NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

Many biological processes depend on the balance between the dynamics and the stability of the actin cytoskeleton. The cellular organization of the microfilament system is therefore characterized by a large variability and flexibility. Proteins of the α-actinin superfamily utilize a double calponin homology domain to arrange actin filaments in bundles or meshworks and to link them to the plasma membrane(Matsudaira, 1994). The modular organization of the proteins of the α-actinin superfamily(Puius et al., 1998) allows the building of extremely long protein chains. The largest proteins mainly represent two families: plakins (plectin and dystonin)(Brown et al., 1995;Wiche, 1998) and spectrins(dystrophin, utrophin and spectrin itself)(Ahn and Kunkel, 1993;Tinsley et al., 1992). Plakins are characterized by a tripartite structure, where a central coiled-coil rod separates two globular domains: the N-terminal plakin domain and the C-terminal domain, which is composed of plectin repeats(Ruhrberg and Watt, 1997). The characteristic feature of spectrins is the rod domain, which is composed of multiple triple helical spectrin repeats(Pascual et al., 1997).

The identification of the Drosophila protein kakapo (also known as groovin) (Gregory and Brown,1998; Prokop et al.,1998; Strumpf and Volk,1998) and its mammalian homologue MACF (also described under the names trabeculin-α and macrophin)(Leung et al., 1999;Okuda et al., 1999;Sun et al., 1999), as well as BPAG1-a and BPAG1-b forms of the BPAG/dystonin locus(Leung et al., 2001b), brought confusion to this classification, since these proteins, which are larger than the other known plakin and spectrin proteins, harbor both domains found in plakin and spectrin and may represent a fusion product of two precursor genes. An alternative hypothesis proposes that the ancestral gene resembled the shortstop/kakapo genes of Drosophila and Caenorhabditis (Leung et al.,2001a). Owing to their multiple binding sites for microtubules,intermediate filaments and microfilaments, these giant proteins are recognized as cytolinkers, integrating the cytoplasmic cytoskeleton with membranes and submembrane complexes (Fuchs and Yang,1999; Karakesisoglou et al.,2000).

The recently described protein calmin, which also possesses a N-terminal actin-binding domain (ABD) of the α-actinin type, does not fall into any of the above categories, as it does not harbor either a coiled-coil rod domain or any other common motifs (Ishisaki et al., 2001). Instead it has a C-terminal transmembrane domain (TMD)that targets cytoplasmic reticular structures and therefore represents the first integral membrane α-actinin-related protein known so far. However,some transcripts of calmin are differentially spliced and lack the TMD. The distribution pattern of the calmin isoforms did not overlap with that of the actin cytoskeleton. Here we report the molecular characterization and cellular localization of NUANCE, the largest known protein of the α-actinin superfamily. The 796 kDa protein harbors multiple dystrophinlike spectrin repeats but lacks domains characteristic for plakins. Hence, it is related to the spectrin family of the α-actinin superfamily. Unlike other related proteins, NUANCE is associated with the nuclear envelope (NE) via the predicted TMD. This enormously large protein may harbor numerous binding sites and serve as a scaffold for the assembly of various protein complexes.

The clones AA480953 and AA761297, containing partial NUANCE cDNA, were obtained from RZPD (Resource Center/Primary Database of German Human Genome Project) and fully sequenced. The full-length cDNA of NUANCE was cloned by using SMART RACE cDNA amplification kit (Clontech) and Advantage 2 polymerase(Clontech) with mRNA from Burkitt's lymphoma cells BL-60 as a template. With 5′-RACE-PCR we extended the cDNA to the putative 5′ end (fragment 1, Fig. 1A). Several rounds of 3′-RACE-PCR extended the cDNA to a total length of 6.1 kb (fragments from 2 to 5, Fig. 1A). Close inspection of the publicly accessible genomic sequence (GenBank) downstream of the identified NUANCE partial cDNA revealed a polyadenylated cDNA of DKFZp434G173 (accession number AL080133), which encoded several spectrin repeats, similar to those found in NUANCE. This suggested that the amplified 6.1 kb fragment of NUANCE and DKFZp434G173 cDNAs represent the 5′ and 3′ end of the same giant transcript. The ESTs matching the genomic DNA from the gap region, which encoded peptides with homology to dystrophin-like spectrin repeats, were used to design supplementary primers. The PCR products were cloned into the pGEM T-Easy vector (Promega) and sequenced. 22 clones containing partial NUANCE cDNAs were used to assemble fulllength NUANCE cDNA(accession number AF435011). The cDNA of the short ABD-S isoform has been deposited under accession number AF435010. Partial mouse NUANCE cDNAs from RT-PCR of kidney RNA with the primers designed from the sequence of EST clones A1747790 and AA498987 were deposited under accession number AF435012.

The expression profile of human NUANCE was studied by probing a human multiple tissue expression array (Clontech). The cDNA probe encompassing nucleotides from 247 to 1443, which corresponds to the NUANCE ABD with the following spectrin repeat, was ³²P-labeled using a random prime labeling kit (Stratagene). As a control, the blot was stripped and reprobed with ubiquitin.

DNA fragments encoding the first 285 (6×His-ABD) and 459 amino acid residues (6×His-ABD-1) were obtained by PCR using fragment 2 as a template and inserted into pQE-30 (Qiagen). Recombinant proteins were induced by IPTG in E. coli M15[pREP4] and purified using a Ni-NTA agarose column according to the manufacturer (Qiagen). mAb K20-478 was produced by immunizing mice with the purified 6×His-ABD-1 protein with ImmunEasy mouse adjuvant (Qiagen) as described earlier(Olski et al., 2001).

The F-actin co-sedimentation assay and the quantification of 6×His-ABD bound to F-actin was performed as described previously(Olski et al., 2001). For high- and low-speed centrifugation assays, samples were pelleted at 125,000 g and at 20,000 g, respectively. Actin polymerization was measured by recording the changes in fluorescence intensity of pyrene-labeledα-actin monomers as described previously(Korenbaum et al., 1998). The fluorescence measurements were made using a Fluoroskan Ascent FL plate reader(Labsystems). Polymerization of 8 μM actin alone or in the presence of various amounts of the ABD was initiated by addition of 2 mM MgCl₂and 100 mM KCl.

The constructs GFP-ABD (residues 1-296) and GFP-ABD-S (residues 1-262 with additional amino acids AYKN from exon 8a) were amplified from fragment 2 and 2a, which code for spliced variants, using primers with extensions for BamHI and KpnI sites and cloned into BglII/KpnI-cut pEGFP-C1 (Clontech). For the GFP-ABDsr1-2 construct (residues 1-531), fragment 2 was digested with BamHI and EcoRI then ligated into BglII/EcoRI-cut pEGFP-C1 vector. For the GFP-sr15-21 (residues 5727-6596), the fragment 13a was excised utilizing the EcoRI sites of pGEM T-Easy (Promega) and ligated into EcoRI site of pEGFP-C1. For the GFP-Cterm1 construct (residues 6571-6885), the cDNA was amplified from clone 13 and ligated into EcoRI-cut pEGFP-C2 vector. For the GFP-Cterm2 constructs, clone 13b was digested with SacI and EcoRI and inserted into corresponding sites of pEGFP-C1. The GFP-Cterm2Δtm (residues 6642-6834 with additional 14 amino acids from the extended exon 111) lacking the putative TMD was generated from the GFP-Cterm2 by PstI digestion and religation. To generate GFP-NUA^Δ460-6643, the GFP-ABDsr1-2 was cut with KpnI and BamHI and ligated with the insert of the GFP-Cterm1 (residues 6643-6885), which was amplified using the primers with added-on KpnI and BglII sites. COS7 cells were transfected by electroporation. The expression of the fusion proteins was controlled by western blotting using a GFP-specific mAb.

COS7 cells were grown in DME medium supplemented with 10% FBS (Sigma), 2 mM glutamine, penicillin and streptomycin. For the propagation of human embryonic kidney cells (293) pyruvate was added to the medium. Human T lymphocytes(Jurkat), Burkitt's lymphoma cells BL-60 and B-JAB were maintained in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, penicillin and streptomycin. For Latrunculin A (Biomol) treatment, 70%-confluent COS-7 cells were fixed 15, 30 and 60 minutes after exposure to 1 μM LatA. The recovery was evaluated by culturing the cells in LatA-free medium for another 30, 60 and 90 minutes. For microtubule depolymerization the cells were incubated in medium containing 1μM vincristine (Sigma) or 5 μg/ml colchicine (Fluka) for 4 hours before processing for immunofluorescence. To promote the formation of lamellipodia,confluent serum-starved monolayers of COS7 cells were wounded by scraping away cells with a P1000 pipet tip. The coverslips were then incubated in complete media for 6 hours prior to fixation.

Adherent cells were allowed to attach onto glass coverslips for 1-16 hours,rinsed with PBS, fixed in 3% paraformaldehyde and permeabilized with 0.5%Triton X-100 in PBS for 5 minutes. Alternatively, cells were fixed with methanol at -20°C for 10 minutes. Generally, no difference was observed between the two fixation protocols except for the cells transfected with the GFP-Cterm2 construct. All figures display cells fixed with paraformaldehyde unless stated otherwise. For permeabilizing with digitonin, fixed cells(3%-paraformaldehyde) were washed in ice-cold PBS and treated with 40 μg/ml digitonin (Sigma) in PBS for 5 minutes on ice. For poorly adherent cells,cover slips were pretreated with poly-L-lysine (1 mg/ml), and cells were allowed to attach for at least 3 hours. Cells were incubated with mAbs against vinculin (Sigma), annexin A7 (Selbert et al., 1995), with the maD mAb, which is specific for β-COP(Pepperkok et al., 1993), with JOL2, which is specific for lamin A/C, with LN43, which is specific for anti-lamin B2 (kind gift from Frans Ramaekers and Jos Broers) or with polyclonal antibodies against Nup358 (kind gift from Elias Coutavas and Günter Blobel) and against NO38/B23 (kind gift from M. Schmidt-Zachmann). Cells washed with PBS were incubated with the appropriate secondary antibodies conjugated to Cy3 (Sigma), Alexa488 or Alexa568 (Molecular Probes) and mounted in Gelvatol/DABCO (Sigma). Factin was detected with TRITC-labeled phalloidin;for nuclear staining the DNA-specific dye DAPI (Sigma) was used. Samples were analyzed by wide-field fluorescence microscopy (DMR, Leica) or confocal laser scanning microscopy (TCS-SP, Leica).

COS7 cells were harvested and washed in PBS, homogenized in lysis buffer(1% SDS, 1 mM sodium vanadate and 100 mM Tris-HCl, pH 7.4), incubated at 95°C for 5 minutes and mixed with the protein sample buffer(Laemmli, 1970) and heated again for 5 minutes to 95°C. Proteins were separated on 3-15% gradient SDS-PAGE then transferred onto PDVF membrane (Millipore). The membranes were treated with mAb K20-478 followed by enhanced chemiluminescence.

For nuclei preparation, COS7 cells were sonicated in hypotonic buffer (10 mM HEPES, pH 7.5, 1.5 mM MgCl₂, 1.5 mM KCl, 0.5 mM DTT, 0.2 mM PMSF) supplemented with Protease Inhibitor Cocktail CompleteTM, Mini(Boehringer Mannheim). Nuclei were sedimented at 1,000 g at 4°C for 15 minutes. For further fractionation the supernatant was centrifuged at 100,000 g for 30 minutes at 4°C. Both pellets were resuspended in hypotonic buffer and analyzed on immunoblots with anti-NUANCE, anti-lamin B2 (LN43) and anti-annexin A7 mAbs.

For alignment of cDNA sequences and database mining, the GCG software package and the BLAST (NCBI) program were used. Protein sequences were aligned using the programs ClustalW and TreeView. Motif predictions and pattern searches were performed with the ExPaSY (SIB) software package.

We searched for new proteins with homology to the ABD of α-actinin by screening the human EST database using the peptide sequence of ABDs of knownα-actinin-related proteins as a query. Two overlapping ESTs, AA480953 and AA761297, were identified that contained a partial cDNA encoding for a novel protein with an ABD that was highly homologous to MACF(Leung et al., 1999) and plectin (Wiche et al., 1991). Both clones were derived from a cDNA library prepared from human tonsillar cells enriched for germinal center B cells. Combining RACE-PCR with the analysis of data available from human non-redundant EST and human genomic GenBank databases, we cloned 21.8 kb of NUANCE cDNA (see Materials and Methods; Fig. 1A). The putative ATG start codon at position 205 together with the surrounding sequence AGAATGG matched the Kozak consensus (Kozak,1987). An in-frame stop codon was located 26 bp upstream, making this ATG the translational start site. The 3′ untranslated region(3′UTR) of 910 bases ends in a poly(A) tail. A polyadenylation signal,ATTAAA, is located 22 nucleotides upstream of the poly(A) addition site.

To analyse the intron-exon organization of NUANCE and to identify the alternatively spliced isoforms, we searched the genomic database and found that the NUANCE gene matched the publicly available working draft sequences of human clones with the accession numbers AL355100.2, AL162832.3,AL359235.2, AL355094.2, AF215937.1, AL352983.2 and AL161756.3 of chromosome 14 in contig NT_025892 mapped to 14q22.1-q22.3(Fig. 1B). The human NUANCE gene spans over 373 kb. About 6 kb downstream the NUANCE gene is the estrogen receptor 2 gene (ESR2).

The NUANCE gene is split into 115 recognizable exons(Fig. 1B,Table 1). All exon-intron boundaries are consistent with the consensus sequence for splice junctions 5′-GT...AG-3′ (Breathnach and Chambon, 1981). The intronic sequences comprise 94% of the total gene length. The 5′UTR is interrupted by the longest 55,954 bp intron located 42 bp upstream of the start codon, therefore translation starts in the second exon. An alternatively spliced form with the exon 1a positioned 228 bp upstream of exon 1 was found in the EST BI026470. The exon lengths vary from 50 nt in exon 114 to 2101 nt in the exon 48 coding for central coiled-coil sequences.

Exons 3 to 9 encode the ABD region of NUANCE. One of the ABD fragments amplified by RACE-PCR (clone 2a) contained an additional exon 8a(Fig. 1C) inserted between exons 8 and 9. This leads to premature termination of the ORF and generation of an isoform comprising a truncated ABD, named ABD-S. Although the physiological relevance of ABD-S is not yet clear, the effect of ABD-S overexpression was analyzed in transfection experiments (see below;Fig. 9B,E). Several differentially spliced cDNAs corresponding to the 3′ end of the NUANCE cDNA were found in the non-redundant NCBI database. The cDNAs with accession numbers AK001876, AL080133, AB023228, which correspond to a protein KIAA1011(Syne-2), may represent a partial sequence of NUANCE, since their ORF is not interrupted by any Stop codons upstream of the methionine proposed as a translation initiation in AL080133. The cDNA with accession number AL117404 appears to contain a short isoform with a distinct 5′ terminus(Fig. 1D). However, it can also result from amplification of unprocessed mRNA.

The NUANCE ORF encodes a protein of 6,885 amino acids with a molecular mass of 796,000 (Fig. 2). The theoretical pI is 5.29. Three distinct structural domains can be distinguished: the N-terminal ABD (Fig. 3A,B), which is followed by a long largely helical rod domain(Fig. 3A,E) and a C-terminal TMD (Fig. 3A,D).

The ABD of NUANCE shares a high homology with the ABDs of the recently identified proteins enaptin (S. Braune, MD Thesis, University of Cologne,2001) and calmin (Ishisaki et al.,2001). The ABDs of these three proteins differ from the conventional ones as they have 30 amino-acids long linkers between two CH domains (Fig. 3B). Across its 255 amino acid ABD region, NUANCE shares about 49% identity with enaptin, 45%with calmin and 43% with β-spectrin, plectin, dystonin and MACF. The phylogenetic analysis of the ABDs suggested that NUANCE, enaptin and calmin form a distinct family within the α-actinin superfamily(Fig. 3C). BLAST searches with human NUANCE cDNA identified several highly homologous mouse EST clones. On the basis of the sequence of EST clones AI747790 and AA498987, we have designed primers and amplified a partial mouse NUANCE cDNA. The amino-acid sequence of the mouse and human NUANCE ABD is well conserved with an identity of 97%.

A central rod domain contains coiled coils interrupted by several fragments of random coils. Furthermore, four nuclear localization signals (amino acids 1188-1205, 1464-1467, 3629-3645 and 6115-6132) and two leucine zippers (amino acids 2127-2148 and 5008-5029) were also predicted by computer analysis within the rod domain (Fig. 3A). Multiple repeated units weakly homologous to the triple-helical spectrin-like repeats of dystrophin and utrophin can be recognized in the regions predicted for two- and three-stranded coiled coils(Fig. 3E). The repeats found in NUANCE are less regular than the ones of dystrophin and utrophin(Winder et al., 1995). The alignment of the 22 best-defined NUANCE repeats are shown inFig. 3E. The helix A is characterized by a highly conserved tryptophan followed by a hydrophobic residue. The first helix (helix A) and the last one (helix C), which are continuous in the following repeats, are relatively well defined, whereas the middle helix B is less ordered. Random coils in the regions 4082-4232,4332-4532 and 6347-6547 (Fig. 3A) may represent hinges providing additional flexibility to the molecule.

In addition, the C-terminal region of NUANCE contains a 62 amino-acid region similar to the Syne-1 and Drosophila Klarsicht proteins, which are involved in nuclear migration and nuclear positioning(Apel et al., 2000;Mosley-Bishop et al., 1999). The highly conserved hydrophobic stretch of 22 amino acids (residues 6848 to 6872) in the Klarsicht-like domain was identified as a transmembrane region(Fig. 3D). It is flanked by the N-terminally positioned neck region and the C-terminal tail.

In a Human Tissue Multiple Expression array containing mRNA from a variety of human tissues and cell lines we found that most of the tissues showed detectable levels of NUANCE mRNA. The highest expression was detected in the kidney, both adult and fetal, liver, stomach and placenta, and the lowest levels were in skeletal muscle and brain(Fig. 4A,Table 2). The corpus callosum and pituitary gland displayed a relatively strong signal in contrast to the generally low levels of expression in other parts of the brain. Expression of NUANCE in lymphatic organs was not uniform either. High levels of mRNA were detected in spleen and lymphatic nodes; however they were relatively low in the thymus and hardly detectable in bone marrow. Taken together with the strong signal seen in peripheral blood lymphocytes, this suggests that NUANCE is characteristic for mature lymphocytes. In the digestive system only stomach, duodenum and salivary gland showed an elevated level of NUANCE mRNA relative to the other parts of the gastrointestinal tract. In addition,significant amounts of mRNA were also noted in the trachea, prostate, gonads,thyroid and adrenal glands. In RNA from cancer-derived cell lines, NUANCE was expressed at hardly traceable amounts with the exception of Daudi Burkitt's lymphoma cells.

mAb K20-478 generated against the ABD of NUANCE recognized a protein of about 800 kDa that cofractionated with nuclei and membranes of COS7 cells(Fig. 4B,C). The mAbs against lamin B2 as nuclear protein and annexin A7 as mainly cytosolic protein were included as a control. Weaker bands below may represent degradation products of NUANCE, although they could also be short isoforms or crossreactive proteins. For size estimation, we included the A chain of EHS-laminin with a molecular mass of about 400,000, which migrated significantly faster than NUANCE (data not shown).

In immunofluorescence studies the mAb yielded a spotted rim-enriched nuclear labeling in Burkitt's lymphoma cells BL-60, human embryonic kidney cells 293 and COS7 cells (Fig. 5). In addition, a dotted pattern was detected in the cytoplasm,which might result from association with vesicular structures or ER(Fig. 5A,C). A similar pattern was observed in C3H/10T1/2 mouse fibroblasts (data not shown). NUANCE is also seen associated with bridges that occasionally connected nuclei in COS7 cells(Fig. 5C,D). The punctate NUANCE staining of the nuclear surface was reminiscent of that of nucleoporins. A comparison of NUANCE and Nup358 distribution(Wu et al., 1995) however showed that both proteins are only partly colocalized(Fig. 6A-C), which implies that NUANCE is targeted to the NE without being localized to nuclear pores.

To clarify whether NUANCE is associated with the inner (INM) or outer nuclear membrane (ONM) we compared the immunostaining of the digitonin- and Triton X-100-permeabilized cells. Digitonin disrupts the plasmalemma leaving the intracellular membranes, including the NE, intact(Adam et al., 1990). As a result, the mAb can access ONM-associated antigens but not the proteins facing the nucleoplasm. The anti-NUANCE mAb stained only the nuclear periphery of the digitonin-permeabilized cells, which was similar to the staining for cytoplasm-facing nucleoporin 358 (Fig. 6J-L). The anti-lamin B2 mAb taken for a control did not yield any staining in digitonin-treated cells in interphasel; instead it labeled mitotic cells on the same cover slip (data not shown). This suggests that NUANCE associates with the ONM with its N-terminus facing the cytoplasm. However, it does not exclude its further association with the inner membrane of the envelope. In the Triton X-100-treated cells we also observed a labeling in the nucleus (Fig. 6D-I). It was especially enriched at nucleoli, which were identified using antiserum against the nucleolus protein NO38/B23 (G-I). Furthermore we always observed a diffuse cytoplasmic NUANCE staining that accumulated at the nuclear invagination harboring the Golgi apparatus (Fig. 6J), as visualized with a β-COP-specific mAb (data not shown).

In mitotic cells NUANCE remains associated with the NE during its breakdown, which marks the end of prophase and beginning of prometaphase(Fig. 5A,B, arrows). This indicates a stable association of NUANCE with the NE. In prometaphase, NUANCE is accumulated at condensed chromosomes(Fig. 5A,B, large arrowheads)and is diffusely present throughout the cytoplasm at later stages(Fig. 5A, small arrowhead).

The distribution pattern of NUANCE did not overlap with that of the actin cytoskeleton in spite of the presence of the predicted conserved ABD(Fig. 7A-C). To test whether disruption of the actin cytoskeleton affects NUANCE localization, we have treated COS7 cells with Latrunculin A (LatA), a drug depolymerizing actin filaments owing to its G-actin-sequestering activity(Yarmola et al., 2000). Already after 15 minutes exposure to 1 μM LatA, the actin filament meshwork was partially disassembled, and actin-rich foci were formed (data not shown). By 30 minutes, actin accumulated in the central area of the cell in which the NUANCE-positive `cloud' was often seen at the invagination of the nuclei(Fig. 7D-F). Along with further disassembly of the actin cytoskeleton, the nuclei became less flattened and acquired an irregular shape with wrinkled invaginations from the side where a diffuse cytoplasmic pool of NUANCE and actin aggregates accumulated(Fig. 7G-I). This effect on nuclei was fully reversible. After 3 hours washout of LatA their shape was completely restored (not shown). This observation implies that the nuclear shape is controlled by the actin cytoskeleton and that NUANCE may have a structural role in maintaining the nuclear architecture.

In wound-healing assays performed on confluent monolayers of COS7 cells,NUANCE was detected at the leading edge of migrating cells colocalized with F-actin (Fig. 7J-L); this indicates partial redistribution of NUANCE in polarized cells.

We have also examined the pattern of NUANCE distribution upon treatment with vincristine and colchicine, drugs that disrupt selectively microtubules in the cell. However, no changes in nuclear morphology or in NUANCE distribution were observed (data not shown).

As our immunofluorescence analysis revealed that association of NUANCE with the actin cytoskeleton is limited to the leading edge in migrating cells and to the perinuclear patches in LatA-treated cells, we examined the functionality and the subcellular localization of the ABD. For biochemical characterization of NUANCE we have used the recombinantly expressed ABD-containing construct 6×His-ABD. The 6×His-ABD associates with F-actin filaments in a high-speed cosedimentation assay(Fig. 8A). Some 6×His-ABD protein was present in the pellet without added actin; however it was always enriched in the pellets in the presence of actin-containing samples. The assay was performed with different concentrations of 6×His-ABD in order to quantify binding to F-actin. The K_d value was determined to be 3.8±1 μM, and saturation was achieved at a 1:1 molar ratio. The presence of 6×His-ABD also had an effect on the polymerization kinetics of pyrene-labeled actin (Fig. 8C). It shortened the elongation time and increased the rate of actin polymerization in a concentration-dependent manner. Moreover, in low-speed F-actin co-sedimentation assays most of the actin was detected in the pellet fraction, suggesting that NUANCE ABD acts to bundle F-actin(Fig. 8B).

We expressed GFP-fused ABD in COS7 cells and examined its subcellular distribution in vivo. In contrast to the endogenous protein, the GFP-ABD containing the 285 N-terminal amino acids of NUANCE was associated with all microfilament structures detectable by TRITC-phalloidin, stress fibers,lamellar meshwork and cortical actin (Fig. 9A,D). The GFP-ABD-S fusion protein, which corresponds to a short alternatively spliced isoform where insertion of an exon 8a introduced a stop codon in the second CH domain, showed a slightly different pattern. The GFP-ABD-S weakly associated with the middle parts of stress fibers but was enriched at their ends, which colocalized with the vinculin-labeled focal adhesions (Fig. 9B,E). Interestingly, the small focal complexes at the extreme edge of lamellipodia,which were not associated with stress fibers yet(Nobes and Hall, 1995), did not recruit the GFP-ABD-S (Fig. 9B,E). The GFP-ABD-S association with cortical actin was mainly confined to the bundles in retracting concave parts but not to the protruding lamellas. We also noted the formation of multiple spikes and filopodia in the cells strongly overexpressing GFP-ABD or GFP-ABD-S (data not shown). The filopodia-rich phenotype was presumably caused by the increased formation of actin bundles, which is in agreement with our in vitro observations. These proteins might therefore exhibit a dominant-negative effect. Moderate GFP-ABD expression did not seem to affect the organization of the actin cytoskeleton in COS7 cells.

To explore the role of the spectrin repeats in actin association, we constructed a fusion protein GFP-ABDsr1-2 harboring a 531 amino acid fragment containing the ABD and the first two repeats(Fig. 9C,F). Costaining the GFP-ABDsr1-2-expressing cells with TRITC-phalloidin showed overall colocalization of the fusion protein with filamentous actin (data not shown). However, the GFP-ABDsr1-2 was strongly attracted to the subplasmalemmal regions, especially in protruding lamellas(Fig. 9C,F). This implies a role for the two spectrin repeats in membrane binding. The targeting of the stress fibers and focal contacts was reduced in comparison with the GFP-ABD and GFP-ABD-S proteins. The anti-NUANCE mAb recognized the GFP-ABDsr1-2 protein (Fig. 9F) as well as the other two ABD-containing chimeras (data not shown) and showed an identical pattern.

GFP-sr15-21 harboring spectrin repeats from 15 to 20 followed by the random coil stretch and a part of repeat 21 were used for the examination of the further spectrin repeats. The distribution pattern of the GFP-sr15-21 suggests its association with the intracellular membranes and with vesicular structures colocalizing with β-COP, a component of the COP coat(Fig. 9G,J). No association with the NE was detected.

The NUANCE staining pattern appeared to be similar to the subcellular distribution of Syne-1, a protein that is associated with the NE in skeletal,cardiac and smooth muscle cells (Apel et al., 2000). As Syne-1 is highly homologous to the C-terminal part of NUANCE, the targeting of NUANCE to the NE could result from the Klarsicht-like domain at the C-terminus. To test our assumption we have prepared two C-terminal constructs, GFP-Cterm1 and GFP-Cterm2, comprising the Klarsicht-like domain and the preceding spectrin repeats. The GFP-Cterm1 protein possesses spectrin repeats 21 and 22, whereas the GFP-Cterm2 harbors only one complete repeat 22 but contains the additional 14 amino acids gained from the alternatively spliced exon 111. Both fusion proteins showed clear nuclear rim staining along with diffuse cytoplasmic staining in the Golgi area(Fig. 9H, shown for GFP-Cterm1). Moreover, both constructs seemed to displace endogenous NUANCE from the NE but did not interfere with the nucleoplasmic staining(Fig. 9K). In heavily overexpressing cells both GFP-Cterm1 and GFP-Cterm2 localized in patches at the NE and also showed a reticular or vesicular pattern in the cytoplasm (data not shown). To inspect whether the overexpression of the GFP-Cterm1 affects the nuclear pore distribution, we stained the transfected cells with the anti-Nup358 antibody. Confocal analysis reveals a mutually exclusive pattern of GFP patches and anti-Nup358 staining(Fig. 10A-C), suggesting the displacement of nuclear pores from the regions of GFP-Cterm1 accumulation. The appearance of lamins A/C and B, detected by the mAbs JOL2 and LN43, in contrast, was not affected in GFP-Cterm1- and GFP-Cterm2-expressing cells(data not shown). The overexpression of the C-terminal constructs did not hamper mitotic progression in COS7 cells. GFP-Cterm2, like the endogenous protein, remained associated with the NE during prophase, whereas the anti-Nup358 staining was largely diffuse(Fig. 10D-F), indicating at least partial disassembly of nuclear pores.

GFP-Cterm2▵tm, which is identical to GFP-Cterm2, but lacks the hydrophobic membrane-spanning sequence and the C-terminal tail, confirmed the importance of the TMD for the nuclear association. This protein was found in all cellular compartments when overexpressed in COS7 cells. However, in cells fixed with methanol, only vesicular staining in the cytoplasm remained(Fig. 9I). No nuclear staining was detected, and the localization of the endogenous NUANCE was not affected by the overexpression of the GFP-Cterm2▵tm(Fig. 9L). Thus, the TMD is necessary for recruitment to the NE. These results suggest that NUANCE is an integral protein of the NE that shares the TMD with the other proteins exhibiting a similar subcellular localization.

Having identified the intracellular locations for N-terminal and C-terminal parts of NUANCE we generated a construct GFP-NUA^▵460-6643where the first 459 amino acids were directly fused to the C-terminal 315 amino acids that correspond to the Cterm1 construct. In transfected COS7 cells, the GFP-NUA^▵460-6643 was arranged in various structures from fine filaments to large aggregates(Fig. 10G-L). In most of the cells, the GFP-NUA^▵460-6643 was observed at the NE envelope,enriched at one side of the nucleus (Fig. 10J-L). Actin was often attracted to the GFP-NUA^▵460-6643 fibers and aggregates; however,GFP-NUA^▵460-6643 was never seen in association with the dynamic submembranous actin cytoskeleton.

Cloning of the NUANCE cDNA yielded the largest protein of theα-actinin superfamily that has so far been characterized. It is conceivable that NUANCE forms dimers or even higher order oligomers since it has an extremely long largely coiled-coil rod with two leucine-zippers. NUANCE is localized at the NE, which is rather unusual for actin-binding proteins. The NUANCE ABD appears to bind to F-actin in vitro and in vivo and promote actin bundling. Moreover, transfection with the GFP-NUA^Δ460-6643 mutant has resulted in significant recruitment of F-actin to the nuclear membrane. This difference between the NUA^Δ460-6643 and wild-type phenotypes indicates that the ABD of the full-length NUANCE could be regulated in vivo, for instance by adopting a conformation with the actin-binding sites being hidden. Two other proteins,calmin and Dictyostelium interaptin, which likewise possess ABDs at the N-termini and TMDs at the C-termini, associate with cytoplasmic reticular structures and, in the case of interaptin, with the NE(Rivero et al. 1998;Ishisaki et al., 2001). The predicted TMDs of these three proteins show very low homology and may be responsible for the differential intracellular localization.

NUANCE appears to be recruited to the NE through the Klarsicht-like C-terminal domain harboring a TMD. Drosophila Klarsicht protein is required for migration of nuclei in developing retinal photoreceptors(Mosley-Bishop et al., 1999). Similarly, a role in the migration of micronuclei in myotubes and/or their anchoring at the postsynaptic apparatus was ascribed to Syne-1(Apel et al., 2000), a protein that is highly homologous to the C-terminal part of NUANCE. Syne-1 is selectively associated with the nuclei that lie beneath the postsynaptic membrane at the neuromuscular junctions in skeletal, cardiac and smooth muscle cells. Syne-1 is strongly expressed in the muscle and brain, tissues with the lowest level of NUANCE expression. Since the N-terminus of the longest Syne-1 isoform has not yet been identified, it is plausible that the full-length Syne-1 protein also possesses an N-terminal ABD and therefore may represent a muscle/brain counterpart of NUANCE. Nuclear positioning and migration are essential for the movement of pronuclei during fertilization, normal mitotic and meiotic cell division and various morphogenetic processes during metazoan development. Nuclear migration has been directly linked to a human disease,the brain developmental disorder lissencephaly(Morris, 2000). Migration of neurons involves nucleokinesis, a process whereby cells acquire an elongated shape or extend a frontal protrusion, and the nucleus migrates to its new position within the cytoplasm. The nuclear translocation within the cell can also be associated with cell motility during tumor cell invasion. In moving fibroblasts, the classical model for cell motility studies, the nucleus remains centrally anchored within the cell despite drastic morphological changes. However, the human lung adenocarcinoma cells, which generally display a fibroblast-like motility, can be switched to the characteristic pattern of cell translocation with nucleokinesis as a distinct step in response to an autocrine motility factor (Klominek et al., 1991). The motility steps of the invasion process have been proposed to comprise protrusion of the tumor cell pseudopodia through surrounding tissues, followed by nucleokinesis and, finally, retraction of the pseudopodia behind the nucleus.

On the basis of the data accumulated on NUANCE, we can envision several roles for it. Rapid shrinkage of the nuclei accompanied by the accumulation of cytoplasmic NUANCE and actin at the side of the nucleus in the LatA-treated cells suggests that the actin cytoskeleton is implicated in controlling nuclear shape and that this process could be mediated by NUANCE. In addition to a role in nuclear positioning and/or migration, NUANCE may also contribute to the mechanical connection between cell surface receptors, cytoskeletal filaments and nuclear scaffolds. Such a coordinated response to mechanical stress was demonstrated by micromanipulation of integrins, which resulted in changes in nuclear shape that may influence gene expression(Chicurel et al., 1998). Maintenance of the nuclear shape and polarity could be essential for many cell types undergoing mechanical stress in vivo, as for instance, for lymphocytes when transgressing the blood vessels.

Eight integral proteins of the NE have so far been identified. Six of them are located at the INM. These are lamina-associated polypepetide (LAP)1(Senior and Gerace, 1988),LAP2/thymopoietin (Foisner and Gerace,1993), p58/lamin B receptor (LBR)(Worman et al., 1988), emerin(Nagano et al., 1996), nurim(Rolls et al., 1999) and MAN1(Lin et al., 2000). Two integral proteins, POM121 and gp210, have been found to be specific for the nuclear pore membrane (Gerace et al.,1982; Soderqvist and Hallberg,1994). All of them are arranged with their N-termini facing the nucleoplasm. The discovery of NUANCE as the first example of a protein, which seems to associate specifically with the ONM, implies that the ONM represents a membrane subdomain that is biochemically and functionally distinct from the peripheral ER. Recent studies demonstrated that the dynein-dynactin complex is recruited to the cytoplasmic face of the NE during late G2 and early prophase prior to the NE breakdown and plays a role in tearing the NE at the beginning of mitosis (Beaudouin et al.,2002; Salina et al.,2002). Our data suggest that the N-terminal part of NUANCE faces the cytoplasm thereby allowing the protein to represent a platform for anchoring the dynein-dynactin complexes at the ONM. Interestingly, the NE wrinkles observed at the sites of invaginations of the `kidney bean' - shaped nuclei in the LatA-treated cells resemble the microtubules containing finger-like projections of the prophase and prometaphase cells. Furthermore,accumulation of F-actin and the cytoplasmic NUANCE at the sites of nuclear invaginations upon LatA treatment imply a link between NUANCE and pericentrosomal astral complex. Although at present we have no evidence of a functional relationship between NUANCE and NE breakdown, it is possible that NUANCE is involved in the spatial organization of microtubule-dependent machinery linking it to the NE.

Our results with digitonin-treated cells do not exclude an association of NUANCE with the INM where it might face the nucleoplasm with its ABD and interact with nuclear actin whose role is still elusive(Rando et al., 2000). NUANCE staining was also observed throughout the nucleoplasm and in nucleoli. These forms might well represent differentially spliced variants lacking the TMD. Such isoforms may build a fibrous meshwork in the nucleoplasm by oligomerizating via leucine zippers of the coiled coil. Although we have not identified TMD-lacking transcripts in our screen yet, their existence is quite plausible since many membrane proteins including calmin are differentially spliced and give rise to soluble isoforms(Ishisaki et al., 2001). It can be speculated that NUANCE plays a role in the organization of the intranuclear space. It was shown that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is involved in nuclear export (Hofmann et al., 2001). A role of NUANCE in nuclear export would explain its partial colocalization with nuclear pores. Actin may also be important for packaging the RNA into a Balbiani ring particles and their translocation through the nuclear pores to the cytoplasm (Percipalle et al.,2001). In a complex with actin, NUANCE may play a role in location and transportation of proteins and RNA within the nucleoplasm. The observation of NUANCE in nucleoli is particularly intriguing. The nucleoli are structures responsible mainly for ribosomal biogenesis; however they also participate in processing or export of some mRNAs and tRNAs(Schneiter et al., 1995;Bertrand et al., 1998) and are involved in sequestering cell cycle regulating proteins(Visintin and Amon, 2000). NUANCE would make an ideal candidate for organizing the nucleoli matrix and ensuring its compartmentalization and integrity.

This work was supported by a grant from the Center for Molecular Medicine Cologne and by a Köln Fortune studentship to T.L. We thank Elias Coutavas and Günter Blobel (The Rockefeller University, USA) for providing anti-Nup358 antibodies, Frans Ramaekers and Jos Broers (University of Maastricht, the Netherlands) for the gift of antilamin A/C and anti-lamin B1 antibodies, Marion Schmidt-Zachmann for the gift of anti-NO38/B23 antiserum,Mats Paulsson (University of Cologne) for laminin, Michael Schleicher(University of Munich, Germany) for sharing reagents and Prof. Diehl(University of Cologne, Germany) for providing the lymphoma cell lines.