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Address correspondence to Larry Gerace, 10550 N. Torrey Pines Rd., Imm10, R209, La Jolla, CA 92037. Tel.: (858) 784-8514. Fax: (858) 784-9132. E-mail: lgerace{at}scripps.edu
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Abstract |
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Key Words: nuclear basket; nuclear foci; nuclear filaments; nuclear transport; nuclear pore complex
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Introduction |
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The NPC has an eightfold radially symmetrical framework that consists of central spokes flanked by nuclear and cytoplasmic rings (for review see Pante and Aebi, 1996). The ringspoke assembly surrounds an operationally defined gated channel involved in receptor-mediated transport. Eight fibrils extend outward from the cytoplasmic and nucleoplasmic rings. The cytoplasmic fibrils are 50-nm long, whereas the nuclear fibrils are
100-nm long and often joined at their distal ends by a terminal ring to form a "nuclear basket" (Jarnik and Aebi, 1991; Ris and Malecki, 1993). The cytoplasmic and nuclear fibrils may comprise the initial docking sites for import and export complexes, respectively, before their movement through the central gated channel.
Mammalian NPCs are thought to be composed of up to 50 or more different polypeptides. Several of these have been molecularly characterized and localized to substructures within the pore. These include Nup358 and Nup214 found at the cytoplasmic filaments, Nup98, Nup93, Nup205, Nup153 and Nup50 localized at the nuclear basket, and the Nup62 complex found near the central channel (Stoffler et al., 1999). Many nucleoporins contain multiple copies of the FG (phenylalanine-glycine) dipeptide repeat, and these FG repeat proteins appear to be interaction sites for karyopherins (for review see Gorlich and Kutay, 1999).
Mammalian Tpr (for translocated promoter region [Mitchell and Cooper, 1992]) is a 265 kD protein associated with the NPC (Byrd et al., 1994). Tpr contains two separate domains: most of the 1,600 residue NH2-terminal domain forms a parallel two-stranded coiled-coil interrupted periodically along its length (Hase et al., 2001), which may result in a hinged rod. The remaining
800 residue COOH-terminal domain is nonhelical and highly enriched in acidic residues. The precise localization of Tpr within the nucleus is controversial. In mammalian cells and Xenopus oocytes, Tpr is found near the nucleoplasmic surface of the NPC and is also proposed to be in filaments or hollow cables, which extend up to 350 nm from the nuclear basket into the nuclear interior (Cordes et al., 1997; Fontoura et al., 2001). In cells of the Drosophila salivary gland, the Tpr homologue localizes to the nucleoplasmic side of the NE and to extrachromosomal areas within the nucleus (Zimowska et al., 1997) and has been proposed to be a component of an intranuclear filamentous scaffold associated with NPCs (Zimowska et al., 1997; Paddy, 1998).
In Saccharomyces cerevisiae, Mlp1p and Mlp2p have predicted secondary structural similarity to Tpr, localize to the nuclear side of the NPC, and are thought to be Tpr homologs (Kolling et al., 1993; Strambio-de-Castillia et al., 1999). A double deletion of MLP1 and MLP2 is viable (Strambio-de-Castillia et al., 1999; Kosova et al., 2000) but causes a loss of telomeric clustering (Galy et al., 2000). Moreover, Mlp2p coimmunoprecipitates with Yku70p, a structural component of yeast telomeres (Galy et al., 2000). This suggests a potential role for Mlp1p and Mlp2p in telomere localization. The overexpression of Mlp1p but not Mlp2p results in a nuclear accumulation of poly-(A)+ RNA (Strambio-de-Castillia et al., 1999; Kosova et al., 2000), suggesting a potential role for Mlps in poly-(A)+ RNA export. In mammalian cells, overexpression of full-length Tpr and several Tpr fragments also results in the nuclear accumulation of poly-(A)+ RNA (Bangs et al., 1998). However, because of the long duration of protein overexpression in these experiments it is not clear whether the poly-(A)+ accumulation reflects a direct involvement of Tpr in mRNA export or an indirect effect due to a role of Tpr in another cellular or nuclear transport function.
In this study, we have reevaluated the localization of Tpr in the nucleus using several light microscopy and EM immunolocalization approaches combined with imaging of GFP-tagged Tpr in living cells. We found that Tpr is concentrated at the nucleoplasmic side of the NPC within 120 nm of the pore midplane similar to well-defined nuclear basket proteins. Although we also found Tpr and several other nucleoporins in discrete foci inside the nucleus, we obtained no evidence that it exists in long filaments that are connected to the nuclear basket or that extend through the nuclear interior. Microinjection of anti-Tpr antibodies into mitotic and interphase cells resulted in inhibition of nuclear export of proteins with a leucine-rich NES but did not affect nuclear import of proteins with a basic amino acidrich NLS. We conclude that Tpr is a nucleoporin that is localized discretely within the nuclear basket of the NPC and has a role in nuclear protein export.
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Results |
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We also investigated the localization of Tpr after immunofluorescence staining of cells using deconvolution microscopy. In contrast to confocal microscopy, which removes out-of-focus light from a z-section by optical methods, deconvolution microscopy removes out of focus light using computational methods. Optical sections of HeLa and NRK cells obtained by deconvolution microscopy (Fig. 2) showed that Tpr occurs in a punctate pattern at the NE and in apparently discrete foci throughout the nuclear interior similar to the findings from confocal microscopy. All three anti-Tpr antibodies gave a similar localization pattern by this method. The nuclear basket proteins Nup98 and Nup153 and the cytoplasmic fibril protein Nup358 also were localized at the NE and in intranuclear foci, although these foci largely did not colocalize with the Tpr-containing foci (Fig. 2; unpublished data). Our finding that some Nup98 is present in intranuclear foci is consistent with previous work (Powers et al., 1995; Fontoura et al., 1999), although we do not find extensive colocalization of intranuclear Nup98 and Tpr as reported (Fontoura et al., 2001).
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Mitotic NRK cells (typically in metaphase) were microinjected with a cocktail containing either antibodies against TprN, TprM, or TprC or control IgG and a marker for injection (Alexa fluor 488conjugated BSA or Alexa fluor 594conjugated BSA). Injected cells then were incubated at 37°C for up to 4 h during which time they completed mitosis. When the cells were fixed at 4 h and examined by immunofluorescence microscopy, pairs of daughter cells resulting from a single cell that had been injected with anti-Tpr antibodies in the preceding mitosis showed a dramatically reduced level of Tpr compared with that in the adjacent noninjected cells. In contrast, cells injected with control IgG showed Tpr staining similar to that in noninjected cells (Fig. 7). Efficient depletion of Tpr was obtained by injection of either anti-TprN, anti-TprM, or anti-TprC antibodies (Fig. 7; unpublished data). At 12 h after antibody injection, Tpr was observed in large aggregates in the nucleus and cytoplasm in most cells (unpublished data), suggesting that the antibody induces the formation of Tpr aggregates, and the aggregated Tpr is subsequently degraded. Sometimes these aggregates persisted at 4 h as seen in the pair of anti-TprCinjected cells in Fig. 7.
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We used this mitotic cell microinjection approach to generate interphase daughter cells with NPCs depleted of Tpr to study the potential role of Tpr in nuclear transport processes. To examine nuclear import, mitotic cells were initially injected with anti-TprN. After 4 h, the resultant daughter cells were then cytoplasmically injected with a fluorescently labeled import cargo containing a basic amino acidrich NLS (GST-NLS). The level of nuclear import was determined 5 and 30 min after cytoplasmic injection of import cargo. Transport in individual cells was classified by visual inspection of fluorescent images and fell into one of three categories: nuclear fluorescence of import cargo was either greater than the cytoplasmic fluorescence (N > C), equal to the cytoplasmic fluorescence (N = C), or less than the cytoplasmic fluorescence (N < C). At 5 and 30 min after cargo injection, nuclear import in the population of cells that had been mitotically injected with anti-Tpr was not significantly different from import in those cells mitotically injected with control IgG (Fig. 8 and Fig. 9 A), suggesting that Tpr is not essential for this nuclear import pathway.
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Microinjection of anti-Tpr antibodies into interphase cells results in the disruption of protein export but not import
As a second approach to address the role of Tpr in nuclear protein transport, we microinjected anti-Tpr antibodies into the nucleus of interphase NRK cells and examined nuclear import and export at short times thereafter. We determined by immunofluorescence staining that there was no apparent loss of Tpr from the NE within the time course of these experiments after the nuclear microinjection of antibodies (unpublished data).
To examine protein import, anti-Tpr antibodies (anti-TprN, anti-TprM, or anti-TprC), or control IgG were microinjected into NRK cell nuclei, and import cargo (GST-NLS) was separately microinjected into the cytoplasm at 2030 min after the initial antibody injection. The GST-NLS import cargo was detected 30 min after its injection by using immunofluorescence microscopy with an antibody to GST. Injected cells were scored for import as above (N > C, N = C, or N < C). We did not observe any significant effect on protein import in cells that had been microinjected with the various anti-Tpr or control (IgG) antibodies (Fig. 10 A).
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Discussion |
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We obtained a consistent picture of the localization of Tpr in the nucleus with all of our different localization approaches. By EM immunolocalization, we found that Tpr is concentrated at the nuclear basket of the NPC within 120 nm of the pore midplane similar to the localization of the nuclear basket proteins Nup98 and Nup153. However, we found no evidence for Tpr in filamentous structures that emanate from the NPC into the nuclear interior. To the contrary, we observed that the zone of Tpr concentration at the nuclear side of the NPC abruptly ends outside the confines of the nuclear basket as is the case for other nuclear basket proteins. By all the light microscope localization methods we used, we observed that Tpr is present throughout the nuclear interior and at the NPC. However, the intranuclear Tpr is present in a diffuse localization and discrete foci that are not connected with the NPC and not part of a continuous intranuclear network. We observed that the well-characterized nucleoporins Nup98, Nup153, and Nup358 also are present in discrete intranuclear foci. Thus, the intranuclear localization pattern seen for Tpr is no different from that observed for several other NPC proteins. In conclusion, our results demonstrate that Tpr is a component of the nuclear basket of the NPC and argue that Tpr is not part of filamentous structures that extend into the nuclear interior.
Since the presence of Tpr in intranuclear filaments that emanate from the NPC is widely accepted in the literature (Stoffler et al., 1999; Allen et al., 2000; Ryan and Wente, 2000), it is important to evaluate the discrepancy between our conclusions and those of previous studies. A general concern is that most of the previous localization studies of Tpr have relied on antipeptide or monoclonal antibodies (Cordes et al., 1997; Zimowska et al., 1997; Strambio-de-Castillia et al., 1999; Fontoura et al., 2001), which have much greater potential for cross-reaction (Lane and Koprowski, 1982) or limited epitope accessibility than the antibodies that we generated. Moreover, most previous studies have not simultaneously examined the localization of other nucleoporins in the same sample preparations. This substantially weakens conclusions about a particular localization being specific to Tpr, since it lacks the frame of reference of how other nucleoporins appear under the conditions used. Cordes et al. (1997) used immuno-EM of mammalian tissues and cultured cells to suggest that Tpr is localized to filaments that extend up to 350 nm from the nuclear side of the NPC. However, in these specimens nuclear ultrastructure is poorly preserved, since NEs are swollen and chromatin is detached from the NE, so it is plausible that the fragile nuclear basket is similarly distorted and distended in these preparations. These authors also found that an anti-Tpr antibody labels long filamentous structures extending from the NPC in isolated NEs of Xenopus oocytes, but a previous study also found the nuclear basket protein Nup153 in similar filaments (Cordes et al., 1993). Since Nup153 is restricted to the nuclear basket in somatic cells (Pante et al., 1994), this raises the question of whether structures found associated with isolated NEs of oocytes are representative of the somatic cell NE.
In other work, Zimowska et al. (1997) used immunogold EM to examine Tpr in Drosophila salivary gland cells and observed concentrated clusters of gold particles outside the chromosomal boundaries. These clusters were suggested to comprise filamentous Tpr (Zimowska et al., 1997). However, whether the Tpr in these interchromosomal spaces is present in filaments or whether it simply represents unassembled intranuclear protein present at a relatively high concentration was not established. An immunogold EM analysis of yeast Mlp1p and Mlp2p showed that they are concentrated at the NE within 120 nm of the pore midplane and are scattered at a lower density throughout the nuclear interior (Strambio-de-Castillia et al., 1999). Although the authors proposed that Mlp1p and Mlp2p form filamentous intranuclear structures that emanate from the NPC, the distribution of intranuclear Mlp1p and Mlp2p is consistent with the possibility that these proteins occur diffusely or in discrete intranuclear foci rather than in filaments as we have found for mammalian Tpr.
Immunofluorescence microscopy in cultured Xenopus cells by Shah et al. (1998) suggested that a COOH-terminal epitope of Tpr was further from the nuclear side of the NE than was an NH2-terminal epitope of Nup153. This observation was taken to support the model that Tpr comprises NPC-associated filaments. However, since labeling was not performed with antibodies against different Nup153 regions no conclusions can be made on the relative dispositions of Tpr and Nup153 as a whole with respect to the NPC midplane, since the proteins may be in an elongated conformation.
The proposal that Tpr is present in intranuclear filaments has been influenced by the results of scanning EM studies, which have suggested the existence of filamentous structures that underlie the NE or that seem to extend into the nucleus from NPCs (Goldberg and Allen, 1993; Ris and Malecki, 1993; Ris, 1997). The long predicted coiled-coil segment of Tpr and Tpr's localization inside the nucleus make it a tempting candidate to be present in these proposed structures. However, neither Tpr nor any other protein has been identified in these structures by immunolabeling. Moreover, concerns have been raised that many apparent intranuclear filaments may be artifacts of sample preparation (Cook, 1988; Pederson, 2000).
We found that the intranuclear foci of Tpr do not colocalize with foci of other nucleoporins that we have examined, suggesting that they are not part of hypothetical preassembled nuclear basket elements. Nonetheless it is possible that the foci represent nucleoporins synthesized in excess and stockpiled inside the nucleus, awaiting the assembly of new NPCs. In this case, NPC assembly could be regulated by the expression of a small number of limiting (or nucleating) nucleoporins. Alternatively, the foci may represent nucleoporins that dock to and are released from the NPC as part of a transport cycle. Some experiments have suggested that certain nucleoporins can shuttle between the nucleus and the cytoplasm (Boer et al., 1997; Nakielny et al., 1999). However, whether this shuttling occurs on the time scale of nuclear transport is not known.
Functional analysis of Tpr
Our anti-Tpr antibody microinjection experiments provide direct evidence for a role of Tpr in nuclear protein export. In these studies, we used two complementary strategies. First, we depleted Tpr from the nucleus by microinjection of antibodies against Tpr into mitotic cells and assessed the transport competence of the resulting interphase daughter cells. In a complementary approach, we microinjected anti-Tpr antibodies into the nuclei of interphase cells. Both techniques yielded the same result: nuclear import of a cargo containing a basic amino acidrich NLS proceeded normally, but the rate of nuclear export of a cargo containing a leucine-rich NES was strongly diminished.
The NPC does not appear to be disrupted dramatically when Tpr is depleted by antibody injection into mitotic cells, since the nuclear basket proteins Nup153 and Nup98 and the RL1 antigens showed apparently normal NPC staining, and the cells retained a normal level of nuclear protein import. The ability of NPCs to assemble in the absence of Tpr is consistent with localization studies, which show that Tpr assembles into the NPC later than most nucleoporins at the end of mitosis. (Bodoor et al., 1999; Haraguchi et al., 2000). Together, these results suggest that Tpr is not the backbone of the nuclear basket onto which Nup98 and Nup153 assemble.
Our mitotic cell microinjection analysis cannot exclude the possibility that inhibition of export is due to loss of a hypothetical NPC protein whose assembly into the NPC depends on the presence of Tpr. By contrast, the interphase cell injection experiments do not suffer from this problem, since Tpr remains assembled in the NPC throughout the experiment. In the latter case, the finding that antibodies to three different regions of Tpr inhibited nuclear export but did not affect nuclear import argues that the antibodies are not simply occluding the NPC. Since all phenotypes were scored at relatively short times after antibody injection, we believe that our microinjection approach effectively minimized the interpretive problems associated with previous functional studies, all of which used overexpression of Tpr or fragments thereof (Bangs et al., 1998; Cordes et al., 1998), and involved long time courses such that the observations could reflect indirect effects (as described in Introduction). Considered together, our data strongly argue that Tpr has a direct role in nuclear protein export.
Since Crm1 is the transport receptor for leucine-rich NES cargo used in our injection studies, our results raise the possibility that Tpr may function as an NPC binding site for Crm1 export complexes. In both of our microinjection approaches, we observed a strong decrease in the rate of protein export but not a total block. This may reflect incomplete depletion of Tpr or incomplete obstruction of Tpr with the antibodies. Alternatively, other functionally redundant nucleoporins may compensate for the absence of Tpr. Candidates include the nuclear basket proteins Nup153 and Nup50 both of which bind to Crm1 in a Ran-dependent manner (Nakielny et al., 1999; Guan et al., 2000). It should be noted that Tpr has been observed to coimmunoprecipitate with the import receptor importin ß in Xenopus oocyte extracts (Shah et al., 1998), and it remains possible that Tpr has a role in some aspect of nuclear import that is not detected by our antibody injection experiments. Our finding that Tpr is a bona fide nucleoporin rather than an NPC-attached intranuclear scaffolding protein provides a new conceptual framework for the analysis of these and other questions.
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Materials and methods |
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SDS-PAGE and immunoblotting
Suspension HeLa cells were pelleted at 250 g, resuspended in PBS with protease inhibitors (1 µg/ml aprotinin, pepstatin, leupeptin, AEBSF, E-64, and Bestatin) (Roche Biochemicals), mixed with an equal volume of boiling SDS sample buffer, and incubated at 100°C for 5 min. Protein separation by SDS-PAGE, electrophoretic transfer onto nitrocellulose, and immunoblotting were by standard methods. The Super Signal system (Pierce Chemical Co.) was used for visualization of proteins.
Antibodies
Antibodies were generated against GST fusions to three fragments of human Tpr: residues 1150 (Tpr N), 500750 (Tpr M), and 2,0952,348 (Tpr C). Rabbits and guinea pigs were immunized with the purified fusion proteins, and antibodies were affinity purified as described (Melchior et al., 1995) on a matrix of the purified GST fusion proteins coupled to CNBr-activated Sepharose (Amersham Pharmacia Biotech) at 0.51.0 mg/ml. Anti-Tpr antibodies used for interphase cell microinjection experiments were further purified on Trisacryl protein A beads (Pierce Chemical Co.) according to the manufacturer's instructions. For those experiments in which multiple anti-Tpr antibodies were used, all antibodies yielded very similar results. Control rabbit IgG was from Sigma-Aldrich. Antibodies to Nup98 were generated against a 6His-tagged fragment of Nup98 (residues 515937) and affinity purified. Antibodies against Nup153 (provided by Brian Burke [The Scripps Research Institute, La Jolla, CA] and Iris Ben-Efraim [University of Florida, Gainesville, FL]), lamin A/C, and Nup358 were described in Pante et al. (1994), Schirmer et al. (2001), and Delphin et al. (1997), respectively.
Immunofluorescence microscopy
Cells were grown to 70% confluency. For standard fixation and permeabilization conditions, cells were fixed in 4% formaldehyde in PBS for 4 min at room temperature, washed with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 4 min. Other conditions tested were fixation of cells with freshly prepared 4% formaldehyde or 4% formaldehyde plus 0.2% glutaraldehyde for 420 min followed by permeabilization in 0.2% Triton X-100 for 410 min. Alternatively, cells were treated with -20°C methanol for 5 min and -20°C acetone for 5 min, or cells were fixed in 0.2% formaldehyde for 30 min and treated with -20°C acetone for 3 min (Fontoura et al., 1999). These alternate methods resulted in higher background but gave similar results as the standard conditions. After fixation and permeabilization, cells were blocked with 3% BSA, incubated with primary antibodies (0.5 µg/ml) in PBS for 1 h at room temperature, washed with PBS, and incubated for 1 h with Cy5-conjugated goat antirabbit or goat antiguinea pig IgG (1:200; Jackson ImmunoResearch Laboratories) or with FITC-conjugated goat antirabbit or goat antiguinea pig IgG (1:200; Jackson ImmunoResearch Laboratories). The monoclonal antibody RL1 was detected with FITC-conjugated donkey antimouse IgM (Jackson ImmunoResearch Laboratories). The specimens were mounted in Slowfade Light mounting medium (Molecular Probes, Inc.) or in Fluoromount G (Electron Microscopy Sciences) and examined with a laser-scanning confocal microscope (model MRC-1024; Bio-Rad Laboratories) or by deconvolution microscopy using a DeltaVision optical sectioning microscope (Model 283; Applied Precision, Inc.). Images were prepared for figures using Canvas 6.0 and Adobe Photoshop® 5.2.
Analysis of GFP-tagged Tpr
The mammalian expression vector containing GFP fused to the COOH terminus of full-length Tpr was generated using pEGFPN1 (CLONTECH Laboratories). Cloning was performed in Stbl2 bacteria (Invitrogen) to prevent recombination events. pGFP-Tpr was transfected into HeLa cells using Fugene 6 (Roche), and live cells were imaged after 4860 h.
Cell microinjection
For microinjection, cells were grown on glass coverslips 1224 h before the procedure. Injection cocktails were prepared by combining a single antibody and an injection marker (Alexa fluor 488conjugated BSA or Alexa fluor 594conjugated BSA) (Molecular Probes) with or without export cargo (GST-NES) in a 0.5 ml Ultrafree concentrator (Millipore) and adding PBS to a final volume of 0.5 ml. The sample was concentrated 10-fold and centrifuged at 13,000 g for 20 min. Antibodies were injected into mitotic cells at a concentration of 5 mg/ml and into interphase cells at 2 mg/ml. Microinjection experiments used exponentially growing cultures, and metaphase cells were selected by their appearance in phasecontrast microscopy. Cells injected at mitosis were allowed to recover at 37°C for 4 h, and those identified by the fluorescence of the injected marker were microinjected again with either export cargo (a GST fusion to the PKI NES; GST-NES) into the nucleus or with import cargo (a GST fusion to the SV40 T-antigen NLS; GST-NLS) into the cytoplasm. After either 5, 30, or 60 min, cells were fixed in 4% formaldehyde for 5 min and permeabilized with 0.2% Triton X-100 for 4 min at room temperature. Cargo was visualized using a polyclonal goat anti-GST antibody (Amersham Pharmacia Biotech), and an appropriate secondary antibody and cells were examined by fluorescence microscopy. Cells receiving nuclear antibody microinjections at interphase were either coinjected with GST-NES or subsequently cytoplasmically injected 2030 min after antibody injection with GST-NLS. Graphs were created using Cricket Graph 3 v1.5.3.
Immunogold EM
Immunogold labeling was performed on isolated rat liver nuclei (Dwyer and Blobel, 1976), HeLa, or NRK cells. To permeabilize cultured cells, cells were pelleted in PBS, frozen in liquid nitrogen, and thawed at room temperature. Samples were incubated with anti-TprN, anti-TprM, anti-TprC, anti-Nup98, or anti-Nup153 antibodies (0.5 µg/ml) for 23 h at room temperature, washed, and incubated for 23 h at room temperature with 5 or 10 nm gold-conjugated antirabbit IgG (Sigma-Aldrich) or 12 nm gold-conjugated antiguinea pig IgG (Jackson ImmunoResearch Laboratories). EM processing was as in Guan et al. (2000).
For cryo-EM, HeLa cells or freshly dissected rat liver were fixed with 4% paraformaldehyde, 0.2% glutaraldehyde, and 4% sucrose in PBS for 1 h at room temperature. Cell pellets and tissues were prepared and sectioned as in Beesley (1993) and Raska et al. (1990). Grids were blocked in 5% FBS for 1 h and then incubated with primary antibodies (0.5 µg/ml) for 1 h. Grids were washed in PBS and blocked in 5% FBS for 30 min and were incubated with secondary antibodies (see above). After washing in PBS for 3060 min, grids were treated with a mixture of 2% methyl cellulose and 3% uranyl acetate (9:1) and air dried. Micrographs were recorded with a Philips EM-208 at 70100 kV.
Online supplemental material
Videos 1 and 2 showing three-dimensional rotations of nuclei in which are detected endogenous Tpr and GFP-Tpr (as described in Results) are available at http://www.jcb.org/cgi/content/full/jcb.200106046/DC1.
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Footnotes |
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* Abbreviations used in this paper: GFP, green fluorescent protein; NE, nuclear envelope; NES, nuclear export signal; NLS, nuclear localization signal; NPC, nuclear pore complex; NRK, normal rat kidney.
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Acknowledgments |
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This work was supported by a grant to L. Gerace (GM41955) and K. Hahn (GM57464) from the National Institutes of Health and by a fellowship to P. Frosst from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.
Submitted: 8 June 2001
Revised: 21 November 2001
Accepted: 17 December 2001
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References |
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