To be or not to be in the nucleolus.

Compartmentalization has long been known to have a key role in regulation of cellular processes. By keeping enzymes and regulatory complexes in compartments where the delivery of substrate or exit of product is controlled, competing reactions can occur simultaneously in different parts of the cell. Moreover, spatial confinement facilitates the working of molecules participating in reaction chains and is crucial for coupling unfavourable with energetically favourable chemical reactions. Although in many cases intracellular compartmentalization relies on boundaries imposed by membranes, several non-membrane-bounded compartments exist in eukaryotic cells. One of these, the nucleolus, has recently attracted much attention. The emerging view is that molecular confinement in the nucleolus actively contributes to the control of cellular survival and proliferation.

Detection of rare and novel EGFR mutations in NSCLC patients: Implications for treatment-decision.

OBJECTIVES: Mutations in the gene that encodes epidermal growth factor receptor (EGFR) are biomarkers that predict how non-small cell lung cancer (NSCLC) patients respond to EGFR-targeted therapies collectively known as tyrosine kinase inhibitors (TKIs). Thus, EGFR genotyping provides crucial information for treatment decision. Both Sanger sequencing and real-time PCR methodologies are used for EGFR genotyping. However, methods based on real-time PCR have limitations, as they may not detect rare or novel mutations. The aim of this study was to determine the prevalence of rare mutations in the tyrosine kinase domain (exons 18-21) of the EGFR gene not targeted by the most frequently used real-time PCR approaches, i.e., the cobas® EGFR Mutation Test, and the Idylla™ EGFR Mutation Assay. METHODS: A total of 1228 NSCLC patients were screened for mutations in exons 18-21 of the EGFR gene using Sanger sequencing. RESULTS: We observed that 252 patients (∼20%) had at least one mutation in the EGFR gene, and 38 (∼3%) carried uncommon genetic alterations that would not be identified by the cobas® or the Idylla™ tests. We further found six new single mutations and seven previously unreported compound mutations. Clinical information and patient outcome are presented for these cases. CONCLUSIONS: This study highlights the value of sequencing-based approaches to identify rare mutations. Our results add to the inventory of known EGFR mutations, thus contributing to improved lung cancer precision treatment.

The coiled body.

The coiled body is a nuclear organelle that contains snRNPs involved in splicing, the non-snRNP splicing factor U2AF and the nucleolar protein fibrillarin. It is highly conserved in evolution and is present in both animal and plant cells. The coiled body is a dynamic structure that can undergo regulated cycles of assembly and disassembly during interphase and mitosis and it may represent a distinct metabolic compartment within the nucleus.

Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis--evidence that the coiled body is a kinetic nuclear structure.

Coiled bodies (CBs) are nuclear organelles in which splicing snRNPs concentrate. While CBs are sometimes observed in association with the nucleolar periphery, they are shown not to contain 5S or 28S rRNA or the U3 snoRNA. This argues against CBs playing a role in rRNA maturation or transport as previously suggested. We present evidence here that CBs are kinetic structures and demonstrate that the formation of snRNP-containing CBs is regulated in interphase and mitosis. The coiled body antigen, p80 coilin, was present in all cell types studied, even when CBs were not prominent. Striking changes in the formation of CBs could be induced by changes in cellular growth temperature without a concomitant change in the intracellular p80 coilin level. During mitosis, CBs disassemble, coinciding with a mitotic-specific phosphorylation of p80 coilin. Coilin is shown to be a phosphoprotein that is phosphorylated on at least two additional sites during mitosis. CBs reform in daughter nuclei after a lag period during which they are not detected. CBs are thus, dynamic nuclear organelles and we propose that cycling interactions of splicing snRNPs with CBs may be important for their participation in the processing or transport of pre-mRNA in mammalian cells.

Nuclear organization of splicing snRNPs during differentiation of murine erythroleukemia cells in vitro.

Murine erythroleukemia (MEL) cells are erythroid progenitors that can be induced to undergo terminal erythroid differentiation in culture. We have used MEL cells here as a model system to study the nuclear organization of splicing snRNPs during the physiological changes in gene expression which accompany differentiation. In uninduced MEL cells, snRNPs are widely distributed throughout the nucleoplasm and show an elevated concentration in coiled bodies. Within the first two days after induction of terminal erythroid differentiation, the pattern of gene expression changes, erythroid-specific transcription is activated and transcription of many other genes is repressed. During this early stage splicing snRNPs remain widely distributed through the nucleoplasm and continue to associate with coiled bodies. At later stages of differentiation (four to six days), when total transcription levels have greatly decreased, splicing snRNPs are redistributed. By six days postinduction snRNPs were concentrated in large clusters of interchromatin granules and no longer associated with coiled bodies. At the end-point of erythroid differentiation, just before enucleation, we observe a dramatic segregation of splicing snRNPs from the condensed chromatin. Analysis by EM shows that the snRNPs are packaged into a membrane-associated structure at the nuclear periphery which we term the "SCIM" domain (i.e., SnRNP Clusters Inside a Membrane).

Differential interaction of splicing snRNPs with coiled bodies and interchromatin granules during mitosis and assembly of daughter cell nuclei.

In the interphase nucleus of mammalian cells the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs), which are subunits of spliceosomes, associate with specific subnuclear domains including interchromatin granules and coiled bodies. Here, we analyze the association of splicing snRNPs with these structures during mitosis and reassembly of daughter nuclei. At the onset of mitosis snRNPs are predominantly diffuse in the cytoplasm, although a subset remain associated with remnants of coiled bodies and clusters of mitotic interchromatin granules, respectively. The number and size of mitotic coiled bodies remain approximately unchanged from metaphase to early telophase while snRNP-containing clusters of mitotic interchromatin granules increase in size and number as cells progress from anaphase to telophase. During telophase snRNPs are transported into daughter nuclei while the clusters of mitotic interchromatin granules remain in the cytoplasm. The timing of nuclear import of splicing snRNPs closely correlates with the onset of transcriptional activity in daughter nuclei. When transcription restarts in telophase cells snRNPs have a diffuse nucleoplasmic distribution. As cells progress to G1 snRNP-containing clusters of interchromatin granules reappear in the nucleus. Coiled bodies appear later in G1, although the coiled body antigen, p80 coilin, enters early into telophase nuclei. After inhibition of transcription we still observe nuclear import of snRNPs and the subsequent appearance of snRNP-containing clusters of interchromatin granules, but not coiled body formation. These data demonstrate that snRNP associations with coiled bodies and interchromatin granules are differentially regulated during the cell division cycle and suggest that these structures play distinct roles connected with snRNP structure, transport, and/or function.

In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

Microinjection of anti-coilin antibodies affects the structure of coiled bodies.

The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies.

We have recently shown that discrete foci are present in the nuclei of mammalian cells in which each of the U1, U2, U4/U6, and U5 snRNPs involved in pre-mRNA splicing, and the non-snRNP-splicing factor U2AF, are concentrated (Carmo-Fonseca, M., D. Tollervey, R. Pepperkok, S. Barabino, A. Merdes, C. Brunner, P. D. Zamore, M. R. Green, E. Hurt, and A. I. Lamond. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:195-206; Carmo-Fonseca, M., R. Pepperkok, B. S. Sproat, W. Ansorge, M. S. Swanson, and A. I. Lamond. 1991 EMBO (Eur. Mol. Biol. Organ.) J. 10:1863-1873). Here, we identify these snRNP-rich organelles as coiled bodies. snRNPs no longer concentrate in coiled bodies after cells are treated with the transcription inhibitors alpha-amanitin or actinomycin D. snRNP association with coiled bodies is also disrupted by heat shock. This indicates that the association of snRNPs with coiled bodies may be connected with the metabolism of nascent transcripts. A novel labeling method is described which shows both the RNA and protein components of individual snRNPs colocalizing in situ. Using this procedure all spliceosomal snRNPs are seen distributed in a nonhomogeneous pattern throughout the nucleoplasm, excluding nucleoli. They are most concentrated in coiled bodies, but in addition are present in "speckled" structures which are distinct from coiled bodies and which contain the non-snRNP splicing factor SC-35. U1 snRNP shows a more widespread nucleoplasmic staining, outside of coiled bodies and "speckled" structures, relative to the other snRNPs. The association of snRNPs with "speckles" is disrupted by heat shock but enhanced when cells are treated with alpha-amanitin.

The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body.

The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.

Targeting of U2AF65 to sites of active splicing in the nucleus.

U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast.

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.

Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies.

The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.

TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture.

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.

The cdk7-cyclin H-MAT1 complex associated with TFIIH is localized in coiled bodies.

TFIIH is a general transcription factor for RNA polymerase II that in addition is involved in DNA excision repair. TFIIH is composed of eight or nine subunits and we show that at least four of them, namely cdk7, cyclin H, MAT1, and p62 are localized in the coiled body, a distinct subnuclear structure that is transcription dependent and highly enriched in small nuclear ribonucleoproteins. Although coiled bodies do not correspond to sites of transcription, in vivo incorporation of bromo-UTP shows that they are surrounded by transcription foci. Immunofluorescence analysis using antibodies directed against the essential repair factors proliferating cell nuclear antigen and XPG did not reveal labeling of the coiled body in either untreated cells or cells irradiated with UV light, arguing that coiled bodies are probably not involved in DNA repair mechanisms. The localization of cyclin H in the coiled body was predominantly detected during the G1 and S-phases of the cell cycle, whereas in G2 coiled bodies were very small or not detected. Finally, both cyclin H and cdk7 did not colocalize with P80 coilin after disruption of the coiled body, indicating that these proteins are specifically targeted to the small nuclear ribonucleoprotein-containing domain.

Chromosomal G-dark bands determine the spatial organization of centromeric heterochromatin in the nucleus.

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric alpha-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric alpha-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that "chromosomal environment" plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.

The dynamics of coiled bodies in the nucleus of adenovirus-infected cells.

The coiled body is a specific intranuclear structure of unknown function that is enriched in splicing small nuclear ribonucleoproteins (snRNPs). Because adenoviruses make use of the host cell-splicing machinery and subvert the normal subnuclear organization, we initially decided to investigate the effect of adenovirus infection on the coiled body. The results indicate that adenovirus infection induces the disassembly of coiled bodies and that this effect is probably secondary to the block of host protein synthesis induced by the virus. Furthermore, coiled bodies are shown to be very labile structures, with a half-life of approximately 2 h after treatment of HeLa cells with protein synthesis inhibitors. After blocking of protein synthesis, p80 coilin was detected in numerous microfoci that do not concentrate snRNP. These structures may represent precursor forms of the coiled body, which goes through a rapid cycle of assembly/disassembly in the nucleus and requires ongoing protein synthesis to reassemble.

Inflammatory cell infiltrate and RANKL/OPG expression in rheumatoid synovium: comparison with other inflammatory arthropathies and correlation with outcome.

OBJECTIVES: To evaluate if the immunofluorescence analysis of synovial tissue (ST) using antibodies against RANKL/OPG, conjugated with the immunophenotyping of lymphocytes and macrophages, could be of diagnostic and prognostic value in rheumatoid arthritis (RA) patients. METHODS: 3-year prospective study of 103 consecutive patients submitted to closed needle biopsy for diagnostic purposes. ST was analyzed with routine histologic techniques and immunofluorescence, using monoclonal antibodies against RANKL, OPG, CD163, CD68, CD4, CD8, interferon-gamma and CD19. Patients were prospectively evaluated with a clinical, laboratorial and radiological protocol. At the end of the follow-up patients were divided according to the final diagnosis. Results of the initial histologic evaluation were compared between the main diagnostic groups and in RA patients histologic data was correlated with clinical and radiologic outcome measures. RESULTS: The RANKL/OPG ratio and the inflammatory infiltrate were significatively higher in RA (n = 25) as compared to the same ratio observed in other inflammatory joint diseases (OIJD, n = 48) and in osteoarthritis (n = 17). The difference between RA and OIJD was specifically confirmed when the comparison involved spondyloarthropathy (n = 26). Final HAQ score and radiologic outcome were correlated with the density of intimal CD68+ macrophages. Radiologic progression was correlated with subintimal CD4+ lymphocytes and CD68+ macrophages and intimal CD68 and CD163+ macrophages. CONCLUSION: The quantification of the RANKL/OPG ratio and of the number of lymphocytes in the ST might be useful to differentiate RA from other inflammatory joint diseases. The ST number of CD4+ lymphocytes and macrophages are probable predictors of radiologic progression in RA patients.

Human nucleoporin p62 and the essential yeast nuclear pore protein NSP1 show sequence homology and a similar domain organization.

NSP1 is an essential nuclear pore protein in yeast. We observed that anti-NSP1 antibodies label mammalian nuclear pore complexes and recognize nucleoporin p62. Also peptide antibodies raised against the NSP1 carboxy-terminal end cross-react with p62, a conserved component of the nuclear pore complex in higher eukaryotes. To further analyze the structural and functional similarity between NSP1 and mammalian nucleoporins, we cloned and sequenced nucleoporin p62 from a HeLa cDNA library. Human p62 consists of a carboxy-terminal domain homologous to the essential yeast NSP1 carboxy-terminal domain and an amino-terminal half resembling the repetitive middle domain of NSP1. The full-length p62 and a fusion protein consisting of cytosolic mouse dihydrofolate reductase (DHFR) and the p62 carboxy-terminal domain were expressed in transfected HeLa cells. Only overexpressed full-length p62, but not the DHFR-C-p62 fusion protein, binds wheat germ agglutinin (WGA). This suggests that modification by N-acetylglucosamine is mainly restricted to the repetitive amino-terminal half of p62 and implies a role of this type of repetitive sequences in nuclear transport. In the transfected HeLa cells, the DHFR-C-p62 fusion protein forms patchy aggregates that accumulate at the nuclear periphery but are also scattered through the cytoplasm. It is suggested that nucleoporin p62 may be targeted and anchored to the pore complex via its carboxy-terminal domain which reveals a hydrophobic heptad repeat organization similar to that found in lamins and other intermediate filament proteins.

Filamentous cross-bridges link intermediate filaments to the nuclear pore complexes.

Intermediate filament-nuclear matrix interactions were studied in cultured rat ventral prostate cells and isolated rat uterine epithelial cells. Cytokeratin filaments were identified by immunoelectron microscopy. In addition to conventional thin section of Triton X-100 treated cells, subcellular residues composed of intermediate filaments and nuclear matrix were critical-point dried and platinum-carbon replicated. The results demonstrate the presence of a previously unrecognized type of filamentous cross-bridges that link intermediate filaments to the nuclear pore complexes.