Identification of a 270-kD Mammalian Protein as a Novel Component of NPC-attached Intranuclear Filaments
mAb 203-37 showed in immunofluorescence microscopy of cultured cells, permeabilized either by methanol/acetone treatment or by use of Triton X-100 after initial formaldehyde fixation, a punctate staining in the nuclear periphery, similar to that obtained by immunostainings of NPCs. Positive cells included diverse human cell types differing in differentiation such as cervix adenocarcinoma cells of line HeLa (Fig. , a and a′), glioblastoma cells U333/CG/343 MG, primary cultures of endothelial cells, hepatocellular carcinoma PLC cells, carcinoma cells of line A-431, colon adenocarcinoma CaCo2 cells, SV-40–transformed fibroblasts (“SV-80 cells”), and keratinocytes of line HaCaT (not shown), demonstrating that the antigen was widespread if not ubiquitous. Bovine mammary gland epithelial BMGE cells (Fig. b) and kidney cells of line MDBK (not shown), porcine kidney cells of line PK(15) (Fig. c), and canine MDCK cells (not shown) were also immunoreactive with this mAb. In some cell cultures, occasional intranuclear speckles were noticeable in addition to the peripheral staining (see also below). Nuclear immunostaining was not observed in rat cells of lines RV and 967-D2, murine cells of lines L-M(TK−) and 3T3, marsupial PtK2 cells, and African green monkey cells of line RC37. Xenopus laevis kidney epithelial cells of line A6 were also negative.
Figure 1 Immunofluorescence microscopy of cultured mammalian cells and tissue cryostat sections after reaction with mAb 203-37. (a) Phase contrast and (a′) epifluorescence optics of human adenocarcinoma cells of line HeLa. (b–e) Epifluorescence (more ...)
When cells were permeabilized with 0.004% digitonin, striking differences of nuclear labeling were noticed when comparing mAb 203-37 with antibodies against RanBP2/ Nup358, a nucleoporin located at the cytoplasmic side of the NPC. While RanBP2 antibodies showed nuclear labeling in all cells, indicating full accessibility of this protein, mAb 203-37 stained only nuclei of some cells while others were negative (not shown). Moreover, double-label immunofluorescence microscopy using antibodies against nucleolar and lamin proteins further showed that the antigen recognized by mAb 203-37 is accessible only in a specific proportion of nuclei in which intranuclear structures such as the lamina and the nucleolus were also labeled. This indicated that the mAb 203-37 antigen is also intranuclear.
Immunofluorescence microscopy performed on cryostat sections of human and bovine tissues confirmed immunolabeling in the nuclear periphery with mAb 203-37 in all tissues examined, including human esophagus (Fig. d), testis (Fig. d, inset), ovary, liver, skin, smooth muscles, cerebrum, fetal cerebellum, erythroblastoma, and colon carcinoma (not shown), as well as bovine liver (Fig. e), testis, and thymus (not shown). In most tissues, we observed occasional intranuclear dot reactions in addition to the nuclear rim staining (an example is shown in Fig. d, inset).
To identify the antigen recognized by mAb 203-37, we performed immunoblotting on total cell proteins of HeLa cells and identified a protein of Mr
~270 kD (Fig. , a
), henceforth called p270. Cell fractionation revealed that p270 remained structure associated after cell extractions with detergent and intermediate salt concentrations (Fig. , b
). Immunoreactive polypeptides of lower molecular weight, detected in fractions representing saltextracted soluble proteins (Fig. b′
, lane 3
), were considered to be degradation products of p270. Indeed, fractionation of cells induced proteolytic degradation of p270 and omission of protease inhibitors, and prolonged fractionation procedures resulted in an increase of lower molecular weight polypeptides reactive with mAb 203-37. Protein p270 was also identified by immunoblottings with mAb 203-37 on total cell proteins of porcine PK(15) and bovine BMGE cells (Fig. , c
), as well as canine MDCK cells (not shown), whereas rodent and marsupial p270 did not cross-react with this antibody. Immunoblottings on African green monkey proteins yielded a weak reaction signal after prolonged exposure (not shown). In essence, our immunoblotting results confirm and extend the study of Bangs et al. (1996)
performed largely in parallel.
Figure 2 Immunoblot detection of a ~270-kD mammalian protein reactive with mAb 203-37. Proteins were separated by SDS-PAGE and then, in parallel, either stained by Coomassie blue (a, b, and c) or transferred to nitrocellulose filters for immunodetection (more ...)
To localize p270 at the EM level, we examined its distribution by preembedding immunogold localization using mAb 203-37 and 5-nm gold–coupled secondary antibodies on cryostat sections of human and bovine tissues and on monolayers of detergent-permeabilized human PLC cells (Fig. , a–f). Specific labeling was found on intranuclear filamentous structures that appeared to be attached to NPCs. In some sections, such gold particle–decorated filament bundles projected into the nuclear interior for up to 200 nm (see, e.g., Fig. , b and c). Double immunogold labeling with antibodies against protein RanBP2/Nup358 (10-nm gold–coupled secondary antibodies) and mAb 20337 (5-nm gold) showed that both margins of the NPC were accessible, and thus confirmed the specificity of the intranuclear localization of p270 (Fig. , g–j). Besides this labeling of NPC-attached intranuclear filaments, we occasionally also noticed specific mAb 203-37 labeling of unknown intranuclear spheroidal structures ~40–200 nm in diam that were located at greater distances from the nuclear envelope (see below).
Figure 3 Immunoelectron microscopy of mammalian cells and tissues using mAb 203-37 in a preembedding technique, showing intense decoration of intranuclear sites near NPCs, often directly seen on the NPC-associated intranuclear filaments. Immunogold localization (more ...)
cDNA Clones Encoding p270 Reveal Sequence Identity to Protein Tpr
Using mAb 203-37, we screened a human fetal brain expression library and isolated a total of six immunoreactive clones (T1–T6) that, on DNA sequencing, were recognized to encode protein Tpr (Fig. a
) recently localized exclusively to the cytoplasmic margin of NPCs (Byrd et al., 1994
; Bangs et al., 1996
). One cDNA clone, T1, contained the entire open reading frame, encoding a 2,363-aa polypeptide with a predicted molecular mass of 267,333 daltons. Sequence comparison between human fetal brain Tpr and the previously published, slightly shorter aa sequence (265,559 daltons) of human Tpr that had been deduced from various cDNAs isolated from fibroblast and fibrosarcoma cDNA libraries (Mitchell, 1992a
; Byrd et al., 1994
) showed three aa exchanges in the amino-terminal domain (positions 779, 906, and 1239) and an additional insertion of 14 mainly acidic aa in the carboxy-terminal domain of fetal brain Tpr (at position 1951) as the only differences between the two sequences (Fig. b
Figure 4 Characterization of p270 cDNA clones isolated from a human expression library using mAb 203-37. (a) Schematic presentation of human p270 cDNA clones isolated from a fetal brain Uni-ZapTMXR cDNA library. Screening of this library with mAb 203-37 yielded (more ...)
In vitro transcription–translation of cDNA clone T1 using the reticulocyte lysate system yielded a product of ~270 kD (Fig. c) that was indistinguishable in mobility from p270 identified by immunoblotting of HeLa cell proteins and was specifically immunoprecipitated by mAb 203-37 (Fig. d), indicating that p270 and at least the human fetal brain variant of Tpr represent the same antigen.
Expression of the human fetal brain tpr gene under control of a CMV promoter in African green monkey cells (Fig. , e–g′) and in rodent cells of lines Faza-967-D2 and L-M(TK−) (not shown) confirmed that this variant of the human Tpr protein is targeted to, and stably associates with, nuclear structures. Immunofluorescence microscopy on transfected cells using mAb 203-37 revealed both the finely punctate staining in the nuclear periphery reminiscent of NPC labeling and the occurrence of intranuclear speckles, which occasionally appeared close to the nucleoli (Fig. , f–g′). A similar nuclear localization of human fetal brain Tpr was also observed when an expression vector containing a major histocompatibility complex class I H-2 promoter was used (not shown). In some transfected cells, with a seemingly higher level of human Tpr protein synthesis, some punctate immunoreaction sites that remain to be characterized were also noticeable in the cytoplasm (not shown).
Antibodies against Different Epitopes of Protein Tpr React with p270
To exclude that mAb 203-37 immunoreacts only with a distinct Tpr variant located at the nucleoplasmic side of the NPC but not with the Tpr protein that had been localized to the cytoplasmic side of the NPC by Byrd et al. (1994)
, we raised antibodies against two peptides representing different regions of human Tpr that were identical to both brain and fibroblast Tpr (Fig. a
). Immunoblotting using the affinity-purified antibodies revealed specific immunoreaction with p270 (Fig. , b–b
). Moreover, both mAb 203-37 and the antibodies against human Tpr peptide no. 1 (Fig. a
) similarly reacted also with the same pattern of lower molecular weight polypeptides considered to represent p270 degradation products (Fig. , b′
). Two-dimensional gel electrophoresis followed by immunoblotting definitively proved that the same mAb 203-37 reactive protein p270 was indeed also detected with the Tpr peptide antibodies (Fig. , c–c″
Figure 5 Immunoblot detection of p270 with affinity-purified guinea pig and rabbit antibodies raised against human Tpr protein. (a) Schematic presentation of human protein Tpr and the relative positions (arrows) of the two Tpr peptides, No. 1 (hTpr-Pep1, 1.) (more ...)
Antibodies against mammalian p270/Tpr either did not react with the Xenopus laevis Tpr orthologue or exhibited additional unspecific cross-reactions with other Xenopus proteins (not shown). We therefore also raised rabbit antibodies against the Xenopus Tpr protein. To this end, we first isolated a series of overlapping PCR products using a Xenopus kidney cDNA library as template and from which assembled a partial DNA sequence of 1,811 bp that encoded the 558 carboxy-terminal aa of the Xenopus Tpr protein (Fig. a). This sequence exhibited a high degree of homology (70% identity) to the human sequence, including uninterrupted stretches of 27–45 identical aa. For immunization, a peptide located near the carboxy terminus (Fig. b) was chosen, yielding antibodies that specifically reacted with the Xenopus p270/Tpr protein on total proteins of Xenopus kidney epithelial cells of line A6 and of manually isolated oocyte nuclei (Fig. , c and c′).
Figure 6 Generation of antibodies immunoreactive with amphibian Tpr. (a) Isolation of PCR products encoding the carboxy-terminal domain of the Xenopus laevis Tpr protein and alignment of human and Xenopus Tpr aa sequences. A Xenopus kidney cDNA library was (more ...)
Antibodies against Human and Xenopus p270/Tpr Label NPC-attached Intranuclear Filaments
Immunofluorescence microscopy using the human p270/ Tpr peptide antibodies on cultured mammalian cells and on cryostat sections of mammalian tissues (liver, esophagus, epidermis, and colon) yielded results (some are shown in Fig. , a–d) that were essentially indistinguishable from those obtained with mAb 203-37 (compare with Fig. ) but revealed broader cross-reactivity between the various mammalian species, including rodents and African green monkey cells (not shown). The antibodies against the Xenopus p270/Tpr sequence equally resulted in punctate staining of the nuclear periphery of cultured Xenopus cells with some occasional intranuclear speckles (Fig. , e–g′). Punctate nuclear periphery staining was further noticed in Xenopus erythrocytes (not shown). On cryostat sections of Xenopus skin, liver, and oocytes (Fig. , h and h′), immunolabeling was seen at the nuclear envelope and occasionally, in somatic cells, also in additional intranuclear dots (not shown).
Figure 7 Immunofluorescence microscopy of cultured cells and tissue cryostat sections after reaction with affinity-purified guinea pig and rabbit antibodies raised against different epitopes of human and Xenopus Tpr. (a) HeLa cells stained with guinea pig antibodies (more ...)
In contrast with several other NPC proteins, p270/Tpr was not detected in the pore complex–containing cytoplasmic annulate lamellae as revealed by double immunofluorescence microscopy on different mammalian and amphibian cells (Fig. ), using the above described antibodies against p270/Tpr and such against NPC proteins that have been demonstrated to represent marker molecules for annulate lamellae (Cordes et al., 1995
Figure 8 Double immunofluorescence microscopy of mammalian and amphibian cells using antibodies against p270/Tpr (a–c), and against other NPC proteins (a′–c′) that represent marker proteins for pore complexes both of the nuclear (more ...)
Immunoelectron microscopy using preembedding immunogold labeling (secondary antibodies coupled to 5-nm colloidal gold) revealed identical results on human liver sections with antibodies against human Tpr peptides Nos. 1 and 2. Both antibodies specifically labeled the NPC- attached intranuclear filaments, whereas the cytoplasmic side of the NPC was devoid of gold particles (Fig. , a–d
). Essentially identical results were obtained on cryostat sections of human epidermis (not shown). As already noticed by immunoelectron microscopy using mAb 203-37 (see above), we occasionally also noticed specific immunolabeling of yet unidentified small intranuclear spheroidal structures (Fig. e
). To control for the accessibility of both sides of the NPC, double immunogold label localizations with antibodies against RanBP2/Nup358 (10-nm gold– coupled secondary antibodies) and p270/Tpr (5-nm gold) were performed. The larger RanBP2 immunogold particles were seen almost exclusively at the outer NPC annulus (Fig. , f–h
), thus confirming the localizations of others (Wu et al., 1995
; Yokoyama et al., 1995
; Wilken et al., 1995
). This differential double label therefore underscores the significance of the intranuclear location of protein p270/Tpr.
Figure 9 Immunoelectron microscopy of human tissues after reaction with affinity-purified antibodies raised against different epitopes of human Tpr revealing exclusive intranuclear localization of protein p270/Tpr. (a–e) Immunogold localization (5-nm-gold) (more ...)
Immunogold labeling on manually isolated stage VI oocytes of Xenopus laevis
, using rabbit antibodies against peptide No. 3 of the Xenopus
p270/Tpr protein and secondary antibodies coupled to 5-nm gold particles (Fig. , a–d
), resulted in a very dense and specific decoration of the NPC-attached intranuclear filament bundles, whereas both the outer aspect of the NPC and the pore complexes of annulate lamellae were free of immunogold label. In some micrographs with structurally well-preserved filament bundles, the gold-decorated filaments projected into the nuclear interior for up to 350 nm. Double label immunogold localization performed as control on manually isolated oocytes (Fig. , e–j
) and on cryostat sections through Xenopus
ovaries (Fig. k
), using antibodies against RanBP2/Nup358 (10-nm gold) and p270/Tpr (5-nm gold), showed the localization of RanBP2 at both the cytoplasmic margin of the NPCs and the cytoplasmic pore complexes of annulate lamellae (Fig. j
; see also Cordes et al., 1996
), whereas the p270/Tpr protein again was localized to the intranuclear filaments.
Figure 10 Immunoelectron microscopy of Xenopus oocytes after reaction with affinity-purified antibodies raised against Xenopus p270/Tpr showing dense labeling of NPC-attached intranuclear filament bundles. (a–d) Immunogold localization (5-nm-gold) of (more ...)