Localization of Tpr to the NE and discrete foci in the nuclear interior by immunofluorescence microscopy
We have used a combination of immunofluorescence microscopy, light microscope imaging of GFP-Tpr in living cells, and immunogold EM to decisively determine the localization of Tpr. In particular, we wanted to investigate whether Tpr forms long filaments that extend into the nucleus from the NPC and/or a filamentous intranuclear network as proposed (Cordes et al., 1997
; Zimowska et al., 1997
; Paddy, 1998
). Part of the controversy regarding the localization of Tpr may stem from the fact that most previous localization studies used monoclonal or antipeptide antibodies, which have potential problems of cross-reaction with other antigens (Lane and Koprowski, 1982
) or limited epitope accessibility. Therefore, we generated affinity purified polyclonal antibodies to 150–250 amino acid fragments from three regions of Tpr in the NH2
terminus, central region, and COOH terminus (anti-TprN, anti-TprM, and anti-TprC, respectively) ( A). TprN and TprM were located in the NH2
-terminal domain of Tpr, which contains the predicted coiled-coil region, and TprC was located at the end of the COOH-terminal acidic domain ( A). We tested the three affinity purified antibodies for their specificity using immunoblot analysis of HeLa cell extracts ( B). All three antibodies reacted strongly with a single major band that migrated at the position expected for full-length Tpr. Anti-TprC also recognized minor apparent proteolytic fragments in addition to the full-length protein, consistent with the finding that Tpr is extremely sensitive to proteolysis (Byrd et al., 1994
; Bangs et al., 1996
Figure 1. Characterization of antibodies to three Tpr fragments. (A) Antibodies against Tpr were made to NH2- and COOH-terminal fragments and to an internal fragment spanning a break in the predicted coiled-coil region as indicated. (B) HeLa cell extracts were (more ...)
Using our three anti-Tpr antibodies for immunofluorescence staining, we first examined the localization of Tpr in HeLa ( C) and normal rat kidney (NRK) (unpublished data) cells by confocal microscopy. In optical sections through the center of the nucleus, all three antibodies labeled the NE in a punctate pattern characteristic of nucleoporins (Snow et al., 1987
) and also yielded staining throughout the nuclear interior as has been observed previously (Bangs et al., 1996
; Cordes et al., 1997
). The intranuclear Tpr occurred in apparently discrete foci rather than in continuous interconnected zones. In some cells, Tpr was also localized in a diffuse intranuclear pattern and in foci. Immunolocalization of Tpr by confocal microscopy of cultured BRL cells (a rat liver line) and 5 μm cryosections of rat intestine and kidney showed Tpr to be localized at the NE and in discrete intranuclear foci (unpublished data) similar to our findings in HeLa and NRK cells.
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 () 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 (; 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
Figure 2. Localization of Tpr in cultured mammalian cells. Immunofluorescence staining followed by deconvolution microscopy was used to visualize Tpr (red) in single optical sections HeLa and NRK cells as indicated. Cells are colabeled with antibodies to either (more ...)
To further investigate whether the intranuclear foci of Tpr we observed in z-sections obtained by deconvolution microscopy are discrete isolated bodies or a two-dimensional cross-sectional view of a three-dimensional network, we constructed volume projections of HeLa cell nuclei from 20 sequential 0.2 μm serial z-sections. In the en-face or edge-on views of the volume projection rotated 90° to each other ( A, left and right, respectively), the intranuclear staining for Tpr shows only discontinuous foci with no evidence of the foci either forming a filamentous intranuclear network or being associated with filaments that emanate from NPCs. This was confirmed by examination of sequential z-sections at 0.4-μm intervals ( B), which shows that individual foci of staining for Tpr (red) and the nuclear basket nucleoporin Nup98 (green) mostly do not overlap, although occasional colocalization of foci is observed. Arrows ( B) indicate foci of Tpr that wax and wane through the sequential sections of the nucleus, showing that foci appear discontinuous. The finding that intranuclear Tpr occurs in discrete intranuclear foci rather than in interconnected networks was obtained with several fixation conditions in addition to the standard method used for the images shown (see Materials and methods), which yielded the lowest background. We note that there are differences in the intensities of Tpr and Nup98 in individual foci at the NE ( B). This may reflect different relative concentrations of Tpr and Nup98 in different NPCs.
Figure 3. Localization of Tpr and Nup98 in HeLa cells by immunofluorescence staining and deconvolution microscopy. (A) Volume projection of 20 sequential 0.2 μm z-sections stained with anti-Tpr antibodies (red). Panels are shown at 0 and 90° to (more ...)
Imaging of GFP-Tpr in living cells
To verify that our localization of Tpr to discrete intranuclear foci rather than a filamentous network is not an artifact of fixation, we expressed a protein consisting of full-length Tpr tagged with GFP in HeLa cells for live cell imaging. Immunoblot analysis of the transfected cells detected a roughly 300 kD GFP fusion protein as expected (unpublished data). When the transfected cells were fixed and analyzed by immunolabeling and confocal microscopy, the GFP-Tpr (detected by GFP fluorescence) showed a very similar distribution to bulk Tpr (detected by anti-Tpr) in these cells whether low or moderate levels of GFP-Tpr were expressed ( A). Not only was GFP-Tpr efficiently targeted to the NE, but the GFP-Tpr remained stably associated with the NE upon extraction of unfixed cells with PBS containing 0.2% Triton X-100 (unpublished data), suggesting that its interactions with the NE are not grossly perturbed by the GFP tag. Consistent with the fixed cell immunolocalization, imaging of living transfected cells by deconvolution microscopy () showed that the GFP-Tpr was localized to both the NE and discrete intranuclear foci and in a diffuse intranuclear pattern (). The presence of Tpr in foci distributed throughout the nuclear interior can be clearly appreciated in volume projections of stacks of sections ( C). As further evidence that the Tpr localization we obtain by antibody labeling and GFP imaging is very similar, we provide two videos depicting nuclei in which Tpr is localized by antibody staining of untransfected HeLa cells (video 1 available at http://www.jcb.org/cgi/
content/full/jcb.200106046/DC1) or by detection of GFP-Tpr in transfected HeLa cells (video 2 available at http://www.jcb.org/cgi/content/full/jcb.200106046/DC1
). Both sets of images were generated from a stack of deconvolved optical sections and show the three-dimensional Tpr distribution that can be appreciated by rotating the nucleus. In conclusion, live cell imaging of GFP-Tpr supports the results of antibody localization, showing that intranuclear Tpr is localized diffusely and in discrete foci and is not present in a filamentous intranuclear network.
Figure 4. Imaging of GFP-Tpr in HeLa cells. Cells were examined 50 h after transfection using confocal (A) or deconvolution (B and C) techniques. (A) Cells were fixed and immunostained with anti-TprC (GFP fluorescence, left, green; anti-TprC, right, red). (more ...)
Localization of Tpr to the nuclear basket of the NPC by immunogold EM
To localize Tpr at high resolution by immunogold EM, we used two complementary techniques: preembedding labeling of cells or isolated nuclei that had been permeabilized to allow antibody access and labeling of ultrathin cryosections of intact cells or tissue in which the entire surface of the section is available for antibody detection. In preembedding analysis of isolated rat liver nuclei ( A) and HeLa cells () with anti-Tpr antibodies, we found that strong labeling occurred at the nucleoplasmic side of the NPC as observed previously (Cordes et al., 1997
). The labeling occurred within ~120 nm of the pore midplane () consistent with a localization to the nuclear basket. It is noteworthy that the strong labeling at the NPC did not continue beyond ~120 nm. Thus, Tpr is unlikely to form filaments or cables that emanate from the nuclear basket into the nuclear interior. In addition to the gold labeling at the NE, single gold particles and small clusters of gold (, asterisks) are also scattered throughout the nucleus. We suggest that the intranuclear clusters of gold labeling reflect the intranuclear Tpr foci observed by light microscopy. In rare cases, clusters of gold particles are seen in the vicinity of an NPC ( A, top asterisk), presumably reflecting the coincidental occurrence of a cluster of intranuclear Tpr near an NPC. We obtained a very similar labeling pattern when we examined ultrathin cryosections of HeLa cells and rat liver by immunogold EM as illustrated by the rat liver samples shown in . Gold is concentrated at the nuclear surface of the NPC within ~120 nm of the pore midplane and is also found inside the nucleus in clusters and single particles.
Figure 5. Immuno-EM localization of Tpr in nuclei of cultured cells and liver. NPCs are indicated by arrows; gold is seen at the nuclear face of NPCs and also either as single particles (arrowheads) or as clusters in the nuclear interior (asterisks). (A) (more ...)
A comparison of the distribution of Tpr, Nup153, and Nup98 in the NPC using preembedding immunogold EM of HeLa cells is shown in where a gallery of high magnification views is presented (left). For all three proteins, the NPC-associated gold particles are broadly distributed throughout a zone that is within a ~120 nm radius of the eightfold symmetry axis of the NPC and within a ~120 nm span from the midplane of the NPC (, left). The distribution of gold particles with respect to the midplane of the NPC is quantified in the histograms shown in (right). The gold particles localizing TprN, TprM, TprC, Nup98, and Nup153 all have broad nucleoplasmic distributions from 20–120 nm of the NPC midplane.
Figure 6. Immunoelectron microscopic localization of Tpr at the NPC in HeLa cells. Indirect immunogold labeling was performed for Tpr and the nuclear basket proteins Nup153 and Nup98 in HeLa cells using the preembedding technique. (Left) Gallery of NPCs (arrows) (more ...)
presents a comparison of the mean distances of gold particles representing Tpr, Nup98, and Nup153 from the midplane of the NPC obtained from analysis of HeLa cells and rat liver by preembedding and cryosectioning techniques. For each of the three antibodies to Tpr, the mean distance from the NPC midplane ranges from 54–77 nm. The mean distance for Nup153 ranges between 75–84 nm, which is further from the NPC midplane than obtained with a monoclonal anti-Nup153 antibody (35–45 nm) presumed to recognize a different epitope (Guan et al., 2000
). The mean Nup98 distance is between 53–67 nm, which is also consistent with previously reported results (Radu et al., 1995
). Since the distribution of Tpr is very similar to that of the well-recognized nuclear basket components Nup153 and Nup98, this strongly suggests that Tpr is an element of the nuclear basket. The broad distribution of gold particles labeling Tpr on the nucleoplasmic side of the NPC also is consistent with the known flexibility of the nuclear basket, which could result in a variable positioning of epitopes.
Distance of Tpr, Nup153, and Nup98 from the midplane of the NPC
Depletion of Tpr from the NE by antibody injection into mitotic cells inhibits nuclear protein export but not import
Until now, the function of Tpr in higher eukaryotes has been studied only by transfection and overexpression of full-length Tpr or fragments of Tpr in cultured cells (Cordes et al., 1998
). However, these studies cannot distinguish whether Tpr overexpression has a direct or indirect effect on nuclear transport, since the phenotypes are determined after the accumulation of overexpressed protein over a 24–48-h period. To more directly examine the role of Tpr in nuclear transport, we used our anti-Tpr antibodies as inhibitory reagents in cell microinjection experiments. This approach has the advantage of being able to examine transport at relatively short time points postinjection. We first investigated whether we could deplete Tpr from the NPC by injecting antibodies into mitotic cells when the NPC is reversibly disassembled.
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 488–conjugated BSA or Alexa fluor 594–conjugated 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 (). Efficient depletion of Tpr was obtained by injection of either anti-TprN, anti-TprM, or anti-TprC antibodies (; unpublished data). At 1–2 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-TprC–injected cells in .
Figure 7. Immunofluorescent localization of nucleoporins 4 h after microinjection of anti-TprN, anti-TprC, or control rabbit IgG into NRK cells at mitosis. The injection marker (Alexa fluor 488–conjugated BSA) and rhodamine-coupled secondary antibodies (more ...)
When we examined the nuclear basket components Nup153 and Nup98, we found that their staining pattern in cells injected with anti-Tpr antibodies was similar to that in noninjected cells (). Furthermore, immunofluorescence labeling of cells with the monoclonal antibody RL1, which recognizes multiple FG repeat nucleoporins, was indistinguishable in control versus anti-Tpr–injected cells (unpublished data). Taken together, this suggests that other proteins of the NPC, including nuclear basket components, can assemble in the NE at the end of mitosis in the absence of Tpr.
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 acid–rich 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 ( and A), suggesting that Tpr is not essential for this nuclear import pathway.
Figure 8. Analysis of nuclear import and export in NRK cells injected with control IgG or depleted of Tpr by mitotic microinjection of anti-TprN. (A) Nuclear import of GST-NLS cargo (as described in Materials and methods) 5 and 30 min after cargo injection. The (more ...)
Figure 9. Analysis of nuclear import and export in interphase NRK cells microinjected with control antibodies or depleted of Tpr by mitotic microinjection of anti-TprN antibodies. Cells subsequently injected with a fluorescently labeled cargo were fixed at the (more ...)
To examine nuclear protein export, fluorescently labeled export cargo bearing a leucine-rich export signal (GST-NES) was injected into the nucleus of interphase cells that had been injected with anti-Tpr or with control IgG during the preceding mitosis. Export was evaluated at 30 and 60 min after nuclear injection of export cargo. At 30 min after cargo injection, substantial export of the cargo had occurred in most cells that had been injected with control IgG, whereas in cells injected with anti-Tpr antibodies export was significantly impaired ( and B). The level of export in cells injected with control IgG at mitosis was unchanged at 60 min compared with 30 min. At 60 min in the anti-Tpr–injected cells, the level of export was greater than at 30 min but still had not reached the level of export seen in control cells in only 30 min ( and B). This indicates that the depletion of Tpr achieved with this approach results in a kinetic impairment of protein export rather than an absolute block.
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 20–30 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 ( A).
Figure 10. Analysis of nuclear import and export in interphase cells injected with anti-Tpr or with control IgG antibodies. Cells were fixed, imaged, and scored for transport as in the legend to . (A) Anti-TprN, anti-TprM, anti-TprC, or control IgG was injected (more ...)
To examine nuclear protein export, anti-Tpr antibodies or control antibodies were mixed with export cargo (GST-NES), and the cocktail was injected into the nucleus of NRK cells. The level of nuclear export was scored after 30 min as noted above. We observed strong inhibition of export with all three antibodies to Tpr ( B). The distribution of cells among the three nuclear transport categories indicates that there were differences in the relative inhibitory efficacy of different anti-Tpr antibodies with anti-Tpr M having the strongest inhibitory effect and anti-TprC having the weakest effect. However, these differences may reflect the heterogeneity of the different antibody populations (i.e., a variable subpopulation of each antibody may be effective at inhibition). These results are in complete agreement with our microinjection studies in which Tpr was depleted by anti-Tpr injection of mitotic cells. Together, the results strongly suggest a direct role for Tpr in the nuclear export of cargo bearing leucine-rich NES sequences.