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Vol. 15, Issue 9, 4261-4277, September 2004
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* Department of Cell and Molecular Biology, Karolinska Institutet, S-17177 Stockholm, Sweden;
Peptide Specialty Laboratories GmbH, D-69120 Heidelberg, Germany
Submitted March 1, 2004;
Accepted June 14, 2004
Monitoring Editor: Pamela Silver
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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30 proteins (Cronshaw et al., 2002
), serves as a gateway for the exchange of material between cytoplasm and nucleus in eukaryotes. Its core structure of eightfold-radial symmetry consists of globular subunits that form a central spoke-ring complex, including a cylindrical structure surrounding the actual translocation channel (Allen et al., 2000; Vasu and Forbes, 2001; Fahrenkrog and Aebi, 2002; Suntharalingam and Wente, 2003). Coaxial ring-like structures flank the spoke-ring complex on both its cytoplasmic and nuclear side.
In addition to this NPC proper, eight fibrils of distinctive shape are attached to each of these rings. Fibers of the cytoplasmic ring look like short tufts 30–35 nm in length with free distal ends. In contrast, the thin fibrils emanating from the nuclear ring are 40–60 nm in length and laterally interconnected at their distal ends, forming another ring-like structure referred to as the terminal ring. Nuclear fibrils and terminal ring are believed to represent a structural and functional entity, called the nuclear basket (Jarnik and Aebi, 1991; Ris, 1991; Goldberg and Allen, 1992).
Several NPC proteins (nucleoporins, Nups), including Nup153 (Sukegawa and Blobel, 1993), Nup98 (Powers et al., 1995; Radu et al., 1995), Nup 50 (Guan et al., 2000), some components of the Nup160 subcomplex (Radu et al., 1994; Fontoura et al., 1999; Belgareh et al., 2001; Vasu et al., 2001), and members of the Nup93 subcomplex (Grandi et al., 1997) have been assigned to the nuclear side of the NPC in higher eukaryotes. The 267-kDa protein Tpr has been located at this side as well (Cordes et al., 1997; Zimowska et al., 1997). However, it has remained uncertain which of these proteins are architectural components of the NPC core, the associated basket fibrils, or the terminal ring.
Nup96, a stable component of the Nup160 subcomplex (Belgareh et al., 2001; Vasu et al., 2001) was reported to occur exclusively at the nuclear side of the NPC in HeLa cells, at an average distance of 100–200 nm from the nuclear envelope (NE) midplane (Fontoura et al., 1999). There it has been considered a distal part of the nuclear basket together with other members of the Nup160 subcomplex (Allen et al., 2000; Vasu and Forbes 2001). Most recently, however, Nup96 was found on both sides of the NPC periphery when using isolated rat liver NEs for immuno-electron microscopy (EM) (Enninga et al., 2003). Two other components of the Nup160 subcomplex, Nup107 and Nup133, were localized on both the cytoplasmic and nuclear side as well, but closer to the horizontal midline passing through the NPC (Radu et al., 1994; Belgareh et al., 2001).
Another Nup96 binding partner, Nup98, also was recently found on both sides of the NPC (Griffis et al., 2003), but other studies have located this Nup exclusively at the nuclear side, close to (Radu et al., 1995) or further away from the NE (Frosst et al., 2002). Nup98 has therefore been regarded as an Nup forming the basket fibers or the terminal ring (Allen et al., 2000; Vasu and Forbes, 2001; Fahrenkrog and Aebi, 2002; Harel et al., 2003). Vertebrate Nup93 again was reported to occur at two distinct sites at the nuclear NPC side, with predominant localization in the terminal ring area (Grandi et al., 1997; Fahrenkrog and Aebi, 2002). In contrast, the yeast homolog Nic96 is symmetrically arranged on both sides of the NPC close to its midplane (Rout et al., 2000).
Nup153 was located in proximity of the nuclear NPC side in somatic mammalian cells (Sukegawa and Blobel, 1993; Cordes et al., 1993; Frosst et al., 2002), but in amphibian oocytes it also has been found associated with NPC-attached filament bundles several hundred nanometers in length that go beyond the dimensions of the nuclear basket (Cordes et al., 1993; Ris, 1997). However, when isolated oocyte NEs stripped of these additional fibers were immunolabeled with antibodies against the N terminus of Nup153, it was located either at the nuclear coaxial ring (Walther et al., 2001, Fahrenkrog et al., 2002) or at the terminal basket ring 50–100 nm away from the NE midplane (Pante et al., 2000). Antibodies against central and C-terminal parts of Nup153 were reported to label both the terminal ring and cytoplasmic side of the NPC in amphibian oocytes (Pante et al., 1994; Fahrenkrog et al., 2002) but not in somatic cells (Sukegawa and Blobel, 1993).
Immuno-EM localizations of Tpr have been similarly diverse. First described as a protein located at the cytoplasmic NPC side (Byrd et al., 1994), it was later found attached to the nuclear NPC side in mammalian cells (Cordes et al., 1997; Frosst et al., 2002). In Xenopus oocytes, Tpr labeling occurred not only within the territory occupied by the nuclear basket but also at the basket-attached fibers that project further into the nucleus (Cordes et al., 1997). In insect cells, Tpr was reported to occur throughout the extrachromosomal space of the nuclear interior (Zimowska et al., 1997). An extensive Tpr filament network crossing the nuclear interior of HeLa cells has been reported as well (Fontoura et al., 2001). However, the specificity of some Tpr antibodies causing such staining patterns in mammals has been disputed (Kuznetsov et al., 2002).
Differences in localization of individual Nups might be due to cell type- or species-specific features. For example, the conspicuous masses of fibrous material attached to the nuclear basket in mature amphibian oocytes (Scheer et al., 1988; Arlucea et al., 1998) have not been identified in other cell types and may be specific for oocytes, perhaps representing a stockpile of Tpr and other proteins required during embryogenesis (Hase and Cordes, 2003). Different types of antibodies, ranging from single epitope monoclonals to polyclonals against large protein segments, and differences in specimen preparation and labeling procedures may have contributed to the diversity of results as well.
In an attempt to minimize variations due to differences in experimental procedures and to allow direct comparisons, we have reinvestigated the localization of several Nups and Tpr in parallel, performing immunogold-EM with peptide-specific antibodies by using a postembedding labeling approach. To gain additional insight into the contribution of certain Nups to NPC architecture, cells were depleted of these proteins by RNA interference (RNAi) and then studied with respect to NPC integrity and composition. The results tempt us to propose a model in which Tpr constitutes the central architectural element of the nuclear basket and other Nups are components of the NPC proper.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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); rabbit (rb) and gp antibodies against hNup358 aa 2285–2314 (Cordes et al., 1996) and hTpr aa 2063–2084, gp 
-hTpr aa 1622–1640, gp and rb -hTpr aa 1–14, 8–21, 604–615, and 2338–2363; monoclonal antibody (mAb) 203–37 against hTpr (Cordes et al., 1997); mAb PF190 x 7A8 against rNup153 (Cordes et al., 1993); gp antibodies for hNup153 aa 22–36 and 391–404 (Ferrando-May et al., 2001), hNup107 aa 33–51, Nup205 aa 1784–1803 (accession no. D86978
[GenBank]
), hNup98 aa 206–220, and 596–618, hNup93 aa 2–203, 350–369, and 371–389, rb -hNup50 aa 126–147, and gp and rb -hNup96 aa 880–900 have been described previously (Hase and Cordes, 2003). The aa numbering for hNup96 relates to the full-length protein of 106 kDa. The corresponding gene encodes the Nup98-Nup96 precursor of Mr 195846 (accession no. AAL56659
[GenBank]
that is autoproteolytically processed to give rise to Nup98, and Nup96 of Mr 105941. A formerly described hNup96 sequence of Mr 96196 (AF071076
[GenBank]
) represents a truncated version resulting from a sequence inversion neither present in the genomic sequence nor in other Nup98-Nup96 mRNAs. Novel gp antibodies were raised against synthetic peptides corresponding to 1) hTpr aa 636–655 and 2) aa 914–932. Peptides were coupled to keyhole limpet hemocyanin via a C-terminal 1) or N-terminal 2) cysteine residue. Affinity-purification of Igs followed standard procedures (Kuznetsov et al., 2002). Rb antibodies against hNup93 aa 2–218 (Grandi et al., 1997) had been kindly provided by Dr. Ed Hurt (University of Heidelberg, Heidelberg, Germany). mAb 414 was from Babco (Richmond, CA). Secondary antibodies coupled to horseradish peroxidase, fluorochromes, and colloidal gold were from Jackson ImmunoResearch Laboratories (West Grove, PA), DakoCytomation Denmark A/S (Glostrup, Denmark), and Sigma-Aldrich (Stockholm, Sweden).
Cell Fractionation and Immunoblotting
Preparation of total protein samples from HeLa cells grown to near confluence (80%), and cell fractionation was performed as described previously (Kuznetsov et al., 2002) with some modifications. Cells were first incubated with phosphate-buffered saline (PBS) plus 0.005% digitonin for 3 min, briefly washed with PBS, and then incubated with PBS plus 0.4% Triton X-100 for 3 min, and washed again. All solutions were recollected and centrifuged at 13,000 x g for 5 min followed by methanol precipitation of proteins. Residual cyto- and karyoskeletons attached to the dishes were scraped off in 95°C SDS protein sample buffer and boiled. SDS-PAGE and immunoblotting were as described previously (Hase et al., 2001).
Posttranscriptional Gene Silencing and Immunofluorescence Microscopy
Transfection of HeLa cells with small interfering RNAs (siRNAs) was as described previously (Elbashir et al., 2001; Kuznetsov et al., 2002; Hase and Cordes, 2003). Synthetic RNA oligonucleotides were from Dharmacon Research (Lafayette, CO) and DNA Technology A/S (Aarhus, Denmark). Antisense strands were complementary to nucleotide (nt) 3732–3712 of the hNup196 cDNA (AF231130
[GenBank]
), nt 530–510 of hNup107 (AJ295745
[GenBank]
), nt 978–958 of hNup93 (NM_014669
[GenBank]
) and nt 440–420 of Nup205 (D86978
[GenBank]
). Cells were analyzed 2, 3, 3.5, 4, and 5 d posttransfection. Immunofluorescence microscopy (IFM) of formaldehyde (FA)-fixed HeLa cells was as described previously (Hase and Cordes, 2003). For digitonin permeabilization of cells before or after fixation, cells were treated with 0.005 or 0.05% digitonin in ice-cold PBS for 10 or 20 min. Confocal microscopy and image analysis was performed with an LSM 510 (Carl Zeiss, Oberkochen, Germany).
Immunoelectron Microscopy and Specimen Evaluation
HeLa cells grown to near confluence in 8-cm dishes were washed with PBS and fixed in 2% FA plus 0.1% glutaraldehyde (GA) in PBS for 1 h. This low GA concentration was found to represent an acceptable compromise between a reasonable degree of NE and NPC structure preservation and maintaining intact target epitopes for most antibodies in this study. For labeling of GA-sensitive epitopes, cells were fixed with 3.7% FA in PBS for 2 h. After washes in PBS, cells were scraped off the dishes and sedimented at 9,000 x g for 20 min. The resulting pellet was cut into small pieces and dehydrated in ethanol (70, 95, 100%). Pellet pieces were then incubated in a mixture of equal parts (vol/vol) of ethanol and LR White (London Resin, Reading, United Kingdom) at 20°C for 30 min, followed by 12 h in pure resin at 4°C. After two further 30-min incubations in fresh resin, the samples were transferred into resin-filled gelatin capsules and allowed to harden in a UV polymerization unit (Agar Scientific, Stansted, United Kingdom) for 12 h. Sections of 60 to 80 nm in thickness were transferred onto nickel grids coated with Formvar film. For immunogold staining, the grids were first placed onto droplets of PBS with 5% bovine serum albumin (BSA) (PBS-BSA) for 30 min, transferred to droplets containing primary antibodies diluted in PBS-BSA, and incubated in a humid box for 2 h. After washes with PBS-BSA, the grids were placed on droplets of gold-labeled secondary antibodies diluted in PBS/0.5% BSA for 1 h, washed in PBS/0.5% BSA, and then PBS alone. Specimen were postfixed with 2% GA in PBS for 5 min, rinsed with PBS and H2O, and air-dried. Staining for contrast was in a saturated aqueous solution of uranyl acetate for 5 min, followed by lead citrate for 5 s. Immunolabelings were performed in duplicate, with four to six primary antibodies against different target proteins in parallel. Before EM analysis, all grids were encoded and only decoded again after data collection and completion of the evaluation process. Some immunolabelings were paralleled by controls without primary antibodies, and with unrelated gp and rb IgGs. Specimens were examined in a Philips CM120 EM at 80 kV. For quantitative evaluation of gold particle distribution, images were collected at a primary magnification of 53,000x with a Kodak MegaPlus charge-coupled device camera. Borders on both sides of the NPC between which gold particles were considered were set at 200-nm distance from the NE midplane. The analySIS system (Soft Imaging Software, Münster, Germany) was used for distance measurements. Mean grain diameters in different batches of commercial 10-nm gold-coupled antibodies were found to be close to 9 nm; individual grain sizes deviated from the mean by up to 25%. Magnification of the EM was calibrated using a 2160 line/mm crossed ruled grating (Agar Scientific).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Whereas preembedding labeling resulted in a peak distribution of gold grains 35 nm away from the NPC midplane, postembedding immuno-EM gave labeling of the NPC center. These differences were explained by limited antibody access to certain NPC components in preembedding approaches, and better accessibility on ultrathin sections through NPCs used for postembedding labeling.
In preembedding studies on isolated NEs, detergent-permeabilized cells or thick cryostat sections, the antibodies encounter NPCs that still represent three-dimensional entities. Steric constraints are then likely to dictate the angle and preferential orientation by which some antibodies bind to certain NPC components. In fact, when using IgGs as primary antibodies, together with gold-coupled secondary antibodies for indirect immunolabelings, the mean distance between target and peak distribution of gold particles can come close to 30 nm (Kellenberger and Hayat, 1991; Mayer and Bendayan, 2001). In contrast, antibodies binding to epitopes only exposed on the surface of ultrathin sections through resin-embedded NPCs can enjoy more rotational freedom. Consequently, the mean distribution of gold grains in postembedding immuno-EM of NPCs may define the actual location of certain target proteins more accurately. This in mind, we set out to reinvestigate the localization of several Nups and Tpr at the NPC.
Postembedding Immuno-EM Locates Nup153, Components of the Nup93 and Nup160 Subcomplexes, and Nup98 at NPC Core Structures
For postembedding immuno-EM, HeLa cells were embedded in the acrylic resin LR White (Newman et al., 1983). On sections through such resin-embedded cells, only antigens exposed at the surface are labeled by gold-coupled antibodies. Penetration into the depth of the section is limited to a few nanometers at most (Bendayan et al., 1987; Stierhof and Schwarz, 1991).
Recently, we have described peptide-antibodies specific for Nup93, 96, 98, 107, 153, and 205 (Ferrando-May et al., 2001; Hase and Cordes, 2003). Relative positions of the antibody target sites on these proteins are shown in Figure 1. Now used for postembedding immuno-EM, several of these antibodies specifically labeled central or peripheral parts of the NPC proper. Even Nup93, which in IFM can only be labeled after preextraction of nonfixed cells with strong detergents (Grandi et al., 1997; Hase and Cordes, 2003), became accessible for antibodies in sectioned NPCs.
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The antibodies against Nup93 and 205 decorated regions near the NPC center, with gold grains found on both sides of the midplane (x-axis). Grains that decorated Nup96 and 107 were located on both sides of the NPC as well. When looking at each of these gold grain pools as a whole, their patterns only slightly diverged from a mirror-symmetrical distribution (see below). In general, gold grains for Nup93 and 205 were located closer to the x-axis than those for Nup96 and 107.
In contrast, antibodies targeting the N-terminal domain of Nup153 predominantly labeled the nuclear margin of the NPC, with far less gold grains on the cytoplasmic side. The majority of gold particles that labeled Nup98 were in turn located much closer to the x-axis and decorated both sides of the midplane. However, the calculated mean distance from the NE midplane pointed at a slight preference for Nup98 labeling at the nuclear NPC side as well (see below). Labelings with other Nup98 peptide antibodies resulted in similar gold grain distribution patterns (Supplemental Figure S1). A selection of representative micrographs is shown in Figure 2A. The histograms in Figure 2B represent gold particle distributions relative to the midplane of the NE, after normalization of values for NE width variations (see below). Calculated mean distances of gold grains from the NE midplane are listed in Table 1.
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The width of the perinuclear lumen between inner and outer NE membrane was often found to vary by several nanometers between neighboring NE segments. Because this affected the position of the NE midplane, the relative position of each gold grain was normalized for such NE width variations as described in Figure 3A. On the whole, resulting histograms (Figure 2B) did not differ much from the nonnormalized versions (Supplemental Figure S2A) but emphasized the existence of a bilateral symmetrical arrangement of some of the Nups. Gold grain positions relative to the NPC were then plotted according to their x/y coordinates onto a scheme of an idealistic NPC in cross section (Figure 4). In addition to y value normalization for NE width variations, x values were normalized for nondiametric perpendicular section planes as described in Figure 3B. Calculated mean distances of gold particles from the central symmetry-axis of the NPC are listed in Table 1.
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In summary, these immuno-EM data were indicative of a bilateral symmetrical arrangement of Nup96 and Nup107 on both NPC sides; a preferential location of the targeted Nup98, Nup93, and Nup205 domains within the NPC core region as well; and a peripheral position for the Nup153 target domain at the nuclear coaxial ring. These conclusions find further support by confocal IFM studies of semipermeabilized HeLa cells outlined in Supplemental Figure 3.
Congruent Locations of Tpr Protein Domains and Different Parts of the Nuclear Basket
The Tpr protein is located near the nuclear side of the NPC in mammalian cells (Cordes et al., 1997; Frosst et al., 2002), forms coiled-coil homodimers of extended rod-like shape (Hase et al., 2001), and is linked to the NPC via direct binding to Nup153 (Hase and Cordes, 2003). These findings made Tpr a likely candidate for an architectural element of the nuclear basket (Hase and Cordes, 2003). To follow up this idea, we determined where different Tpr domains are located relative to the NPC and basket, by using a panel of peptide-specific antibodies raised against various parts of Tpr. These antibodies comprise such that have been described previously (Cordes et al., 1997; Kuznetsov et al., 2002; Hase and Cordes, 2003) and novel ones raised for this study (see below).
Immunolocalization of Tpr's C-terminal domain on LR White sections of GA-fixed HeLa cells was performed with antibodies against aa 2063–2084 and the actual C-terminus (aa 2338–2363). Both specifically decorated a region in proximity to the nuclear NPC side, at normalized mean distances of 74 nm (-Tpr 2063–2084) and 76 nm (-Tpr 2338–2363) from the NE midplane (Figures 5 and 6 and Table 2). This region coincides with the approximate position of the terminal ring (Goldberg and Allen, 1992).
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Previously, we had raised several monoclonal and peptide-specific polyclonal antibodies against epitopes that flank Tpr's NPC binding domain (NBD) between aa 400 and 600 (Hase et al., 2001; Kuznetsov et al., 2002; unpublished data). However, these antibodies were found to be of no use for postembedding immuno-EM, either because of pronounced in situ cross-reactions with Tpr-unrelated proteins (Kuznetsov et al., 2002) or because some epitopes apparently had become blocked during the fixation and embedding procedure. Therefore, we screened the Tpr segments that flank the NBD for other antigenic sites that would remain intact upon specimen preparation. This resulted in the identification of two Tpr segments against which we raised peptide-specific antibodies (Figure 7). The one site was located close to the NBD (aa 636–655) and the other more distal (aa 914–932). Tpr specificity was controlled by immunoblotting (Figure 7A). In postembedding immuno-EM, both -Tpr 636–655 and -Tpr 914–932 decorated regions in the proximity of the nuclear NPC side (Figure 5A). However, whereas the normalized mean distance of immunogold label with -Tpr 914–932 was found to be 62 nm from the NE midplane, labeling with -Tpr 636–655 was closer to the NPC core, at an approximate mean distance of 27–35 nm (Figures 5B and 6 and Table 2).
To also determine the relative positions of the N terminus and the distal end of the rod domain, we tested a collection of antibodies against sites located between Tpr aa 1–135 and between aa 1370–1640 (Cordes et al., 1997; Kuznetsov et al., 2002; our unpublished data) but had to realize that none of the corresponding epitopes tolerated fixation with GA. However, when GA was omitted and cells were fixed with FA only, several of these antibodies were found to be useful for immuno-EM. Although the NEs in such FA-fixed cells sometimes seemed bloated, with NE membranes less well structurally preserved than in GA-fixed specimens, the direct comparison of both fixation methods, by using several GA-insensitive Tpr and Nup antibodies in parallel, revealed rather similar gold grain distributions relative to the NE midplane. Only slight differences in the calculated means were observed (Figure 8 and Table 3; our unpublished data).
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Under these conditions, the peak of gold grains after labeling with -Tpr 2338–2363 was 70 nm from the NE midplane (Table 3, nonnormalized data). Using a peptide antibody against Tpr aa 1622–1640, the distal end of the rod domain at aa 1630 was found closer to the midplane (61 nm). However, with antibodies against aa 8–21, the N terminus was localized similarly far from the midplane (73 nm) as the C terminus. Nonnormalized positions of these gold particles from FA-fixed specimen were then summarized by superimposing them onto schemes of nuclear pores in cross section (Figure 8B).
Recently, we have shown that Nup153 segment aa 228–439 includes the binding site for the NBD of Tpr (Hase and Cordes, 2003). To complement our immunolocalization of Tpr's NBD relative to the NPC, we labeled the corresponding Nup153 binding site for Tpr as well, by using FA-fixed cells and a peptide-specific antibody raised against Nup153 aa 391–404. This antibody specifically decorated a region corresponding to the position of the nuclear coaxial ring, at a mean distance of 19–23 nm from the NE midplane (Figure 8 and Table 3).
Distinct Effects on NPC Integrity and Docking of Tpr after In Vivo Depletion of Individual Nup160 and Nup93 Subcomplex Components
Recently, we have used siRNAs to posttranscriptionally silence the genes for Tpr and Nup153 in HeLa cells, revealing that Tpr is dispensable for NPC assembly and does not act as a scaffold for presumptive nucleoporins of the nuclear basket. Similarly, in vivo depletion of Nup153 had no immediate effect on NPC assembly and incorporation of various other Nups, but it caused mislocalization of both Tpr and the mobile nucleoporin Nup50, another binding partner of Nup153, to the nuclear interior (Hase and Cordes, 2003). Targeted disruption of the gene encoding Nup98 again did not prevent NPC-binding of either Nup153 or Tpr (Wu et al., 2001).
To also assess the roles of the Nup93 and 96 subcomplex components for NPC architecture and docking of Tpr and Nups to the NPC, our immuno-EM study was paralleled by RNAi experiments in which we targeted the transcripts for Nup93, 107, 205, and 96, the latter encoding the Nup98-Nup96 precursor protein of 196 kDa (Fontoura et al., 1999). The effects after in vivo depletion of Nup96 or 107 (Figure 9, A and B; Supplemental Figures S4 and S5) clearly differed from those in Tpr- or Nup153-depleted cells and pointed at essential roles in NPC assembly or maintenance, and in anchorage of Nup153 and Tpr to the NPC. Steady loss of Nup96 or 107 over a period of several days went along with seemingly simultaneous decrease in NE labeling for other Nups and Tpr. At 3–4 d after treatment with siRNAs, when transfected cells were largely depleted of the target protein, NE labeling for Tpr and Nup153 was largely diminished as well. Instead, both were occasionally found mislocalized to intranuclear and cytoplasmic aggregates.
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Cells transfected with Nup205 siRNAs were more rapidly depleted of Nup205 protein. Already 48 h after the initial transfection, many cells were essentially devoid of Nup205. At this point, NE labeling with mAb 414, an antibody against Nup62 and other FxFG-repeat Nups, was often strongly reduced as well (Figure 9C). In contrast, NE staining for Tpr and several other Nups, including Nup153 and 50, was generally much less affected (Figure 9C'). In fact, different from Nup96- and 107-depleted cells, reduction of NE staining for these Nups and Tpr trailed Nup205 reduction with obvious delay. Even at day 3.5 posttransfection, with no or only traces of Nup205 detectable, NE staining for these proteins was often still only moderately reduced (Supplemental Figure S6).
Furthermore, Tpr and Nup153 were found incorporated into the newly assembled NEs of postmitotic cells early in G1 despite these being largely devoid of Nup205. In contrast, Nup205 deficiency in such postmitotic NEs went along with a strong reduction of Nup93 staining (Figure 9C''). Although RNAi-mediated knockdown of the rather abundant Nup93 protein itself (Cronshaw et al., 2002) was less complete than for Nup205, we noted a similarly rapid reduction of mAb 414 signal from such Nup93-reduced NPCs, but again no immediate defection of Nup153 or Tpr (Supplemental Figure S6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The Nup160 and Nup93 Subcomplexes as Architectural Elements of NPC Core Structure
The Nup160 subcomplex consists of six proteins, Nup85, 96, 107, 133, 160, and Sec13 (Belgareh et al., 2001; Vasu et al., 2001; Harel et al., 2003), whereas the Nup93 subcomplex comprises Nup93, 188, and 205 (Miller et al., 2000). To map their positions relative to the NPC, we used peptide-specific antibodies against Nup93, 96, 107, and 205.
Our localizations of Nup96 and 107 are in line with other immuno-EM studies of cultured cells in which Nup107 and 133 were found on both the cytoplasmic and nuclear side of NPCs (Radu et al., 1994; Belgareh et al., 2001). IFM of semipermeabilized cells supports these EM data, showing Nup96 and 107 labeling at the cytoplasmic NPC side when the nuclear interior is inaccessible (Belgareh et al., 2001; this study). Further support for an NPC core position of the Nup160 subcomplex comes from our RNAi experiments, demonstrating that Nup96 and 107 are essential for NPC assembly and docking of Nup153 and Tpr. Recent studies on the roles of Nup85, 107, and 133 in NPC assembly have reported similar findings (Boehmer et al., 2003; Harel et al., 2003; Walther et al., 2003). In contrast, Nup153 and Tpr are dispensable for NPC assembly and neither acts as an NPC anchor for Nup96 or 107 (Hase and Cordes, 2003).
The distribution of the Nup96 and 107 immunogold label assigns at least parts of the Nup160 subcomplex to both of the coaxial ring structures. However, we do not exclude that the spoke complex sandwiched between these rings may harbor other domain segments of Nup96 and 107, or other members of this subcomplex.
The immuno-EM localization of Nup205 is the first reported and rates this Nup as another NPC core component. These data differ from those for Nup192p, the yeast homolog of Nup205, which has been located exclusively within the nuclear interior 60 nm from the NPC midplane (Kosova et al., 1999). They are, however, in line with another immuno-EM study in which Nup192p was shown symmetrically positioned on both sides of the yeast NPC (Rout et al., 2000). Moreover, the localization of Nup205 close to the NPC midplane is in harmony with the occurrence of its binding partner Nup93 at a similar position. Because Nup93 in intact NPCs is not accessible for any of the Nup93 antibodies used in this study, we believe it to be embedded deep within the NPC core, perhaps as part of the central spoke-ring complex sandwiched between the coaxial rings. This view finds support by our RNAi experiments as well as by NPC reconstitution studies (Grandi et al., 1997), demonstrating that the Nup93 subcomplex is essential for NPC stability and correct assembly of some of its parts. However, because siRNA-mediated loss of Nup93 or 205 did not prevent reincorporation of Nup153 and Tpr into NPCs in early G1, we conclude that the Nup93 subcomplex is not directly required for their mooring to the NPC. Nup153 and Tpr themselves are dispensable for NPC binding of Nup93 and 205 (Hase and Cordes, 2003).
We are aware of the variance between our Nup93 immuno-EM data and a study in which Nup93 was located exclusively at the nuclear NPC side, with peak distributions at 35–55 nm from the NE midplane (Grandi et al., 1997). However, using similar antibodies against a recombinant hNup93 polypeptide comprising aa 2–203, as well as another Nup93 peptide antibody against aa 371–389 for immuno-EM (unpublished data), we obtained staining patterns on both sides of the NPC close to the midplane that were very similar to the one obtained with the representative Nup93 peptide antibody shown in this study. None of our immunogold labelings allowed us to conclude that components of the Nup93 subcomplex may be structural components of the basket terminal ring. However, they are compatible with immuno-EM data for Nic96p, the yeast homolog of Nup93 symmetrically positioned on both sides of the NPC (Rout et al., 2000).
In future immuno-EM studies a higher resolution will be required to clarify whether the Nup93 subcomplex is located in a single plane at or near the NPC midplane or symmetrically on both sides of this axis. This might be achieved by using primary antibodies or Fab fragments coupled directly to colloidal gold, which will reduce signal spread and certain distortion effects that can arise as a consequence of secondary antibody rotation (Supplemental Figure S7).
Using an antibody against the central part of Nup98, immunogold label was mainly found within the NPC core region, on both sides of the NPC midplane. Similar gold grain distributions were found with other Nup98 peptide antibodies as well (Supplemental Figure S1), in keeping with the localization of Nup98 on both sides of the NPC by preembedding immuno-EM (Griffis et al., 2003). Future studies will need to seek an explanation that can harmonize these Nup98 immuno-EM results with reports showing that Nup98 is a protein that shuttles between nucleus and cytoplasm (Zolotukhin and Felber, 1999; Griffis et al., 2002, 2004). Immunogold label for such a mobile Nup may have been anticipated being more spread over NPC and basket, rather than mainly found in the NPC core region.
However, these and other observations unanimously speak against a direct role for Nup98 in basket formation. In HeLa cells depleted of Nup153 and Tpr, NPC binding of Nup98 is not impaired (Hase and Cordes, 2003), and in Nup98-deficient cells not only Nup93 and 96 but also Nup153 and Tpr remain bound to the NPC (Wu et al., 2001). In contrast, targeted disruption of the Nup98 gene leads to impaired NPC-binding of Nup88, 214, and 358 (Wu et al., 2001), Nups that are located only at the cytoplasmic NPC side. Although Nup98 was shown to be a binding partner of Nup88 (Griffis et al., 2003), it still remains to be understood why Nup98 deficiency selectively impairs binding of Nups to the cytoplasmic side. Clearly, however, none of these data support models in which Nup98 is located exclusively at the nuclear side of the NPC as an architectural element of the nuclear basket fibers.
Nup153 as the Proposed Link between NPC Core Structures and Nuclear Basket
By using two antibodies against the N-terminal domain (aa 22–36, 391–404), Nup153 was located at the nuclear NPC side, in an area occupied by the nuclear coaxial ring. Using the same LR White-embedded cells and mAb PF190 x 7A8 that targets an epitope between aa 439–611 of Nup153 (Hase and Cordes, 2003), we observed labeling of the same area (unpublished data).
These localizations are in harmony with a study in which Nup153 was located at the nuclear NPC side on ultrathin cryosections of BRL cells, by using antibodies against both N- and C-terminal Nup153 segments. Although the authors proposed Nup153 to be a component of basket fibers or terminal ring, the micrographs presented show gold grains mainly at the nuclear margin of the NPC proper (Sukegawa and Blobel, 1993). Furthermore, in a scanning EM study with antibodies against aa 1–149 of Xenopus Nup153 (Walther et al., 2001) and a transmission electron microscopy (TEM) study with antibodies against aa 436–655 of the same protein (Fahrenkrog et al., 2002), gold grains were directly attached to the nuclear coaxial ring on manually isolated Xenopus oocyte NEs. Interestingly, yeast Nup60p, which has been proposed to be the yeast version of Nup153 (Hase and Cordes, 2003), was located exclusively at the nuclear side of the NPC as well (Rout et al., 2000).
These localizations of Nup153 are compatible with RNAi experiments in which cellular depletion of Nup153 was shown to have no direct effect on the integration of Nup160 and Nup93 subcomplexes into the NPC but prevented NPC binding of Tpr (Hase and Cordes, 2003). Together, RNAi and immunolocalization data support a model in which Nup153 is proposed to act as the suspender of the nuclear basket at the NPC (Figure 10).
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This notion clearly differs from our former proposal of Nup153 being a fibrous basket protein in somatic cells. That conclusion was built on a preembedding immuno-EM study in which thick mouse liver cryostat sections were exposed to mAb PF190 x 7A8, resulting in Nup153 labeling exclusively at the nuclear NPC side at a mean distance of 49 nm from the NE midplane (Cordes et al., 1993). We now interpret that gold grain distribution as affected by steric constraints that have dictated the angle by which primary and gold-coupled secondary antibodies bound to their targets. However, we cannot explain the variance between our present Nup153 immuno-EM data and a recent study in which mammalian Nup153 was located far from the NPC, at a distance of 75–84 nm from the NE midplane (Frosst et al., 2002).
Our present data do not rule out the possibility that other parts of the Nup153 protein, not targeted by the antibodies used here, may be located in different areas of the NPC. Using antibodies against the C-terminal FxFG-repeat domain for preembedding immuno-EM on manually isolated Xenopus oocyte nuclei, this Nup153 segment was located both at the terminal basket ring and the NPC proper (Fahrenkrog et al., 2002). Such FxFG-repeat sequences have little regular secondary structure and are highly flexible (Bayliss et al., 2000; Denning et al., 2003). Therefore, they are unlikely candidates for stable architectural elements but might well be capable of transiently interacting with different parts of the NPC and basket (Fahrenkrog et al., 2002). Future postembedding immuno-EM studies by using peptide-antibodies against these and other parts of Nup153 will aim at immunolocalizing these domains in somatic cells as well.
Tpr Proposed as the Central Architectural Element of the Nuclear Basket
Using peptide-antibodies against different Tpr segments, we located this protein in an area at the nuclear side of the NPC occupied by the nuclear basket. This is in line with other studies in which Tpr was found at the nuclear NPC side in human cells (Cordes et al., 1997; Frosst et al., 2002). Added to our knowledge of Tpr's ultrastructural properties and dimensions (Hase et al., 2001), the finding that Tpr binding to the NPC is mediated by Nup153 and that Tpr does not act as a scaffold onto which other Nups need to assemble (Hase and Cordes, 2003), the present immuno-EM data tempt us to outline a model in which Tpr acts as the main architectural element of the nuclear basket.
The mean positions determined for different parts of Tpr's C-terminal tail domain correlate well with the position of the basket's distal end 60–80 nm from the NPC midplane (Jarnik and Aebi, 1991; Goldberg and Allen, 1992; Ris, 1997). In contrast, the Tpr-binding domain of Nup153, and an epitope flanking the NBD of Tpr itself, are both located in an area 23–35 nm from the midplane. The distance of 40–50 nm between this NPC anchor site of Tpr and its C terminus correlates well with a basket fiber length of 40–60 nm (Goldberg and Allen, 1992; Ris, 1997). This distance can easily be bridged by the rod-shaped Tpr coiled-coil that can reach >100 nm when fully extended in solution in the absence of Nup153 (Hase et al., 2001; our unpublished data).
Remarkably, the mean position of Tpr's N terminus was not found at the nuclear coaxial ring but close to the basket's distal end. This tempts us to suggest that the basket fibers are formed by Tpr coiled-coils in which one part of the dimer folds back onto another part of itself, forming a shorter but thicker fiber in which both N and C termini are oriented toward the nuclear interior. Folding of the initially soluble and rectilinear Tpr dimer, with a Stokes radius of 160 Å and a sedimentation coefficient of 7.5 S (Hase et al., 2001; our unpublished data), would occur at the NBD upon binding to Nup153, and intradimer interactions would take place between the first two thirds of Tpr's rod domain. The last one-third of the rod, which tends to bifurcate (see below), would instead branch off toward the distal rod ends of adjoining Tpr polypeptides that constitute the neighboring basket fibers. Together with the C-terminal tail domains, these distal rod segments would then constitute the terminal ring structure (see below).
Apart from the immunolocalization data, also some of Tpr's structural properties speak for this model. First, the coiled-coil segment of 51 nm formed by the Tpr sequences upstream (aa 1–398) of the NBD (aa 400–600) is strictly rectilinear as are the rod segments of 350–400 amino acids directly downstream. In contrast to these segments that form stable homodimers, rod segments closer to the tail domain engage only in weak homodimeric interactions and often seem kinked, bent, or bifurcated. Second, the first two thirds of the rod domain tend to interact with each other, but neither of them does so with the last third (Hase and Cordes, 2001; our unpublished data).
At present, we cannot exclude the possibility that the small-sized Nups recently identified (Cronshaw et al., 2002) might include a linker protein that helps connecting the neighboring basket fibers. Future postembedding immuno-EM of these Nups will show whether any of them are basket-associated proteins. In the meantime, we favor a model in which the branched-off distal rod segments of Tpr interdigitate and can loosely catenate neighboring basket fibers without need for further linker proteins. Predicted basket flexibility and the different conformations a basket in action may adopt (Kiseleva et al., 1998) could then reflect dynamic and alternating interactions between these distal Tpr rod segments. In this model, the Tpr tail domains that do not homodimerize and are mainly composed of 
-sheets, -turns, and random coils (Hase et al., 2001) would remain flexible and have free C termini.
A basket in which the neighboring fibers are interconnected also would allow to harmonize the role of Nup153 in tethering Tpr to the NPC with studies in which the major of at least two nuclear pools of Nup153 was found to represent a highly mobile form that dynamically interacts with the NPC (Daigle et al., 2001; Griffis et al., 2004<
, respectively: 
. R stands for the real NPC radius, i.e., the distance between mid axis and membrane boundary of an ideal NPC (here set at 40 nm), r for the measured distance between apparent mid-axis and membrane boundary of the cross-sectioned NPC, and d for the measured distance between apparent mid-axis and gold grain. Grains on grazing sections of the pore where r was <25 nm were not considered.
5%). Red dots mark the NPC center, flanked by the NPC core structures, including the coaxial rings, and the NE membrane (yellow). Attached to the nuclear ring, only two of the eight rod-like fibers of the nuclear basket, and the distal terminal ring (dashed circle) are depicted. Pore diameter in this scheme corresponds to 80 nm, NE width to 32 nm, and distance between NE midplane and terminal ring to 80 nm.
