the narrow time window in nuclear assembly during which LAP2
associates at chromosomes.
LAP2 forms a stable structure at chromatin cores but turns over rapidly in interphase
We next investigated how stably LAP2 was associated with chromosomes during nuclear reassembly by fluorescence recovery after photobleaching (FRAP) analysis in cells expressing GFP-LAP2
(Fig. 2C). In interphase cells, FRAP revealed that photobleached regions in the nucleoplasm rapidly recovered to almost their prebleach intensity within 8 seconds. This suggested that LAP2
structures in the nucleoplasm turn over very rapidly as also shown previously for certain proteins in the NPC (e.g. Nup153) (Daigle et al., 2001
) and heterochromatin (HP1) (Cheutin et al., 2003
). In anaphase, when LAP2
structures first detectable on chromosomes were bleached, fluorescence still recovered to more than half of the prebleach intensity within 8 seconds. However, part of this recovery was probably due to additional LAP2
binding to chromosomes during ongoing nuclear assembly in the recovery time (see unbleached half of the cell in Fig. 2C) rather than recovery to a steady-state situation. In telophase, when the majority of LAP2
was already associated with core structures of chromatin, bleached areas recovered to only about 10% of their prebleach intensity, suggesting that LAP2
was stably associated with these core structures.
Differential localization of LAP2 and lamins during assembly
Unlike B-type lamins, A-type lamins interact directly with LAP2 in the nucleoplasm, particularly during G1 phase (Dechat et al., 2000a
). To compare the kinetics of LAP2
redistribution to chromosomes with those of A- and B-type lamins, we transiently expressed CFP-tagged lamin C or lamin B1 in stable YFP-LAP2
-expressing cells and performed live cell imaging. Both lamins localized primarily at the nuclear periphery in interphase and were dispersed throughout the cytoplasm in mitosis (Fig. 3A). During nuclear reassembly, telomeric LAP2
structures (arrowheads) were clearly detectable before the accumulation of lamins C or B at the chromatin surface, although small fractions of lamins may surround chromatin throughout all stages of assembly. Lamin C and lamin B (arrows) were both detected at higher concentrations on chromatin approximately 1 minute after the first occurrence of telomeric LAP2
, but their localization was different (Moir et al., 2000
). A small subfraction of lamin C colocalized with LAP2
at core structures during early stages, but the majority of lamin C was translocated to the nuclear interior at later stages when LAP2
relocated to the nucleoplasm. Although the translocation of A-type lamins into the nucleoplasm during telophase/G1 has been described (Moir et al., 2000
), the early association of a subfraction of A-type lamins with chromosomes in anaphase was not observed. To make sure that our observation was not an artifact due to lamin C overexpression, we performed immunofluorescence microscopy of untransfected, fixed cells using antibodies to lamin C. Endogenous lamin A/C behaved in a manner similar to the ectopic protein, partially overlapping with LAP2
structures at chromosomes during anaphase (Fig. 3B).
|
In contrast to lamin C, the first enrichment of lamin B on chromosomes did not overlap with telomeric LAP2. Lamin B accumulated initially at chromatin surfaces next to the spindle pole, forming a cap-like structure opposite of the major telomeric LAP2
structures. From there lamin B spread over the entire surface of decondensing chromatin and formed a continuous envelope only when LAP2
translocated to the nucleoplasm (Fig. 3A).
LAP2 association with chromosomes differs from that of nuclear membrane proteins
To test where and when during assembly nuclear membrane proteins associated with chromosomes in relation to LAP2, we transiently expressed CFP fusion proteins of LBR and LAP2ß, both binding partners of B-type lamins (Foisner and Gerace, 1993
; Furukawa et al., 1998
; Meier and Georgatos, 1994
; Worman et al., 1988
; Ye and Worman, 1994
) and emerin, which binds A-type lamins (Lee et al., 2001
; Sakaki et al., 2001
). As expected, all proteins localized to the nuclear periphery in interphase (Fig. 4). LAP2ß was first detectable at chromosomes slightly after LAP2
during nuclear reassembly (Vlcek et al., 2002
) whereas LBR appeared at chromosomes at detectable levels slightly before LAP2
(see Movies 2 and 3 in supplementary material). However, both LAP2ß and LBR mostly localized to more peripheral sites of the chromatin bulk, distinct from the LAP2
-containing core regions (see also Dabauvalle et al., 1999
; Haraguchi et al., 2000
). In contrast to LBR, LAP2ß also accumulated on chromatin core structures, before both LBR and LAP2ß spread over the entire surface of decondensing chromatin at later stages of assembly. Emerin showed a mixed appearance in terms of its association sites. Although the first emerin staining on chromosomes was detected on the peripheral chromatin regions like LBR and LAP2ß, the majority of emerin accumulated subsequently at the LAP2
core structures. Altogether, we observed a temporally and spatially highly coordinated accumulation of lamina proteins with chromosomes during nuclear assembly. LAP2
and LBR became concentrated on anaphase chromatin first, whereas other membrane proteins and lamins were detectable at significant levels slightly later. Furthermore, two groups of lamina proteins with different preferred chromosome association sites could be distinguished. LAP2
, followed by emerin and A-type lamins formed the core region of chromatin, whereas LBR followed by LAP2ß and lamin B bound to more peripheral chromatin regions initially.
|
LAP2 and BAF colocalize at chromatin-associated core structures
The initial binding of LAP2 to telomere patches and formation of chromatin-associated LAP2
core structures could indicate an important role of LAP2
early in nuclear assembly. Consistent with this hypothesis, in in vitro nuclear assembly studies we have previously shown that C-terminal LAP2
fragments bind to chromosomes and dominantly inhibit assembly of nuclear membranes and lamin A around chromosomes (Vlcek et al., 2002
). As the dominant-negative LAP2
mutants lacked the LEM motif, which mediates binding to the DNA crosslinking protein BAF, and BAF mutants deficient in binding to the LEM domain were found to inhibit the assembly of emerin and lamin A in vivo (Haraguchi et al., 2001
), we reasoned that complexes of LAP2
and BAF may be involved in early stages of nuclear assembly. To test this hypothesis, we performed immunofluorescence microscopy of HeLa cells at different mitotic stages using antibodies to LAP2
and BAF. Although the majority of BAF was uniformly localized at the chromosomes in early anaphase, endogenous LAP2
was predominantly cytoplasmic (Fig. 5a). However, when LAP2
was first detectable at the chromosomes, a small fraction of endogenous BAF was also enriched at these structures (Fig. 5b, arrowheads) while still present on the entire chromatin and to some extent also in the cytoplasm. Interestingly, upon formation of telomeric LAP2
structures (Fig. 5c) and core structures in telophase (Fig. 5d), BAF also localized to these structures, whereas it was barely detectable throughout the chromatin or cytoplasm at this time. Thus, cytoplasmic and/or chromosome-associated BAF apparently relocalized to core structures on chromatin with similar timing as LAP2
during nuclear assembly. However, we cannot exclude the fact that BAF is still present in the nuclear interior, but epitopes were masked owing to reorganization of chromatin. Other lamina proteins, such as LAP2ß, did not strictly colocalize with BAF at core structures at this stage of assembly, but showed a broader distribution over the entire surface of chromatin (Fig. 5e).
|
BAF and LAP2 form a complex in vitro and in post-metaphase cell lysates
To test whether LAP2 and BAF may indeed bind to each other, we investigated the interaction of these proteins in vitro. Although LAP2
was expected to bind BAF via its N-terminal LEM motif, it was important to demonstrate the binding directly, because previous studies using different Xenopus LAP2ß isoforms indicated that their slightly different C-termini modulated binding to BAF at the N-terminal LEM motif (Shumaker et al., 2001
). Therefore, BAF was produced by in vitro transcription/translation in reticulocyte cell lysates and mixed with purified recombinant LAP2
. Immunoprecipitation of LAP2
significantly coprecipitated BAF (Fig. 6A). Control experiments in the absence of LAP2
or with a C-terminal LAP2
fragment lacking the LEM domain did not precipitate BAF. Thus, BAF is able to interact with the LEM motif in LAP2
in vitro.
|
In order to confirm the existence of LAP2-BAF complexes in cells at early stages of assembly, we performed coimmunoprecipitation assays. We synchronized NRK cell cultures using a thymidine block, harvested metaphase cells by mechanical shake-off and lysed cells in a metal ball homogenizer. As up to 50% of total BAF (Holaska et al., 2003
; Lin and Engelman, 2003
) and the majority of LAP2
(Dechat et al., 1998
; Vlcek et al., 2002
) are found in the cytoplasm in metaphase cells, we spun out chromosomes from cell lysates and incubated the supernatant for 5-10 minutes at room temperature in order to allow partial post-metaphase assembly of soluble LAP2
complexes. Immunoprecipitation of these LAP2
structures from the lysates precipitated BAF, whereas neither LAP2
nor BAF were precipitated with control beads lacking antibodies (Fig. 6B). Interestingly, BAF in cell lysates and in soluble fractions behaved as a monomeric protein in SDS-PAGE, whereas BAF coprecipitated with LAP2
was primarily detectable as a putative dimer or oligomer (or a posttranslationally modified species) that migrated at approximately 20 kDa on SDS-PAGE (Fig. 6B). We speculate that LAP2
binding to BAF may change the conformation of BAF (see also Forne et al., 2003
) and stabilize dimeric or oligomeric forms that become oxidatively crosslinked during sample preparation, and migrate aberrantly on SDS-PAGE (Zheng et al., 2000
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We also demonstrate an in vitro interaction between LAP2 and BAF via the N-terminal region of LAP2
containing the LEM domain, and provide evidence for the existence of LAP2
-BAF complexes in early post-metaphase stages in situ. Accordingly, a large fraction of BAF colocalized with LAP2
at chromatin core regions. It is still unclear whether BAF at LAP2
core structures originated from the cytoplasmic pool of BAF (Holaska et al., 2003
; Lin and Engelman, 2003
), which could translocate to core regions in a complex with LAP2
, or whether chromosome-bound BAF redistributed transiently to core structures upon binding of LAP2
. The lack of BAF detection on chromatin outside the core regions in telophase would argue for the latter possibility, however, the epitopes of BAF in the internal chromatin structure may be masked at this time. As we found that various N- and C-terminally tagged BAF forms did not behave exactly as endogenous BAF in terms of homo-oligomerization, DNA binding and interaction with LEM-domain proteins (our unpublished data), we could not test GFP-tagged proteins to overcome potential problems of epitope masking.
Other lamina and NE proteins did not colocalize with LAP2 at telomere patches, but a subset of them, including a subfraction of lamin C, emerin, and to some extent also LAP2ß, were targeted to the core regions slightly after LAP2
-BAF (Fig. 7). Targeting of these proteins to chromatin cores may be mediated by direct interaction of lamin C with LAP2
or by binding of the LEM-domain proteins, emerin and LAP2ß, to BAF. Another group of lamina proteins showed a different pattern of assembly. LBR association with chromatin occurred at more peripheral sites, distinct from the LAP2
-BAF core complexes. LBR targeting to chromosomes may be the trigger for attaching membranes to chromatin and may initiate nuclear membrane assembly. Other membrane proteins, such as emerin and LAP2ß may then diffuse within the lipid bilayer and form stable complexes at the chromatin surface by specific interactions with BAF, HA95 (Martins et al., 2003
), heterochromatin protein 1 (HP1, Ye and Worman, 1996
), histones (Goldberg et al., 1999
; Polioudaki et al., 2001
; Taniura et al., 1995
) or DNA (for a review, see Vlcek et al., 2001
).
Significance of telomere association of LAP2 and of LAP2
-BAF interaction
Our findings show that LAP2 does not bind uniformly to chromosomes during assembly, as one might have expected from our previous in vitro binding studies (Vlcek et al., 1999
). Instead, LAP2
initially associates with telomeres and subsequently forms larger complexes, before it relocates into the nuclear interior in late telophase. It is unclear whether the transient interaction of LAP2
with telomeres is mediated by a telomere-associated protein complex or by a direct interaction with DNA. Based on its previously reported properties as a nucleoskeletal component (Dechat et al., 1998
), we hypothesize that the specific targeting of LAP2
to telomeres provides a mechanism by which telomeres are positioned within the reforming nucleus during the establishment of higher order chromatin structure after cell division.
As LAP2 and BAF localized at core structures simultaneously during nuclear assembly, potential functions of this interaction in nuclear assembly and possibly higher order chromatin organization may be envisaged. First, the association of LAP2
with chromosome-bound BAF may target LAP2
from the cytoplasm to chromosomes during early stages of assembly. This, however, seems very unlikely based on our observations that: (1) BAF localized uniformly at chromosomes in metaphase and early anaphase, whereas LAP2
was targeted preferentially to patches in the vicinity of telomeres; (2) the constant LAP2 N-terminus, which is common to all LAP2 isoforms and contains the LEM domain, did not accumulate at chromosomes at any stage of nuclear reassembly in vivo or in vitro (Vlcek et al., 1999
; Vlcek et al., 2002
); (3) LAP2
did not require its N-terminal LEM domain for chromosome targeting, instead its C-terminus (that does not bind BAF) was found to be essential and sufficient for chromosome interaction (Vlcek et al., 1999
). Second, LAP2
may target the cytoplasmic pool of BAF to chromosomes during nuclear reassembly, which would be consistent with our observations. Third, both proteins could be targeted independently to chromosomes and associate in specific subregions of chromosomes. Alternatively, chromosome-bound BAF may be the predominant binding partner for independently targeted LAP2
, whereas cytoplasmic BAF may not be involved in the assembly at all.
We hypothesize that, independent of the targeting mechanisms of LAP2 and BAF, the complex may help to re-organize chromatin during decondensation and nuclear assembly by various mechanisms: (1) the complex may transiently tether telomeres to stable structures favoring ordered chromatin reorganization. This model is supported by low exchange rates of LAP2
in core structures; (2) the binding of BAF to LAP2
may regulate DNA crosslinking activity of BAF, thus resulting in different DNA (de-)compaction states at defined chromosomal subregions; (3) the formation of LAP2
-BAF complexes may provide docking sites for other lamina proteins favoring their ordered assembly.
The essential role of BAF in cell cycle-dependent chromatin organization has been described previously by means of in vivo and in vitro studies in various systems. Drosophila baf null mutants were lethal at the larval-pupal transition and showed grossly aberrant nuclear structures with chromatin clumps (Furukawa et al., 2003). BAF-depletion in Caenorhabditis elegans by RNA interference caused defects in chromatin segregation (Zheng et al., 2000
). Furthermore, BAF mutants defective in binding the LEM domain and DNA did not accumulate at chromosomes and inhibited assembly of lamin A, emerin and LAP2ß in HeLa cells (Haraguchi et al., 2001
). In vitro, BAF favored or inhibited chromatin decondensation during assembly of nuclei in Xenopus extracts depending on its concentration (Segura-Totten et al., 2002
). Finally, our previous observations have indirectly shown a role of LAP2
-BAF complexes in assembly. C-terminal LAP2
mutants that bound chromosomes but lacked the N-terminal LEM domain (Vlcek et al., 2002
) and were thus unable to interact with BAF, dominantly inhibited chromatin decondensation and nuclear membrane assembly in in vitro assays.
In summary, we suggest a model for nuclear assembly in which transient accumulation of LAP2 and BAF at telomeres and/or chromatin cores mediates chromatin reorganization and NE assembly. However, given that BAF is an evolutionarily conserved protein found in metazoans (Cai et al., 1998
; Lee and Craigie, 1998
), whereas LAP2
has only been detected in vertebrates (Dechat et al., 2000b
; Vlcek et al., 2001
), one has to assume that this mechanism is not essential for nuclear assembly in general, but may contribute to vertebrate-specific chromatin organization.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beaudouin, J., Gerlich, D., Daigle, N., Eils, R. and Ellenberg, J. (2002). Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108, 83-96.[Medline]
Berger, R., Theodor, L., Shoham, J., Gokkel, E., Brok-Simoni, F., Avraham, K. B., Copeland, N. G., Jenkins, N. A., Rechavi, G. and Simon, A. J. (1996). The characterization and localization of the mouse thymopoietin/lamina-associated polypeptide 2 gene and its alternatively spliced products. Genome Res. 6, 361-370.[Abstract]
Bodoor, K., Shaikh, S., Salina, D., Raharjo, W. H., Bastos, R., Lohka, M. and Burke, B. (1999). Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis. J. Cell Sci. 112, 2253-2264.
Broccoli, D., Smogorzewska, A., Chong, L. and de Lange, T. (1997). Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat. Genet. 17, 231-235.[Medline]
Burke, B. and Ellenberg, J. (2002). Remodelling the walls of the nucleus. Nat. Rev. Mol. Cell Biol. 3, 487-497.[CrossRef][Medline]
Burke, B. and Stewart, C. L. (2002). Life at the edge: the nuclear envelope and human disease. Nat. Rev. Mol. Cell Biol. 3, 575-585.[CrossRef][Medline]
Cai, M., Huang, Y., Zheng, R., Wei, S. Q., Ghirlando, R., Lee, M. S., Craigie, R., Gronenborn, A. M. and Clore, G. M. (1998). Solution structure of the cellular factor BAF responsible for protecting retroviral DNA from autointegration. Nat. Struct. Biol. 5, 903-909.[CrossRef][Medline]
Cai, M., Huang, Y., Ghirlando, R., Wilson, K. L., Craigie, R. and Clore, G. M. (2001). Solution structure of the constant region of nuclear envelope protein LAP2 reveals two LEM-domain structures: one binds BAF and the other binds DNA. EMBO J. 20, 4399-4407.
Cao, H. and Hegele, R. A. (2003). LMNA is mutated in Hutchinson-Gilford progeria (MIM 176670) but not in Wiedemann-Rautenstrauch progeroid syndrome (MIM 264090). J. Hum. Genet. 48, 271-274.[Medline]
Chaudhary, N. and Courvalin, J. C. (1993). Stepwise reassembly of the nuclear envelope at the end of mitosis. J. Cell Biol. 122, 295-306.[Abstract]
Cheutin, T., McNairn, A. J., Jenuwein, T., Gilbert, D. M., Singh, P. B. and Misteli, T. (2003). Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 299, 721-725.
Clements, L., Manilal, S., Love, D. R. and Morris, G. E. (2000). Direct interaction between emerin and lamin A. Biochem. Biophys. Res. Commun. 267, 709-714.[CrossRef][Medline]
Cohen, M., Lee, K. K., Wilson, K. L. and Gruenbaum, Y. (2001). Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina. Trends Biochem. Sci. 26, 41-47.[CrossRef][Medline]
Dabauvalle, M. C., Loos, K., Merkert, H. and Scheer, U. (1991). Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin. J. Cell Biol. 112, 1073-1082.[Abstract]
Dabauvalle, M. C., Muller, E., Ewald, A., Kress, W., Krohne, G. and Muller, C. R. (1999). Distribution of emerin during the cell cycle. Eur. J. Cell Biol. 78, 749-756.[Medline]
Daigle, N., Beaudouin, J., Hartnell, L., Imreh, G., Hallberg, E., Lippincott-Schwartz, J. and Ellenberg, J. (2001). Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J. Cell Biol. 154, 71-84.
De Sandre-Giovannoli, A., Bernard, R., Cau, P., Navarro, C., Amiel, J., Boccaccio, I., Lyonnet, S., Stewart, C. L., Munnich, A., le Merrer, M. et al. (2003). Lamin A truncation in Hutchinson-Gilford progeria. Science 300, 2055.
Dechat, T., Gotzmann, J., Stockinger, A., Harris, C. A., Talle, M. A., Siekierka, J. J. and Foisner, R. (1998). Detergent-salt resistance of LAP2alpha in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembly implies functions in nuclear structure dynamics. EMBO J. 17, 4887-4902.
Dechat, T., Korbei, B., Vaughan, O. A., Vlcek, S., Hutchison, C. J. and Foisner, R. (2000a). Lamina-associated polypeptide 2alpha binds intranuclear A-type lamins. J. Cell Sci. 113, 3473-3484.
Dechat, T., Vlcek, S. and Foisner, R. (2000b). Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics. J. Struct. Biol. 129, 335-345.[CrossRef][Medline]
Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L., Presley, J. F., Worman, H. J. and Lippincott-Schwartz, J. (1997). Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138, 1193-1206.
Eriksson, M., Brown, W. T., Gordon, L. B., Glynn, M. W., Singer, J., Scott, L., Erdos, M. R., Robbins, C. M., Moses, T. Y., Berglund, P. et al. (2003). Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. Nature 423, 293-298.[CrossRef][Medline]
Foisner, R. (2001). Inner nuclear membrane proteins and the nuclear lamina. J. Cell Sci. 114, 3791-3792.
Foisner, R. (2003). Cell cycle dynamics of the nuclear envelope. ScientificWorldJournal 3, 1-20.[Medline]
Foisner, R. and Gerace, L. (1993). Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation. Cell 73, 1267-1279.[Medline]
Forne, I., Carrascal, M., Martinez-Lostao, L., Abian, J., Rodriguez-Sanchez, J. L. and Juarez, C. (2003). Identification of the autoantigen HB as the barrier-to-autointegration factor. J. Biol. Chem. 278, 50641-50644.
Furukawa, K. (1999). LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction. J. Cell Sci. 112, 2485-2492.
Furukawa, K., Fritze, C. E. and Gerace, L. (1998). The major nuclear envelope targeting domain of LAP2 coincides with its lamin binding region but is distinct from its chromatin interaction domain. J. Biol. Chem. 273, 4213-4219.
Furukawa, K., Sugiyama, S., Osouda, S., Goto, H., Inagaki, M., Horigome, T., Omata, S., McConnell, M., Fisher, P. A. and Nishida, Y. (2003). Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila. J. Cell Sci. 116, 3811-3823.
Gant, T. M., Harris, C. A. and Wilson, K. L. (1999). Roles of LAP2 proteins in nuclear assembly and DNA replication: truncated LAP2beta proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts. J. Cell Biol. 144, 1083-1096.
Gerlich, D. and Ellenberg, J. (2003). 4D imaging to assay complex dynamics in live specimens. Nat. Cell. Biol. Suppl. S14-S19.
Gerlich, D., Beaudouin, J., Gebhard, M., Ellenberg, J. and Eils, R. (2001). Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells. Nat. Cell Biol. 3, 852-855.[CrossRef][Medline]
Goldberg, M., Harel, A., Brandeis, M., Rechsteiner, T., Richmond, T. J., Weiss, A. M. and Gruenbaum, Y. (1999). The tail domain of lamin Dm0 binds histones H2A and H2B. Proc. Natl. Acad. Sci. USA 96, 2852-2857.
Goldman, R. D., Gruenbaum, Y., Moir, R. D., Shumaker, D. K. and Spann, T. P. (2002). Nuclear lamins: building blocks of nuclear architecture. Genes Dev. 16, 533-547.
Haraguchi, T., Koujin, T., Hayakawa, T., Kaneda, T., Tsutsumi, C., Imamoto, N., Akazawa, C., Sukegawa, J., Yoneda, Y. and Hiraoka, Y. (2000). Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes. J. Cell Sci. 113, 779-794.
Haraguchi, T., Koujin, T., Segura-Totten, M., Lee, K. K., Matsuoka, Y., Yoneda, Y., Wilson, K. L. and Hiraoka, Y. (2001). BAF is required for emerin assembly into the reforming nuclear envelope. J. Cell Sci. 114, 4575-4585.[Medline]
Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. and Weber, K. (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114, 4557-4565.[Medline]
Harris, C. A., Andryuk, P. J., Cline, S., Chan, H. K., Natarajan, A., Siekierka, J. J. and Goldstein, G. (1994). Three distinct human thymopoietins are derived from alternatively spliced mRNAs. Proc. Natl. Acad. Sci. USA 91, 6283-6287.
Heald, R. and McKeon, F. (1990). Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell 61, 579-589.[Medline]
Holaska, J. M., Lee, K. K., Kowalski, A. K. and Wilson, K. L. (2003). Transcriptional repressor germ cell-less (GCL) and barrier to autointegration factor (BAF) compete for binding to emerin in vitro. J. Biol. Chem. 278, 6969-6975.
Holt, I., Ostlund, C., Stewart, C. L., Man-Nt, N., Worman, H. J. and Morris, G. E. (2003). Effect of pathogenic mis-sense mutations in lamin A on its interaction with emerin in vivo. J. Cell Sci. 116, 3027-3035.
Hozak, P., Sasseville, A. M., Raymond, Y. and Cook, P. R. (1995). Lamin proteins form an internal nucleoskeleton as well as a peripheral lamina in human cells. J. Cell Sci. 108, 635-644.
Hutchison, C. J. (2002). Lamins: building blocks or regulators of gene expression? Nat. Rev. Mol. Cell Biol. 3, 848-858.[CrossRef][Medline]
Jagatheesan, G., Thanumalayan, S., Muralikrishna, B., Rangaraj, N., Karande, A. A. and Parnaik, V. K. (1999). Colocalization of intranuclear lamin foci with RNA splicing factors. J. Cell Sci. 112, 4651-4661.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Laguri, C., Gilquin, B., Wolff, N., Romi-Lebrun, R., Courchay, K., Callebaut, I., Worman, H. J. and Zinn-Justin, S. (2001). Structural characterization of the LEM motif common to three human inner nuclear membrane proteins. Structure 9, 503-511.[Medline]
Lee, K. K., Haraguchi, T., Lee, R. S., Koujin, T., Hiraoka, Y. and Wilson, K. L. (2001). Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF. J. Cell Sci. 114, 4567-4573.[Medline]
Lee, M. S. and Craigie, R. (1998). A previously unidentified host protein protects retroviral DNA from autointegration. Proc. Natl. Acad. Sci. USA 95, 1528-1533.
Lenz-Bohme, B., Wismar, J., Fuchs, S., Reifegerste, R., Buchner, E., Betz, H. and Schmitt, B. (1997). Insertional mutation of the Drosophila nuclear lamin Dm0 gene results in defective nuclear envelopes, clustering of nuclear pore complexes, and accumulation of annulate lamellae. J. Cell Biol. 137, 1001-1016.
Lin, C. W. and Engelman, A. (2003). The barrier-to-autointegration factor is a component of functional human immunodeficiency virus type 1 preintegration complexes. J. Virol. 77, 5030-5036.
Lin, F., Blake, D. L., Callebaut, I., Skerjanc, I. S., Holmer, L., McBurney, M. W., Paulin-Levasseur, M. and Worman, H. J. (2000). MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin. J. Biol. Chem. 275, 4840-4847.
Liu, J., Ben-Shahar, T. R., Riemer, D., Treinin, M., Spann, P., Weber, K., Fire, A. and Gruenbaum, Y. (2000). Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes. Mol. Biol. Cell 11, 3937-3947.
Lopez-Soler, R. I., Moir, R. D., Spann, T. P., Stick, R. and Goldman, R. D. (2001). A role for nuclear lamins in nuclear envelope assembly. J. Cell Biol. 154, 61-70.
Lourim, D. and Krohne, G. (1994). Lamin-dependent nuclear envelope reassembly following mitosis. Trends Cell Biol. 4, 314-318.[CrossRef][Medline]
Manilal, S., Thi-Man, N., Sewry, C. A. and Morris, G. E. (1996). The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein. Hum. Mol. Genet. 5, 801-808.
Martins, S., Eikvar, S., Furukawa, K. and Collas, P. (2003). HA95 and LAP2 beta mediate a novel chromatin-nuclear envelope interaction implicated in initiation of DNA replication. J. Cell Biol. 160, 177-188.
Meier, J. and Georgatos, S. D. (1994). Type B lamins remain associated with the integral nuclear envelope protein p58 during mitosis: implications for nuclear reassembly. EMBO J. 13, 1888-1898.[Abstract]
Moir, R. D., Yoon, M., Khuon, S. and Goldman, R. D. (2000). Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells. J. Cell Biol. 151, 1155-1168.
Mounkes, L. C., Kozlov, S., Hernandez, L., Sullivan, T. and Stewart, C. L. (2003). A progeroid syndrome in mice is caused by defects in A-type lamins. Nature 423, 298-301.[CrossRef][Medline]
Nikolakaki, E., Meier, J., Simos, G., Georgatos, S. D. and Giannakouros, T. (1997). Mitotic phosphorylation of the lamin B receptor by a serine/arginine kinase and p34(cdc2). J. Biol. Chem. 272, 6208-6213.
Ostlund, C., Ellenberg, J., Hallberg, E., Lippincott-Schwartz, J. and Worman, H. J. (1999). Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein. J. Cell Sci. 112, 1709-1719.
Polioudaki, H., Kourmouli, N., Drosou, V., Bakou, A., Theodoropoulos, P. A., Singh, P. B., Giannakouros, T. and Georgatos, S. D. (2001). Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1. EMBO Rep. 2, 920-925.
Pyrpasopoulou, A., Meier, J., Maison, C., Simos, G. and Georgatos, S. D. (1996). The lamin B receptor (LBR) provides essential chromatin docking sites at the nuclear envelope. EMBO J. 15, 7108-7119.[Abstract]
Sakaki, M., Koike, H., Takahashi, N., Sasagawa, N., Tomioka, S., Arahata, K. and Ishiura, S. (2001). Interaction between emerin and nuclear lamins. J. Biochem. 129, 321-327.[Abstract]
Salina, D., Bodoor, K., Eckley, D. M., Schroer, T. A., Rattner, J. B. and Burke, B. (2002). Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell 108, 97-107.[Medline]
Segura-Totten, M., Kowalski, A. K., Craigie, R. and Wilson, K. L. (2002). Barrier-to-autointegration factor: major roles in chromatin decondensation and nuclear assembly. J. Cell Biol. 158, 475-485.
Shumaker, D. K., Lee, K. K., Tanhehco, Y. C., Craigie, R. and Wilson, K. L. (2001). LAP2 binds to BAF-DNA complexes: requirement for the LEM domain and modulation by variable regions. EMBO J. 20, 1754-1764.
Simos, G. and Georgatos, S. D. (1992). The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins. EMBO J. 11, 4027-4036.[Abstract]
Steen, R. L. and Collas, P. (2001). Mistargeting of B-type lamins at the end of mitosis: implications on cell survival and regulation of lamins A/C expression. J. Cell Biol. 153, 621-626.
Stuurman, N., Heins, S. and Aebi, U. (1998). Nuclear lamins: their structure, assembly, and interactions. J. Struct. Biol. 122, 42-66.[CrossRef][Medline]
Sullivan, T., Escalante-Alcalde, D., Bhatt, H., Anver, M., Bhat, N., Nagashima, K., Stewart, C. L. and Burke, B. (1999). Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell Biol. 147, 913-920.
Taniura, H., Glass, C. and Gerace, L. (1995). A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones. J. Cell Biol. 131, 33-44.[Abstract]
Thompson, L. J., Bollen, M. and Fields, A. P. (1997). Identification of protein phosphatase 1 as a mitotic lamin phosphatase. J. Biol. Chem. 272, 29693-29697.
van Steensel, B., Smogorzewska, A. and de Lange, T. (1998). TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401-413.[Medline]
Vaughan, O. A., Malvarez-Reyes, M., Bridger, J. M., Broers, L. V., Ramaekers, F. C. S., Wehnert, M., Morris, G., Whitfield, W. G. F. and Hutchison, C. J. (2001). Both emerin and lamin C depend on lamin A for localization at the nuclear envelope. J. Cell Sci. 114, 2577-2590.[Medline]
Vlcek, S., Just, H., Dechat, T. and Foisner, R. (1999). Functional diversity of LAP2alpha and LAP2beta in postmitotic chromosome association is caused by an alpha-specific nuclear targeting domain. EMBO J. 18, 6370-6384.
Vlcek, S., Dechat, T. and Foisner, R. (2001). Nuclear envelope and nuclear matrix: interactions and dynamics. Cell. Mol. Life Sci. 58, 1758-1765.[Medline]
Vlcek, S., Korbei, B. and Foisner, R. (2002). Distinct functions of the unique C-terminus of LAP2alpha in cell proliferation and nuclear assembly. J. Biol. Chem. 277, 18898-18907.
Worman, H. J., Yuan, J., Blobel, G. and Georgatos, S. D. (1988). A lamin B receptor in the nuclear envelope. Proc. Natl. Acad. Sci. USA 85, 8531-8534.[Abstract]
Worman, H. J., Evans, C. D. and Blobel, G. (1990). The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains. J. Cell Biol. 111, 1535-1542.[Abstract]
Ye, Q. and Worman, H. J. (1994). Primary structure analysis and lamin B and DNA binding of human LBR, an integral protein of the nuclear envelope inner membrane. J. Biol. Chem. 269, 11306-11311.
Ye, Q. and Worman, H. J. (1996). Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1. J. Biol. Chem. 271, 14653-14656.
Zheng, R., Ghirlando, R., Lee, M. S., Mizuuchi, K., Krause, M. and Craigie, R. (2000). Barrier-to-autointegration factor (BAF) bridges DNA in a discrete, higher-order nucleoprotein complex. Proc. Natl. Acad. Sci. USA 97, 8997-9002.
Related articles in JCS: