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IntroductionThe nuclear lamina, a constituent of the nuclear envelope (NE),and intranuclear lamin complexes are major structural elementsin nuclei of multicellular eukaryotes (Goldman et al., 2002;Hutchison, 2002). The nuclear lamina lines the inner nuclearmembrane and forms a meshwork of tetragonally organizedfilaments of type V intermediate filament proteins, the lamins(Stuurman et al., 1998). Vertebrates have three lamin genesencoding seven distinct isoforms (Cohen et al., 2001). B-typelamins are constitutively expressed throughout developmentand are essential for cell viability (Harborth et al., 2001; Lenz-Bohme et al., 1997; Liu et al., 2000). A-type lamins are notessential, but may have functions in tissue organization andhomeostasis in the adult organism (Mounkes et al., 2003;Sullivan et al., 1999). Inherited mutations in the lamin A genein humans give rise to a heterogeneous group of diseases,termed laminopathies, which affect heart, muscle, adipose,bone and nerve cells (Burke and Stewart, 2002), or lead topremature aging (Cao and Hegele, 2003; De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). Interestingly, A-type lamins in particular are not restricted to the nuclearperiphery but exist throughout the nuclear interior (Hozak etal., 1995; Jagatheesan et al., 1999; Moir et al., 2000).

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 functionof lamin complexes. Lamin B receptor (LBR) is a protein of theinner membrane with eight transmembrane domains (Wormanet al., 1990) and binds B-type lamins (Simos and Georgatos,1992; Ye and Worman, 1994). Lamina-associated polypeptide2 (LAP2) is a family of six alternatively spliced proteins, ofwhich four, LAP2β, -γ, -δ and -ε are type II membrane proteins(Berger et al., 1996; Dechat et al., 2000b; Harris et al., 1994).LAP2α is structurally and functionally different, sharing onlythe N-terminus with the other isoforms. LAP2β binds lamin B(Foisner and Gerace, 1993; Furukawa et al., 1998) at the NEwhereas LAP2α interacts with A-type lamins in thenucleoplasm (Dechat et al., 1998; Dechat et al., 2000a). Emerinis an inner nuclear membrane protein (Manilal et al., 1996) thatbinds 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 NEby A-type lamins (Holt et al., 2003; Sullivan et al., 1999;Vaughan et al., 2001). All LAP2 isoforms, emerin and the innermembrane protein, MAN1 (Lin et al., 2000) share a ~40 aminoacid-long structural motif, the lamina-associated polypeptideemerin MAN1 or LEM domain (Cai et al., 2001; Laguri et al.,

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Lamina-associated polypeptide (LAP) 2α is a LEM(lamina-associated polypeptide emerin MAN1) familyprotein associated with nucleoplasmic A-type lamins andchromatin. Using live cell imaging and fluorescencemicroscopy we demonstrate that LAP2α was mostlycytoplasmic in metaphase and associated with telomeres inanaphase. Telomeric LAP2α clusters grew in size, formed‘core’ structures on chromatin adjacent to the spindle intelophase, and translocated to the nucleoplasm in G1phase. A subfraction of lamin C and emerin followedLAP2α to the core region early on, whereas LAP2β, laminB receptor and lamin B initially bound to more peripheralregions of chromatin, before they spread to core structureswith different kinetics. Furthermore, the DNA-crosslinking

protein barrier-to-autointegration factor (BAF) bound toLAP2α in vitro and in mitotic extracts, and subfractionsof BAF relocalized to core structures with LAP2α. Wepropose that LAP2α and a subfraction of BAF form definedcomplexes in chromatin core regions and may be involvedin chromatin reorganization during early stages of nuclearassembly.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/117/25/6117/DC1

Key words: Chromosomes, Lamins, Mitosis, Nuclear assembly,Nuclear envelope

Summary

LAP2α and BAF transiently localize to telomeres andspecific regions on chromatin during nuclearassemblyThomas Dechat1, Andreas Gajewski1, Barbara Korbei1, Daniel Gerlich2, Nathalie Daigle2, Tokuko Haraguchi3,Kazuhiro Furukawa4, Jan Ellenberg2 and Roland Foisner1,*1Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna,1030 Vienna, Austria2Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory, 69117 Heidelberg, Germany3CREST of JST Kansai Advanced Research Center, Kobe 651-2492, Japan4Department of Chemistry, Faculty of Sciences, Niigata University, Niigata 950-2181, Japan*Author for correspondence (e-mail: [email protected])

Accepted 10 September 2004Journal of Cell Science 117, 6117-6128 Published by The Company of Biologists 2004doi:10.1242/jcs.01529

Research Article

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2001), which mediates binding to BAF (Furukawa, 1999;Shumaker et al., 2001). BAF is a small highly conserved proteinin multicellular eukaryotes that binds double stranded DNAwithout sequence specificity (Zheng et al., 2000).

In higher eukaryotes the nucleus is transiently disassembledduring mitosis because of a phosphorylation-dependentdisassembly of the lamina and pore complexes (Burke andEllenberg, 2002; Foisner, 2003), a process facilitated by amicrotubule/dynein-mediated deformation and disruption ofthe nuclear envelope (Beaudouin et al., 2002; Salina et al.,2002). Mitosis-specific phosphorylation of lamins was foundto be essential for lamina disassembly (Heald and McKeon,1990) and lamin-binding proteins are also phosphorylated inmitosis (Dechat et al., 1998; Foisner and Gerace, 1993;Nikolakaki et al., 1997).

After sister chromatid separation the NE and nuclearstructure reassemble in a tightly regulated manner, ensuring thatthe interphase chromatin organization can be re-established indaughter nuclei (Cohen et al., 2001). Nuclear reassemblyrequires phosphatase activity and, at least for B-type lamins, hasbeen shown to involve the membrane-associated phosphatasePP1 (Steen and Collas, 2001; Thompson et al., 1997). Thekinetics of the association of lamins and some lamin-bindingproteins with the reforming nucleus have been studied in thepast years. LBR and emerin are targeted to chromosomes about5 minutes after metaphase-anaphase transition, followed bynuclear pore complex assembly (Haraguchi et al., 2000).LAP2β was also found to accumulate at around the same timeas emerin (Bodoor et al., 1999; Dabauvalle et al., 1999; Foisnerand Gerace, 1993). B- and A-type lamins follow differentpathways (Moir et al., 2000). Stable B-type lamin structures areseen 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 thataccumulates in the nuclear interior and at the NE later. Additionof protein mutants or antibodies to in vitro nuclear assemblyassays or transient expression of mutants in cells has implicatedlamins, LBR, LAP2β and LAP2α in nuclear assembly (Gant etal., 1999; Lopez-Soler et al., 2001; Lourim and Krohne, 1994;Pyrpasopoulou et al., 1996; Vlcek et al., 2002), but themolecular mechanisms remain unclear.

Here we perform detailed analyses of the dynamics ofLAP2α during nuclear assembly in relation to other NE andchromatin proteins and show several major new findings.Following initial binding to telomeres, LAP2α is the firstamong a group of lamina proteins, including lamin C andemerin, detectable in the ‘core’ structures on chromatin thathave been described previously (Haraguchi et al., 2001). Incontrast, LBR, LAP2β and lamin B initially bind to distinct,peripherally located regions of the chromatin bulk, and spreadto core structures later. In support of previous studiesimplicating BAF in NE assembly (Haraguchi et al., 2001;Segura-Totten et al., 2002), we provide evidence that asubfraction of BAF relocalizes to core structures together withLAP2α. We propose that these structures may define chromatinorganization in the reassembling nucleus.

Materials and MethodsDNA constructsGFP-LAP2α, H2B-CFP and CFP-lamin B1 have been described

previously (Beaudouin et al., 2002; Daigle et al., 2001; Ellenberg etal., 1997; Vlcek et al., 2002). YFP-LAP2α was constructed like GFP-LAP2α except that pEYFP-C1 was used instead of pEGFP-C1(Clontech Laboratories, Palo Alto, CA). For construction of CFP-lamin C, oligonucleotides (5′-TCGAGACTAGTGCCGGCGA-ATTCG-3′ and 5′-GATCCGAATTCGCCGGCACTAGTC-3′) wereinserted into pECFP-C3 via XhoI and BamHI sites, introducing SpeIand EcoRI restriction sites. cDNA encoding lamin C (a gift from G.Krohne, Würzburg, Germany) was cloned into modified pECFP-C3via SpeI and EcoRI. Emerin-CFP was generated by cloning a cDNAencoding emerin amino acids 3-254 fused to a Flag-tag at its N-terminus (Ostlund et al., 1999) into pECFP-N1 (Clontech) via SacIand KpnI sites.

Cell culture, transfection and synchronizationHeLa and NRK cells were routinely maintained in high glucose DMEmedium supplemented with 10% FCS, 10 mM HEPES, pH 7.0 and50 µg/ml penicillin and streptomycin (Life Technologies, Paisley,UK) at 37°C and 5% CO2. For generation of stable HeLa cell clonesexpressing GFP- or YFP-LAP2α, cells were transfected with plasmidsusing the standard calcium phosphate method and clones wereselected in medium containing 700 µg/ml G418 (Life Technologies)and maintained in medium plus 100 µg/ml G418. Transienttransfections were performed with Fugene 6 (Roche, Mannheim,Germany). For live cell microscopy, cells were cultured in two-wellLabTek chambers with glass bottoms (Nunc, Rochester, NY) and cellgrowth was synchronized by arresting cells 24 hours post transfectionin 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/mlacetylsalicylic acid was used.

Immunofluorescence microscopyCells 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 andsecondary antibodies were applied in gelatin/PBS for 1 hour each atroom temperature. Primary antibodies were goat antiserum N18against lamins A/C (Santa Cruz Biotechnology, Santa Cruz, CA),rabbit antiserum against LAP2α (Vlcek et al., 2002), antiserum toBAF (Furukawa, 1999), monoclonal antibody 4A794 to TRF2(Upstate Biotechnology, Lake Placid, NY) and monoclonal antibodies15-2 and 17 to LAP2α and LAP2β, respectively (Dechat et al., 1998);secondary antibodies used are donkey anti-goat IgG conjugated to Cy3and goat anti-mouse IgG conjugated to Texas Red (Jackson Immuno-Research, West Grove, PA) and goat anti-rabbit IgG conjugated toAlexa488 (Molecular Probes, Leiden, The Netherlands). DNA wasstained with 1 µg/ml Hoechst dye 33258 (Calbiochem-Behring) for 10minutes. Samples were mounted in Mowiol and viewed in a ZeissLSM 510 confocal microscope (Carl Zeiss, Jena, Germany).

4D multicolor live cell imaging and image processingLive cell imaging was performed at 37°C maintained by an air streamincubator (ASI 400; Nevtek, Burnsville, VA) in conjunction with anobjective heater (Bioptechs, Butler, PA) on a customized LSM 510confocal microscope fitted with a z-scanning stage, selectedphotomultiplier tubes (Carl Zeiss), a Kr 413 nm laser (Coherent,Dieburg, Germany) and custom dichroics and emission filters(Chroma, Brattleboro, VT) for fluorescent protein imaging asdescribed elsewhere (Daigle et al., 2001; Gerlich et al., 2001). Mitoticevents were monitored with a PlanApochromat 63× N.A. 1.4 oil DICobjective (Carl Zeiss) every 10 seconds for 3 minutes. Image series

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from a single confocal z-plane representing selected time points wereassembled and single confocal z-stacks of each time point wereprojected with the LSM 510 2.3 software (Carl Zeiss) and combinedinto movies using ImageJ software (http://rsb.info.nih.gov/ij/). Falsecolors for optimal presentation were used: mainly green for YFP andred for CFP. Rendering of four-dimensional images was by graphicalreconstruction. Noise levels were reduced using an anisotropicdiffusion filter on individual image slices (Gerlich et al., 2001).Isosurface reconstruction and rendering was carried out using theAmira 2.3 software package (TGS, San Diego, CA, USA).Photobleaching was done as described previously (Daigle et al., 2001).

Chromosome spreadsMitotic cells harvested from an unsynchronized culture wereincubated in 0.075 mM KCl for 20 minutes at room temperature andlysed by adding 0.1% Tween 20. Samples were spun onto coverslipsat 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 forimmunofluorescence microscopy.

ImmunoprecipitationMitotic NRK cells were lysed in KHM buffer and chromosomes spunout as described (Vlcek et al., 2002). The lysate was pre-cleared byaddition of 50 µl 50% protein G-sepharose beads and centrifugationat 400 g for 5 minutes with a tabletop centrifuge. 10 µl protein G-sepharose beads, preincubated in 1 ml of hybridoma supernatantcontaining antibody to LAP2α or 10 µl untreated beads (control) wereadded to the lysates and harvested by centrifugation following a 2 hourincubation. Beads were washed in KHM buffer and proteinssolubilized in 3× SDS-PAGE sample buffer.

LAP2α-BAF in vitro bindingBAF was in vitro translated using the TNT® Quick CoupledTranscription/Translation System (Promega) from a pET15b vector(Novagene) containing full-length BAF (Lee and Craigie, 1998). Thetranslation product was diluted sevenfold in binding buffer (50 mMHEPES, 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 co-immunoprecipitation. Recombinant LAP2α was expressed (Vlceket al., 1999; Vlcek et al., 2002), renatured by dialyzing into bindingbuffer and centrifuged at 1500 g for 5 minutes. 100 µl BAF samplewas mixed with 100 µl recombinant LAP2α or 100µl binding bufferas a control and transferred to 50 µl protein G-sepharose beads,preincubated in 1 ml hybridoma supernatant containing antibody toLAP2α. Following a 30-minute incubation, beads were underlaid with30% sucrose and harvested by centrifugation.

Polyacrylamide gel electrophoresis and immunoblottingSDS-PAGE was performed according to Laemmli (Laemmli, 1970).For solubilizing BAF, 6 M urea was added to sample buffer. Forimmunoblotting, primary antibodies were hybridoma supernatants ofLAP2 antibodies (Dechat et al., 1998) and BAF antiserum 3273(Haraguchi et al., 2001); secondary antibodies, alkaline phosphataseor peroxidase-conjugated goat antibodies. Proteins were detected withthe Protoblot Immunoscreening System (Promega) or the SuperSignal ECL (Pierce, Rockford, CA, USA).

ResultsLAP2α dynamics during nuclear reassemblyTo investigate the cell cycle-dependent dynamic behavior of

LAP2α we performed 3D time-lapse live cell imaging (4Dimaging) (Gerlich and Ellenberg, 2003) of HeLa cells stablyexpressing a yellow fluorescent protein (YFP) fusion constructof LAP2α. Transient co-expression of a cyan fluorescent protein(CFP) fusion construct of histone 2B allowed detection ofchromosomes in living cells. Like endogenous LAP2α (Dechatet al., 1998), YFP-LAP2α was localized throughout the nuclearinterior sparing only the nucleoli in interphase (Fig. 1A). Duringmetaphase only a small fraction of LAP2α was detected on thechromosomes: most of the protein was found dispersedthroughout the cytoplasm. After the onset of anaphase (Fig. 1B;Movie 1 in supplementary material), YFP-LAP2α translocatedto distinct regions at the tips of lagging chromosome endsprotruding from the bulk of segregating sister chromatidstowards the midspindle region (Fig. 1B,C, arrowheads).Isosurface reconstruction of individual 3D data sets confirmedthe generation and accumulation of LAP2α-containing spot-likestructures on the surface of chromatin protrusions (Fig. 1D).LAP2α-enriched patches were first detected approximately 4minutes after anaphase onset and then increased significantly insize and intensity as mitosis progressed. The majority of cellularLAP2α was redistributed from the cytoplasm to the chromatinwithin about 6 minutes after metaphase exit, which wasaccompanied by the retraction of lagging chromosome ends intoa compact chromatin mass. During the next minute the proteinconcentrated into two brightly stained YFP-LAP2α ‘core’structures at the inner and the outer side of newly forming nuclei,facing the midspindle and spindle pole region, respectively (Fig.1B, arrows). When cytokinesis was nearly complete andchromosomes partially decondensed, LAP2α redistributed fromthese core structures back to the nuclear interior where itremained throughout interphase.

LAP2α associates with telomeric regions ofchromosomesThe preferred association sites of LAP2α at the tips of laggingchromosomes observed by live cell imaging suggestedlocalization at telomeric structures. To address this possibilitywe performed double immunofluorescence microscopy offixed cells using antibodies to LAP2α and to the telomericTTAGGG repeat binding factor 2 (TRF2) (Broccoli et al.,1997; van Steensel et al., 1998). Although in interphase cellsTRF2 antibody stained dot-like telomeres throughout thenucleoplasm, the antibody had a complex dotted pattern ontelophase chromosomes. Here, we observed partial overlapof TRF2 structures with LAP2α on chromatin (Fig. 2A),consistent with a targeting of LAP2α to telomeric regions inthe early phases of nuclear reassembly.

To identify LAP2α on individual chromosomes we preparedmitotic chromosome spreads. Both a mouse monoclonalantibody (Fig. 2Ba) and a rabbit antiserum to LAP2α (Fig. 2Bb-d) detected LAP2α at the tips of chromosomes, whereasantibodies to LAP2β (Fig. 2Be), or to lamins (data not shown)stained dots scattered throughout the sample without a preferredtelomere association. The LAP2α structures on chromosomesdiffered significantly in size ranging from small dots (Fig. 2Bb)to larger clusters (Fig. 2Bc,d). Furthermore, most of thechromosomes in the preparations with LAP2α localized at thetips contained two chromatids, representing a metaphase stage,whereas in living cells LAP2α patches were first detected at

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chromosomes in anaphase after sister chromatid separation.From previous studies we knew that binding of LAP2α tochromosomes is controlled by phosphorylation (Dechat et al.,1998; Vlcek et al., 2002) and that incubation of mitotic cellextracts induces dephosphorylation-dependent association ofLAP2α with chromosomes. Thus, we hypothesized that duringchromosome spread preparation the cells proceeded partially topost-metaphase stages without chromatid separation owing tothe lack of a functional spindle. This may inducedephosphorylation and chromosome association of LAP2α. Inline with this model, spreads prepared in the presence ofphosphatase inhibitors rarely showed LAP2α at telomeres (datanot shown). Thus, the preparation of mitotic chromosome

spreads in the absence of phosphatase inhibitors may representthe narrow time window in nuclear assembly during whichLAP2α associates at chromosomes.

LAP2α forms a stable structure at chromatin cores butturns over rapidly in interphaseWe next investigated how stably LAP2α was associated withchromosomes during nuclear reassembly by fluorescencerecovery after photobleaching (FRAP) analysis in cellsexpressing GFP-LAP2α (Fig. 2C). In interphase cells, FRAPrevealed that photobleached regions in the nucleoplasm rapidlyrecovered to almost their prebleach intensity within 8 seconds.


Fig. 1. LAP2α dynamics during nuclear assembly. (A-D) HeLa cells stably expressing YFP-LAP2α were transiently transfected withconstructs encoding CFP-histone 2B and analyzed by live cell imaging. Confocal images of cells in interphase and metaphase (A) or at variousstages during anaphase to G1 phase progression (B) or anaphase (C) are shown. Arrowheads indicate LAP2α at the tips of chromatinextensions; arrows indicate outer core regions. See also Movie 1 in supplementary material. (D) Isosurface reconstruction of z-stacks of images.Non-chromosome-associated clusters were excluded from the isosurface reconstruction. Numbers in images indicate elapsed time fromanaphase onset (B) or from first image (D). Bars, 10 µm.


This suggested that LAP2α structures in the nucleoplasmturn over very rapidly as also shown previously for certainproteins in the NPC (e.g. Nup153) (Daigle et al., 2001) andheterochromatin (HP1) (Cheutin et al., 2003). In anaphase,when LAP2α structures first detectable on chromosomes werebleached, fluorescence still recovered to more than half of theprebleach intensity within 8 seconds. However, part of thisrecovery was probably due to additional LAP2α binding tochromosomes during ongoing nuclear assembly in the recoverytime (see unbleached half of the cell in Fig. 2C) rather than

recovery to a steady-state situation. In telophase, when themajority of LAP2α was already associated with core structuresof chromatin, bleached areas recovered to only about 10% oftheir prebleach intensity, suggesting that LAP2α was stablyassociated with these core structures.

Differential localization of LAP2α and lamins duringassemblyUnlike B-type lamins, A-type lamins interact directly with

Fig. 2. LAP2α associates with telomeres in stable structures. (A) HeLa cells were fixed and processed for immunofluorescence using antibodiesto LAP2α (green) and TRF2 (red). Arrows indicate areas shown at higher magnifications in inserts. (B) Mitotic HeLa cells were incubated for10 minutes (a-c) or 20 minutes (d,e) in hypotonic buffer, followed by preparation of chromosome spreads. Samples were processed forimmunofluorescence microscopy using monoclonal (a) or polyclonal (b-d) antibodies to LAP2α or monoclonal antibody to LAP2β (e) and theHoechst DNA stain. Confocal images are shown. (C) FRAP analyses. The boxed areas were bleached to background levels (0 s) and recoverymonitored after indicated time points. Fluorescence recovery after 8 seconds as a percentage of the prebleach intensity was corrected forintensity changes in unbleached zones and plotted. Bars, 10 µm.

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LAP2α in the nucleoplasm, particularly during G1 phase(Dechat et al., 2000a). To compare the kinetics of LAP2αredistribution to chromosomes with those of A- and B-typelamins, we transiently expressed CFP-tagged lamin C or laminB1 in stable YFP-LAP2α-expressing cells and performed livecell imaging. Both lamins localized primarily at the nuclearperiphery in interphase and were dispersed throughout thecytoplasm in mitosis (Fig. 3A). During nuclear reassembly,telomeric LAP2α structures (arrowheads) were clearlydetectable before the accumulation of lamins C or B at thechromatin surface, although small fractions of lamins maysurround chromatin throughout all stages of assembly. LaminC and lamin B (arrows) were both detected at higherconcentrations on chromatin approximately 1 minute after thefirst occurrence of telomeric LAP2α, but their localization was

different (Moir et al., 2000). A small subfraction of lamin Ccolocalized with LAP2α at core structures during early stages,but the majority of lamin C was translocated to the nuclearinterior at later stages when LAP2α relocated to thenucleoplasm. Although the translocation of A-type lamins intothe nucleoplasm during telophase/G1 has been described (Moiret al., 2000), the early association of a subfraction of A-typelamins with chromosomes in anaphase was not observed. Tomake sure that our observation was not an artifact due tolamin C overexpression, we performed immunofluorescencemicroscopy of untransfected, fixed cells using antibodies tolamin C. Endogenous lamin A/C behaved in a manner similarto the ectopic protein, partially overlapping with LAP2αstructures at chromosomes during anaphase (Fig. 3B).

In contrast to lamin C, the first enrichment of lamin B on


Fig. 3. LAP2α dynamics in relation to lamins. (A) HeLa cells stably expressing YFP-LAP2α were transiently transfected with constructs encoding either CFP-lamin C orCFP-lamin B1 and analyzed by live cell imaging. Confocal images of cells in interphaseand various stages during anaphase to G1 phase progression are shown. Time points showelapsed time relative to first image. Arrowheads denote LAP2α in the inner core region;arrows, lamin structures. (B) Immunofluorescence microscopy of fixed GFP-LAP2α-expressing cells using antibodies to lamins A/C. Bars, 10 µm.


chromosomes did not overlap with telomeric LAP2α. LaminB accumulated initially at chromatin surfaces next to thespindle pole, forming a cap-like structure opposite of the majortelomeric LAP2α structures. From there lamin B spread overthe entire surface of decondensing chromatin and formed acontinuous envelope only when LAP2α translocated to thenucleoplasm (Fig. 3A).

LAP2α association with chromosomes differs from thatof nuclear membrane proteinsTo test where and when during assembly nuclear membraneproteins associated with chromosomes in relation to LAP2α,we transiently expressed CFP fusion proteins of LBR andLAP2β, both binding partners of B-type lamins (Foisner andGerace, 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 nuclearperiphery in interphase (Fig. 4). LAP2β was first detectable atchromosomes slightly after LAP2α during nuclear reassembly(Vlcek et al., 2002) whereas LBR appeared at chromosomes atdetectable levels slightly before LAP2α (see Movies 2 and 3in supplementary material). However, both LAP2β and LBRmostly localized to more peripheral sites of the chromatin bulk,distinct from the LAP2α-containing core regions (see alsoDabauvalle et al., 1999; Haraguchi et al., 2000). In contrast toLBR, LAP2β also accumulated on chromatin core structures,before both LBR and LAP2β spread over the entire surface ofdecondensing chromatin at later stages of assembly. Emerinshowed 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 andLAP2β, the majority of emerin accumulated subsequently atthe LAP2α core structures. Altogether, we observed atemporally and spatially highly coordinated accumulation oflamina proteins with chromosomes during nuclear assembly.LAP2α and LBR became concentrated on anaphase chromatinfirst, whereas other membrane proteins and lamins weredetectable at significant levels slightly later. Furthermore, twogroups of lamina proteins with different preferred chromosomeassociation sites could be distinguished. LAP2α, followed byemerin and A-type lamins formed the core region of chromatin,whereas LBR followed by LAP2β and lamin B bound to moreperipheral chromatin regions initially.

LAP2α and BAF colocalize at chromatin-associated corestructuresThe initial binding of LAP2α to telomere patches andformation of chromatin-associated LAP2α core structurescould indicate an important role of LAP2α early in nuclearassembly. Consistent with this hypothesis, in in vitro nuclearassembly studies we have previously shown that C-terminalLAP2α fragments bind to chromosomes and dominantlyinhibit assembly of nuclear membranes and lamin A aroundchromosomes (Vlcek et al., 2002). As the dominant-negativeLAP2α mutants lacked the LEM motif, which mediatesbinding to the DNA crosslinking protein BAF, and BAFmutants deficient in binding to the LEM domain were foundto inhibit the assembly of emerin and lamin A in vivo(Haraguchi et al., 2001), we reasoned that complexes ofLAP2α and BAF may be involved in early stages ofnuclear assembly. To test this hypothesis, we performed

Fig. 4. LAP2α dynamics in relation to membrane proteins. HeLa cells stably expressing YFP- LAP2α were transiently transfected with CFP-LAP2β or CFP-LBR or CFP-emerin constructs and analyzed by live cell imaging. Confocal images were taken of cells in interphase andvarious stages during telophase to G1 phase progression. Arrowheads denote LAP2α structures in the inner core region. Bars, 10 µm. Movies 2and 3 in supplementary material show dynamics of LAP2α-LAP2β and LAP2α-LBR, respectively.

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immunofluorescence microscopy of HeLa cells at differentmitotic stages using antibodies to LAP2α and BAF. Althoughthe majority of BAF was uniformly localized at thechromosomes in early anaphase, endogenous LAP2α waspredominantly cytoplasmic (Fig. 5a). However, when LAP2αwas 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 tosome extent also in the cytoplasm. Interestingly, upon formationof telomeric LAP2α structures (Fig. 5c) and core structures intelophase (Fig. 5d), BAF also localized to these structures,whereas it was barely detectable throughout the chromatin orcytoplasm at this time. Thus, cytoplasmic and/or chromosome-associated BAF apparently relocalized to core structures onchromatin with similar timing as LAP2α during nuclearassembly. However, we cannot exclude the fact that BAF is stillpresent in the nuclear interior, but epitopes were masked owingto reorganization of chromatin. Other lamina proteins, such asLAP2β, did not strictly colocalize with BAF at core structuresat this stage of assembly, but showed a broader distribution overthe entire surface of chromatin (Fig. 5e).

BAF and LAP2α form a complex in vitro and in post-metaphase cell lysatesTo test whether LAP2α and BAF may indeed bind to eachother, we investigated the interaction of these proteins in vitro.Although LAP2α was expected to bind BAF via its N-terminalLEM motif, it was important to demonstrate the bindingdirectly, because previous studies using different XenopusLAP2β isoforms indicated that their slightly different C-termini modulated binding to BAF at the N-terminal LEMmotif (Shumaker et al., 2001). Therefore, BAF was producedby in vitro transcription/translation in reticulocyte celllysates and mixed with purified recombinant LAP2α.Immunoprecipitation of LAP2α significantly coprecipitatedBAF (Fig. 6A). Control experiments in the absence of LAP2αor with a C-terminal LAP2α fragment lacking the LEMdomain did not precipitate BAF. Thus, BAF is able to interactwith the LEM motif in LAP2α in vitro.

In order to confirm the existence of LAP2α-BAF complexesin cells at early stages of assembly, we performed co-immunoprecipitation assays. We synchronized NRK cellcultures using a thymidine block, harvested metaphase cellsby mechanical shake-off and lysed cells in a metal ballhomogenizer. As up to 50% of total BAF (Holaska et al., 2003;Lin and Engelman, 2003) and the majority of LAP2α (Dechatet al., 1998; Vlcek et al., 2002) are found in the cytoplasm inmetaphase cells, we spun out chromosomes from cell lysatesand incubated the supernatant for 5-10 minutes at room


Fig. 5. LAP2α and BAF colocalize at core regions. (a-e) HeLa cellsat various stages of mitosis were analyzed by immunofluorescencemicroscopy using antibodies to LAP2α (a-d) and LAP2β (e) (green),BAF (red) and Hoechst DNA stain (blue). Arrowheads show firstBAF accumulation at sites of LAP2α association. Confocal imagesare shown with merged images in right hand panels. Bar, 10 µm.

Fig. 6. LAP2α interacts with BAF. (A) Invitro translated BAF (input) was mixed withbuffer (–), recombinant full length LAP2α (1-693) or LAP2α C-terminal fragment (410-693) and complexes were precipitated withimmobilized monoclonal antibodies toLAP2α. Panels show immunoblots usingantibodies to BAF (upper) and to LAP2α(lower). (B) LAP2α was immunoprecipitatedfrom metaphase cell lysates. Controls wereperformed in the absence of antibodies.Supernatant (S) and pellet (P) fractions wereanalyzed by immunoblotting using antiserumto BAF and to LAP2α. IgG, immunoglobulinheavy chain.


temperature in order to allow partial post-metaphase assemblyof soluble LAP2α complexes. Immunoprecipitation of theseLAP2α structures from the lysates precipitated BAF, whereasneither LAP2α nor BAF were precipitated with control beadslacking antibodies (Fig. 6B). Interestingly, BAF in cell lysatesand in soluble fractions behaved as a monomeric protein inSDS-PAGE, whereas BAF coprecipitated with LAP2α wasprimarily detectable as a putative dimer or oligomer (or aposttranslationally modified species) that migrated atapproximately 20 kDa on SDS-PAGE (Fig. 6B). We speculatethat LAP2α binding to BAF may change the conformation ofBAF (see also Forne et al., 2003) and stabilize dimeric oroligomeric forms that become oxidatively crosslinked duringsample preparation, and migrate aberrantly on SDS-PAGE(Zheng et al., 2000).

DiscussionA novel model for nuclear assemblyIn this study, we investigated the dynamics of LAP2α duringnuclear assembly in relation to major components of the NE,which led us to propose a novel model for the molecularmechanisms in initial phases of post-mitotic nuclear assembly(Fig. 7): LAP2α becomes enriched on telomere patches duringanaphase and rapidly accumulates at the initial docking sites.The assembly of LAP2α structures at telomeres and chromatincould then give rise to core structures on the inner and outerregions of anaphase chromatin adjacent to the midspindle andspindle poles, respectively. We cannot rule out, however, thatthe binding of LAP2α to telomeres and its assembly at corestructures are independent events mediated by binding ofLAP2α to different chromosomal proteins or complexes.

We also demonstrate an in vitro interaction between LAP2α

and BAF via the N-terminal region of LAP2α containing theLEM domain, and provide evidence for the existence ofLAP2α-BAF complexes in early post-metaphase stages in situ.Accordingly, a large fraction of BAF colocalized with LAP2αat chromatin core regions. It is still unclear whether BAF atLAP2α core structures originated from the cytoplasmic poolof BAF (Holaska et al., 2003; Lin and Engelman, 2003), whichcould translocate to core regions in a complex with LAP2α, orwhether chromosome-bound BAF redistributed transiently tocore structures upon binding of LAP2α. The lack of BAFdetection on chromatin outside the core regions in telophasewould argue for the latter possibility, however, the epitopes ofBAF in the internal chromatin structure may be masked at thistime. As we found that various N- and C-terminally taggedBAF forms did not behave exactly as endogenous BAF in termsof homo-oligomerization, DNA binding and interaction withLEM-domain proteins (our unpublished data), we could nottest GFP-tagged proteins to overcome potential problems ofepitope masking.

Other lamina and NE proteins did not colocalize withLAP2α at telomere patches, but a subset of them, including asubfraction of lamin C, emerin, and to some extent alsoLAP2β, were targeted to the core regions slightly after LAP2α-BAF (Fig. 7). Targeting of these proteins to chromatin coresmay be mediated by direct interaction of lamin C with LAP2αor by binding of the LEM-domain proteins, emerin andLAP2β, to BAF. Another group of lamina proteins showed adifferent pattern of assembly. LBR association with chromatinoccurred at more peripheral sites, distinct from the LAP2α-BAF core complexes. LBR targeting to chromosomes may bethe trigger for attaching membranes to chromatin and mayinitiate nuclear membrane assembly. Other membrane proteins,such as emerin and LAP2β may then diffuse within the lipidbilayer and form stable complexes at the chromatin surface byspecific 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; Taniuraet al., 1995) or DNA (for a review, see Vlcek et al., 2001).

Significance of telomere association of LAP2α and ofLAP2α-BAF interactionOur findings show that LAP2α does not bind uniformly tochromosomes during assembly, as one might have expectedfrom our previous in vitro binding studies (Vlcek et al., 1999).Instead, LAP2α initially associates with telomeres andsubsequently forms larger complexes, before it relocates intothe nuclear interior in late telophase. It is unclear whether thetransient interaction of LAP2α with telomeres is mediatedby a telomere-associated protein complex or by a directinteraction with DNA. Based on its previously reportedproperties as a nucleoskeletal component (Dechat et al., 1998),we hypothesize that the specific targeting of LAP2α totelomeres provides a mechanism by which telomeres arepositioned within the reforming nucleus during theestablishment of higher order chromatin structure after celldivision.

As LAP2α and BAF localized at core structuressimultaneously during nuclear assembly, potential functionsof this interaction in nuclear assembly and possibly higherorder chromatin organization may be envisaged. First, the

2. Lamin B(subfraction)

LAP2 , LAP / BAFEmerinLAP2βLamin C (subfraction)

α α

1. LBR2. LAP2β

2.

Microtubules

TelomeresTelomeres

Microtubules

Spindle pole

Outer core region

Periphery

Inner core region

Periphery

LAP2LAP / BAF?

αα

1.

1. LBR2. LAP2β

2. Lamin B(subfraction)

Fig. 7. Model depicting the major association sites (arrows) oflamina proteins on chromatin during assembly. Red and green boxesrepresent the two major protein groups preferentially associating attelomeres/core regions and peripheral chromatin regions,respectively. Numbers denote the temporal sequence of detectablechromatin association. For details, see text.

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association of LAP2α with chromosome-bound BAF maytarget LAP2α from the cytoplasm to chromosomes duringearly stages of assembly. This, however, seems very unlikelybased on our observations that: (1) BAF localized uniformly atchromosomes in metaphase and early anaphase, whereasLAP2α was targeted preferentially to patches in the vicinity oftelomeres; (2) the constant LAP2 N-terminus, which iscommon to all LAP2 isoforms and contains the LEM domain,did not accumulate at chromosomes at any stage of nuclearreassembly in vivo or in vitro (Vlcek et al., 1999; Vlcek et al.,2002); (3) LAP2α did not require its N-terminal LEM domainfor chromosome targeting, instead its C-terminus (that does notbind BAF) was found to be essential and sufficient forchromosome interaction (Vlcek et al., 1999). Second, LAP2αmay target the cytoplasmic pool of BAF to chromosomesduring nuclear reassembly, which would be consistent withour observations. Third, both proteins could be targetedindependently to chromosomes and associate in specificsubregions of chromosomes. Alternatively, chromosome-bound BAF may be the predominant binding partner forindependently targeted LAP2α, whereas cytoplasmic BAFmay not be involved in the assembly at all.

We hypothesize that, independent of the targetingmechanisms of LAP2α and BAF, the complex may help tore-organize chromatin during decondensation and nuclearassembly by various mechanisms: (1) the complex maytransiently tether telomeres to stable structures favoringordered chromatin reorganization. This model is supported bylow exchange rates of LAP2α in core structures; (2) thebinding of BAF to LAP2α may regulate DNA crosslinkingactivity of BAF, thus resulting in different DNA(de-)compaction states at defined chromosomal subregions; (3)the formation of LAP2α-BAF complexes may provide dockingsites for other lamina proteins favoring their ordered assembly.

The essential role of BAF in cell cycle-dependent chromatinorganization has been described previously by means of in vivoand in vitro studies in various systems. Drosophila baf nullmutants were lethal at the larval-pupal transition and showedgrossly aberrant nuclear structures with chromatin clumps(Furukawa et al., 2003). BAF-depletion in Caenorhabditiselegans by RNA interference caused defects in chromatinsegregation (Zheng et al., 2000). Furthermore, BAF mutantsdefective in binding the LEM domain and DNA did notaccumulate at chromosomes and inhibited assembly of laminA, emerin and LAP2β in HeLa cells (Haraguchi et al., 2001).In vitro, BAF favored or inhibited chromatin decondensationduring assembly of nuclei in Xenopus extracts depending onits concentration (Segura-Totten et al., 2002). Finally, ourprevious observations have indirectly shown a role of LAP2α-BAF complexes in assembly. C-terminal LAP2α mutants thatbound 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 nuclearmembrane assembly in in vitro assays.

In summary, we suggest a model for nuclear assembly inwhich transient accumulation of LAP2α and BAF at telomeresand/or chromatin cores mediates chromatin reorganization andNE assembly. However, given that BAF is an evolutionarilyconserved protein found in metazoans (Cai et al., 1998; Leeand Craigie, 1998), whereas LAP2α has only been detected invertebrates (Dechat et al., 2000b; Vlcek et al., 2001), one has

to assume that this mechanism is not essential for nuclearassembly in general, but may contribute to vertebrate-specificchromatin organization.

We thank Katherine Wilson, Johns Hopkins School of Medicine,Baltimore, USA, for providing BAF antiserum and plasmids. Thisstudy was supported by grants from the Austrian Science ResearchFund (FWF, P15312), from the Jubiläumsfonds of the AustrianNational Bank (9006) and from the ‘ÖsterreichischeMuskelforschung’ to R.F. and the Japan Science and TechnologyCorporation to T.H. A.G. was a recipient of a fellowship from theDeutsche Forschungsgemeinschaft (DFG), D.G. of an EMBO long-term fellowship and T.D. of a short-term travel fellowship (EurALMF,EC Transnational Access to Major Research InfrastructuresProgramme).

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