ARTICLE |
Correspondence to: Alberto M. Martelli, Dipartimento di Morfologia Umana Normale, Università di Trieste, via Manzoni 16, I-34138 Trieste, Italy.
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Summary |
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The distribution of three nuclear scaffold proteins (of which one is a component of a particular class of nuclear bodies) has been studied in intact K562 human erythroleukemia cells, isolated nuclei, and nuclear scaffolds. Nuclear scaffolds were obtained by extraction with the ionic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of divalent cations (metal-depleted nuclei) and stabilized either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ ions at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffected by all the stabilization procedures performed on isolated nuclei. However, LIS extraction and scaffold preparation procedures markedly modified the distribution of the polypeptide seen in intact cells, unless stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other antigens (Mr 125 and 126 kD), whereas in heat-stabilized nuclei we detected dramatic changes. In nuclear scaffolds reacted with antibodies to 125- and 126-kD proteins, the fluorescent pattern was always disarranged regardless of the stabilization procedure. These results, obtained with nuclei prepared in the absence of Mg++ ions, indicate that heat treatment per se can induce changes in the distribution of nuclear proteins, at variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly because of its specific association with the nuclear scaffold. (J Histochem Cytochem 45:295-305, 1997)
Key Words: nuclear scaffold, confocal microscopy, heat stabilization, Cu++ ions, immunocytochemistry, erythroleukemia cells
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Introduction |
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The nuclear scaffold is a mainly proteinaceous insoluble framework that remains after isolated nuclei are treated with non-ionic detergents, nucleases (DNAse, restriction enzymes, RNAse A) and solutions of high ionic strength (
Exposure of isolated nuclei to 37C was deliberately used by
Our present interest is to establish the extent to which the techniques routinely employed for preparing the nuclear scaffold can maintain in the final structures the same distribution of nuclear polypeptides as seen in intact cells (
However, those results have been obtained using nuclei heat-stabilized in the presence of Mg++. It is commonly believed that Mg++ ions create artifacts, including ultrastructural changes seen in nuclear structure, after a 37C incubation (
We demonstrate that, even without Mg++ ions, a redistribution of nuclear scaffold polypeptides occurs during a 37C or 42C incubation of isolated nuclei. Conversely, treatment with Cu++ at 0C did not lead to changes in the immunofluorescent pattern given by the antibodies. Overall, the LIS extraction and scaffold isolation procedures affected quite dramatically the spatial distribution of the nuclear proteins. These data emphasize the need for a comprehensive reevaluation of the methods now used for preparing the nuclear scaffold.
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Materials and Methods |
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Cell Culture
K562 human erythroleukemia cells were grown in RPMI-1640 medium supplemented with 10% newborn calf serum at 37C in a humidified atmosphere containing 5% CO2. They were seeded at a density of 105/ml and used 4 days later when they reached a density of 106/ml.
Preparation of Isolated Nuclei and Nuclear Scaffold
Cells were washed once in Dulbecco's PBS, pH 7.4, free of Mg++ and Ca++. Nuclei (metal-depleted nuclei) were isolated as reported by
Extraction was carried out for 10 min at room temperature (RT) (
Source of Antibodies
Monoclonal antibodies (MAbs) to inner nuclear scaffold proteins of 160 kD (referred to as p160) and 126-kD component of nuclear bodies (clone 5E10) were kindly donated by Drs. R. van Driel and L. de Jong (E. C. Slater Institute for Biochemical Research; Amsterdam, The Netherlands). The MAbs have been described in previously published papers (
SDS-PAGE and Western Blotting
Nuclei and nuclear scaffolds were briefly digested with DNAse I to improve their solubilty in sample buffer (30 U/100 µl of suspension). Protein was then precipitated with 25% (w/v) trichloroacetic acid (TCA) at 4C. After two washes in acetone and one in diethyl ether, protein was dissolved in electrophoresis sample buffer (
Immunofluorescent Staining
Cells in PBS, nuclei in nuclear washing buffer, and scaffolds in digestion buffer were plated onto 0.1% poly-L-lysine-coated glass slides and adhesion was allowed to proceed for 30 min at 37C for cells or at room temperature for nuclei and nuclear scaffolds. By using a temperature of 37C for adhesion of intact cells, we ensured that the immunofluorescent patterns we detected were representative of the distribution of the nuclear antigens in vivo. Moreover, by employing the same temperature (22C) for adhesion of both isolated nuclei and scaffolds, we ensured that the changes possibly detected in these two fractions were due exclusively to treatments performed before attachment. Finally, it should be stressed that isolated nuclei and scaffolds were used for immunocytochemical staining immediately after their preparation and were not stored for any period of time.
Whole cells were fixed in freshly made 2%, 3%, or 4% paraformaldehyde for p125, p160, and 5E10, respectively, in PBS for 30 min at RT and then permeabilized for 10 min at RT in PBS containing 0.5 % Triton X-100. Isolated nuclei and scaffold samples were fixed without any additional treatment. Isolated nuclei were fixed with the same concentration of paraformaldeheyde as above, prepared in washing buffer, while for nuclear scaffold preparations the fixation buffer was digestion buffer. Both of these buffers were supplemented with 50 mM sodium cacodylate, pH 7.4. After several washes with PBS, nonspecific binding of antibodies was blocked by a 30-min incubation at 37C with PBS, 2% BSA, 5% NGS. Slides were then incubated for 3 hr at 37C with the appropriate primary antibody diluted in PBS, 2% BSA. Slides were then washed three times in PBS and reacted with a fluorescein-conjugated anti-mouse IgG or IgM (Sigma), diluted 1:45 or 1:80, respectively, in PBS, 2% BSA, 5% NGS for 1 hr at 37C. Samples were subsequently washed three times in PBS, stained with 1 µg/ml 4'-6-diamidino-2-phenylindole (DAPI) in PBS, and mounted in 20 mM Tris-HCl, pH 8.2, 90% glycerol containing 2.3% of the antifading agent 1,4-diazobicyclo-[2.2.2]-octane.
Confocal Laser Scanning Microscope (CLSM) Analysis
Samples were imaged by a PHOIBOS 1000-SARASTRO (Molecular Dynamics; Sunnyvale, CA) CLSM mounted on a Nikon Optiphot microscope (Nikon; Tokyo, Japan). This confocal system was coupled with a 25-mW multiline Argon ion laser as a light source. This laser produces two major lines at 488 and 514 nm. The first one was selected with a bandpass filter and was used to reveal FITC signal. The laser power was tuned at 10 mW to obtain the highest light stability and the laser beam was attenuated to 30% of transmission with a neutral density filter to limit bleaching of the FITC fluorescence. Samples were observed with a x 100, 1.4 numerical aperture (NA) planapochromat objective lens. The optical resolution of the confocal microscope is dependent on the wavelength of the incident light and on the NA of the objective. To obtain the highest resolution, we employed the lowest laser light wavelength suitable and the highest NA objective [(0.461)/NA]. The refractive index of the immersion oil was 1.518 (Nikon). The oil was dissolved up to a concentration of 20% in the mounting medium to minimize distortion of the confocal spot during the laser beam penetration inside the specimen (Carlsson 1991). In the detection path, the emitted fluorescent light was focused on a back pinhole aperture with a diameter of 50 µm in front of the detector, a photomultiplier tube (PMT). To block any unwanted contribution signal, for observing FITC a 515 OG longpass filter was inserted before the PMT as a barrier filter. The PMT was set at 864 mV. Settings were rigorously maintained for all experiments. Images were acquired, frame by frame, with a scanning mode format of 512 x 512 pixels. Pixel values were recorded in the range of 0-255 (8 bits). Images were reconstructed as follows. Slides were scanned from left to right on the x-y plane moving down with steps of 0.3 µm from top to bottom in the z direction of samples, using the motor drive focusing system, to a position unaffected by the last horizontal pass. Each frame had a scan time of 0.5 sec and every optical section was the result of a single scan. The microscope table was set manually so that the first z section was collected just at the top of the structures to be analyzed.
Image Processing Analysis
Digitalized optical sections, i.e., z series of confocal data ("stacks") were transferred from the CLSM to the graphics workstation Indigo Irix XS24 (Silicon Graphics; Mountain View, CA) and stored on the graphics workstation with a scanning mode format of 512 x 512 pixels and 256 gray levels. The image processing was performed using the ImageSpace software (Molecular Dynamics). To reduce the unwanted background noise generated by the photomultiplier signal amplification, all the image stacks were treated with a two dimensional filter (Gaussian filtering) that was carried out on each voxel, with a mask of 3 pixels in the x, y, and z direction (3 x 3 x 3).
The FITC signal was elaborated to optimize the contrast, the brightness, and the intensity of the images. Photographs were taken by a digital video recorder Focus ImageCorder Plus (Focus Graphics; Foster City, CA) using 100 ASA T-Max black and white film (Kodak; Rochester, NY).
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Results |
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Western Blotting Analysis
By Western blotting analysis we verified whether or not the antigens we studied were retained in the nuclear scaffolds prepared by LIS extraction. As shown in Figure 1A, antibody to the nuclear body protein (clone 5E10) stained a band with a molecular weight of approximately 126 kD among proteins from K562 cell nuclei. This is consistent with the report by
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CLSM Analysis
-kD Component of Nuclear Bodies.
In intact cells, nine to 14 irregular spots of different size and shape were distributed in the inner nucleoplasm and nuclear periphery. In this case, it should be emphasized that we selected the equatorial plane section through the nucleus that contains the majority but not all the spots. These dots were often associated with smaller points located in the same regions (Figure 2A). A very similar aspect was displayed by nuclei exposed to 0C (control or unstabilized nuclei) (Figure 2B) or stabilized with Cu++ (Figure 2C). Nuclei incubated at 37C were characterized by the fusion of many spots into a few (two or three) larger fluorescent areas, very irregular in shape and located mainly at the nuclear periphery, in association with some smaller spots and points (Figure 2E). A similar condensation of the immunoreactivity was caused by the 42C stabilization, but in this case it was distributed mainly in the center of the nucleus instead of its periphery. Some very tiny dots were dispersed irregularly in the nucleoplasm and at the periphery (Figure 2G). Preparations of nuclear scaffolds stabilized with Cu++ at 0C displayed a strongly condensed fluorescence that resulted in only two or three very large and rounded masses located at the periphery of the structure. Very rarely (in about 5-10% of isolated scaffolds), some extremely tiny dots were observable at the periphery (Figure 2D). In scaffolds derived from nuclei exposed to 37C, the larger masses were located at the nuclear periphery, whereas smaller fluorescent areas were also in the interior. Their number was very limited (three to five), and tiny dots in between were almost absent (Figure 2F). Nuclear scaffolds prepared from nuclei incubated at 42C showed not only condensation of the fluorescence in a few masses, as seen at 37C, but also their accumulation in the center of the structure. The size was smaller and the shape more irregular than at 37C (Figure 2H).
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p160. A fibrogranular fluorescent meshwork characterized by fine, punctate, and regularly ordered staining was observable in intact cells. The nuclear interior appeared more intensely labeled by tiny, round fluorescent points than the nuclear periphery. Negative nucleoli were clearly distinguishable (Figure 3A). All nuclear preparations showed the same pattern (Figure 3C, Figure 3E, and Figure 3G) that was seen also in Cu++-stabilized scaffold (Figure 3D). Nuclear scaffolds prepared from nuclei exposed to 37C showed a condensation of the fluorescence in the interior accompanied by disappearance of the negative nucleolar areas (Figure 3F). A similar pattern was observable after 42C stabilization of the nuclear scaffold. Homogeneous fluorescence covered the structure, with a partial condensation of the fluorescent dots in its center. Nucleoli were not detectable (Figure 3H).
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p125. Cells reacted with anti-p125 antibody showed large, irregular masses of fluorescence that stained the nuclear periphery and interior except for nucleolar areas, identified by phase-contrast microscopy (not shown). Some tiny dots or punctate fluorescence were dispersed in between the larger masses (Figure 4A). Incubation of isolated nuclei at 0C was characterized by similar fluorescent masses, accompanied by many irregular dots of various shapes at the periphery and in the nuclear interior. Nucleoli still appeared as negative areas (Figure 4B). Stabilization with Cu++ gave a pattern very similar to that seen in nuclei kept at 0C (control or unstabilized nuclei), even when the labeling was clearly decreased at the nuclear periphery (Figure 4C). In nuclei exposed to 37C, except for the negative nucleolar areas, the immunostaining assumed a punctate pattern, distributed especially in the nuclear interior. Some large masses were still observable, accompanied by smaller areas of irregular shape (Figure 4E). The larger masses were detectable after 42C stabilization. They were located mainly in the inner nucleoplasm and emerged from a finer and fainter fluorescence (Figure 4G). Nuclear scaffolds stabilized with Cu++ were characterized mainly by large fluorescent areas of irregular shape, located both at the periphery and in the center of the structure. The fine fluorescence almost disappeared and nucleoli were well detectable as negative areas (Figure 4D). Rounded fluorescent areas of different size were detectable in scaffolds after 37C exposure. Both the center and the periphery of the scaffolds were labeled, excluding nucleolar regions (Figure 4F). Scaffolds obtained after 42C incubation of isolated nuclei showed large, irregular fluorescent areas clumped in the center of the structures, accompanied by fine granular fluorescence (Figure 4H).
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Quantitative Analysis
Table 1 shows the results of quantitative analysis performed on the various samples we analyzed, immunostained for the three antigens studied. For each condition, 400 cells, nuclei, and nuclear scaffolds were manually counted and the staining patterns examined, using five different experiments. A statistical test was performed by comparing the number of structures exihibiting the typical immunofluorescent pattern identified in intact cells with the number of those showing a different pattern. The data revealed that highly significant differences were detected in several samples after different treatments, thus demonstrating that the changes in the distribution of antigens we have illustrated above were indeed present in the great majority of isolated nuclei and scaffolds.
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Discussion |
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The method for preparing the nuclear scaffold that we employed in this study was originally devised by
An important issue that has often been neglected in the nuclear scaffold field is to assess whether the harsh treatments nuclei undergo during the preparations can induce changes in the distribution of nuclear scaffold components. This is particularly true for the scaffold prepared from cells growing as suspension cultures, whereas when the preparation starts from adherent growing cells (the so-called in situ matrix) these controls have been generally performed (see, e.g.,
Given also the fact that LIS is an ionic detergent as well as a chaotropic agent, it is surprising that thus far no morphological investigation has been devoted to study of possible modifications induced by the chemical on the distribution of nuclear scaffold polypeptides. Our results have clearly shown that exposure of isolated nuclei to moderate heat for a brief time causes dramatic changes in the original fluorescent pattern given by antibodies to two different nuclear scaffold proteins (p125 and 126 kD). A third antigen, p160, was insensitive to this kind of incubation.
On the other hand, Cu++ ions did not cause such a dramatic redistribution and preserved quite well the original immunostaining of the three antigens as seen in intact cells.
Therefore, a major conclusion that can be drawn is that heat and Cu++ stabilization cannot be considered equivalent as far as the preservation of the distribution of at least some nuclear antigens is concerned. In the future it will be important to ascertain whether or not this also applies to other nuclear antigens.
Our data also show unequivocally that heat treatment, even when performed in the absence of Mg++ ions, induces dramatic changes in the immunostaining of nuclear scaffold proteins. Therefore, a buffer containing spermine/spermidine/KCl can also have negative effects on nuclear structure and function.
Finally, it should be noted that all the three antigens were severely perturbed by LIS extraction and scaffold isolation procedures, except for p160 when stabilized by Cu++.
The possible role(s) performed by the three proteins we have analyzed is at present unclear. However, our unpublished experiments have shown that p125 is a DNA-binding protein. In this connection, it should be recalled that
Exposure of isolated nuclei to a temperature of 37C or more might cause changes in the structure of nuclear scaffold constituents, by mechanisms similar to those that occur when intact cells are subjected to heat shock in vivo (see, e.g.,
Nevertheless, a temperature of 37C, which is physiological for cells, was able to induce alterations of two of the examined antigens, 5E10 and p125, in isolated nuclei. In any case, these changes were more marked when the samples were stabilized with a 42C exposure, showing a correspondence with the patterns observed in heat shock experiments for the p125 nuclear scaffold protein (
The three proteins we have studied belong to the ill-defined inner scaffold network. However, their behavior after different forms of stabilization is clearly different. Conceivably, such behavior is a direct consequence of their specific type of association with the scaffold. The definition of inner network is clearly inadequate, but it reflects our very limited knowledge about its organization. In the future it will be interesting to examine the behavior of other proteins under the same conditions as described here, such as the 240-kD matrix antigen referred to as NuMA (nucleus-mitotic apparatus; see, e.g.,
Although there is quite strong evidence indicating the existence of a nuclear framework also in vivo, our knowledge about the molecules that are the basic constituents of this network is only in its infancy. Indeed, the methods presently used to isolate the scaffold entail progressive stripping of isolated nuclei. However, at the end, a structure is obtained that retains many more components than the molecules of the network proper. Because of this, we believe that morphological studies, monitoring the distribution of nuclear antigens at all stages, might make an important contribution to defining which nuclear proteins are the key structural components of a nuclear framework existing in vivo.
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Acknowledgments |
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We thank Giovanna Baldini and Aurelio Valmori for the illustrations.
Supported by Italian CNR grant PF ACRO and 94. 00413.CT12, by Fondi AIRC 1995 (Associazione Italiana Ricerca sul Cancro) to AMM, and by Italian MURST 60% grants to Università di Trieste and Ferrara.
Received for publication May 8, 1996; accepted September 26, 1996.
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