Visualization of the Nuclear Lamina in Mouse Anterior Pituitary Cells and Immunocytochemical Detection of Lamin A/C by Quick-freeze Freeze-substitution Electron Microscopy
Department of Anatomy I, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
Correspondence to: Takao Senda, Department of Anatomy I, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan. E-mail: tsenda{at}fujita-hu.ac.jp
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Summary |
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(J Histochem Cytochem 53:497507, 2005)
Key Words: nuclear lamina lamin A/C quick freezing freeze substitution deep etching anterior pituitary cell immunogold electron microscopy
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Introduction |
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The nuclear lamina was first described in invertebrates by transmission electron microscope observation (Pappas 1956; Beams et al. 1957
; Mercer 1959
; Gray and Guillery 1963
; Coggeshall and Fawcett 1964
). Although the corresponding layer had been identified in the nuclei of some vertebrate cells, its morphological characteristics differed among the cells (Fawcett 1966
). Actually, an ultrastructurally distinct nuclear lamina is not visible in many eukaryotic cells by conventional electron microscopy.
The localization of lamins in a variety of mammalian cells and tissues has been studied by immunofluorescence microscopy (Ely et al. 1978; Gerace et al. 1978
; Krohne et al. 1978
). Electron microscopic localization of lamins was further investigated by immunoperoxidase labeling in isolated rat liver nuclei (Gerace et al. 1978
) and rat liver tissue cryosections (Krohne et al. 1978
). These studies demonstrated that the lamins are principally present at the nuclear periphery and are absent from more internal regions of the nucleus.
This discrepancy between ultrastructural and immunocytochemical investigations suggests that the nuclear lamina composed of lamin proteins cannot be visualized successfully by thin-section electron microscopic preparation. This may be because the nuclear lamina is a ubiquitously widespread component of the nuclear envelopes and is too thin to be visualized in ultrathin sections (Aaronson and Blobel 1975; Gerace et al. 1978
; Krohne et al. 1978
). Moreover, highly electron-dense heterochromatin would obscure the thin layer of the nuclear lamina.
To clearly visualize the nuclear lamina, various attempts have been made on the specimens, and some novel electron microscopic techniques have been employed. Dwyer and Blobel (1976) observed isolated rat liver nuclear envelopes by electron microscopy and could clearly visualize the nuclear lamina. In such preparations, the nuclear lamina exhibited a thin fibrous layer attached to the nucleoplasmic surface of the inner nuclear membrane. When Triton X-100-extracted, freeze-dried/metal-shadowed nuclear envelopes of Xenopus oocytes were observed with a transmission electron microscope, the nuclear lamina was clearly shown to comprise a meshwork of 10-nm-diameter intermediate filaments (Aebi et al. 1986
). The meshwork structure of the nuclear lamina was also revealed in Xenopus oocytes by field-emission scanning electron microscopy (Ris 1997
) and in Necturus by cryo-electron microscopy (Akey 1989
).
Quick-freeze freeze substitution is a unique method for electron microscopic preparation, in which the fresh samples removed from animals are quickly fixed by freezing them with liquid helium (Heuser et al. 1979). This technique cannot only provide real in vivo subcellular ultrastructure and capture dynamic biological phenomena but can also avoid the possible artifacts caused by prolonged chemical fixation in aqueous fixatives (Hirokawa and Kirino 1980
; Hirokawa and Heuser 1981
).
In this study we employed quick-freeze freeze-substitution electron microscopy to visualize the nuclear lamina in mouse anterior pituitary cells and to detect and localize lamin A/C in their nuclei. Although the layer of the nuclear lamina was not visible in the cells fixed conventionally with glutaraldehyde, it could be visualized in the cells processed by the quick-freeze freeze-substitution technique. Furthermore, by immunogold electron microscopy, lamin A/C was detected and localized in the nucleus of the quickly frozen and freeze-substituted cells, in spite of the failure in detection of lamin A/C in the chemically fixed cells.
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Materials and Methods |
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Quick-freeze Freeze-substitution Electron Microscopy
Fresh unfixed anterior pituitary glands removed from decapitated mice were quickly frozen with liquid helium as described previously (Senda and Fujita 1987). The frozen glands were transferred into 2% OsO4 in acetone at 80C for 3 days and then warmed stepwise to 20C for 2 hr, to 4C for 2 hr, and finally to RT. The specimens were washed in 100% acetone and then in 100% ethanol and block stained for 2 hr with 5% uranyl acetate in 100% methanol. After washing in methanol, they were passed through propylene oxide and embedded in Epok812 epoxy resin (Oken Shoji; Tokyo, Japan). Semi-thin (700 nm) and thin (90 nm) sections were cut perpendicular to the slammed surface with a diamond knife. The semi-thin sections were stained with toluidine blue and examined with a BX50 light microscope (Olympus; Tokyo, Japan). The thin sections were stained with uranyl acetate and lead citrate and observed with a JEM-1010 transmission electron microscope (JEOL; Tokyo, Japan).
Conventional Thin-section Electron Microscopy
Anterior pituitary glands removed from decapitated mice were fixed by incubating with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 hr and subsequently with 1% OsO4 in the buffer for 1 hr. After block staining for 2 hr with 2% uranyl acetate in distilled water, they were dehydrated in graded concentrations of ethanol, incubated with propylene oxide, and embedded in Epok812 epoxy resin. The thin sections were stained with uranyl acetate and lead citrate and observed with the electron microscope.
Quick-freeze Deep-etch Electron Microscopy
Both glutaraldehyde-fixed and fresh unfixed anterior pituitary glands were quickly frozen, processed for freeze-fracture, deep-etch technique using a JFD-9000 freeze-fracture apparatus (JEOL) as described previously (Senda and Fujita 1987), and examined with the electron microscope.
Immunofluorescence Microscopy
Anterior pituitary glands removed from the decapitated mice were fixed with 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4C for 2 hr. The samples were washed thoroughly in 20% sucrose in PBS overnight, embedded in optimal cutting temperature (O.C.T.) compound (Sakura Finetechnical; Tokyo, Japan), an embedding medium composed of 10% (w/w) polyvinyl alcohol and 4% (w/w) polyethylene glycol in non-reactive ingredients, and frozen by immersing them in isopentan cooled with liquid nitrogen. Some pituitary glands were embedded immediately after being removed from the mice and frozen in the same manner. Cryosections as thick as 10 µm were cut with a cryostat (Leica; Vienna, Austria) at 20C and mounted on glass slides. The sections of the unfixed pituitary glands were fixed with 100% acetone at 20C for 10 min. All sections were treated with 0.1% Triton X-100 in PBS (phosphate-buffered saline) for 10 min and, after a brief wash, with 5% normal goat serum in PBS for 30 min. The sections were then incubated in polyclonal rabbit anti-lamin A/C antibody (H-110) (Santa Cruz Biotechnology; Santa Cruz, CA) and diluted 1:30 at 4C overnight. After washing in PBS, the sections were incubated for 30 min with the mixture containing AlexaFluor 488-labeled anti-rabbit IgG antibody (Molecular Probes; Eugene, OR) diluted 1:200 and TOPRO-3 (Molecular Probes) diluted 1:1000 for nuclear labeling. All antibodies were diluted in 5% normal goat serum in PBS. The sections were then washed in PBS, mounted in Mowiol containing 2.5% DABCO (1,4-diazabicyclo[2.2.2]octane) as an antifade, and observed with a confocal laser scanning microscope (LSM510; Carl Zeiss, Göttingen, Germany). Control sections were incubated with non-immune rabbit serum in place of the antibody.
Immunogold Electron Microscopy
For chemically fixed samples, mice anterior pituitaries were fixed with the fixative containing 3% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 hr. After washing in distilled water containing 5% sucrose, they were dehydrated in ethanol and embedded in Lowicryl K4M resin (TAAB; Berks, England) as described previously (Senda et al., 1991). Lowicryl thin sections cut with the ultramicrotome were picked up on uncoated nickel grids. The sections pretreated with 3% bovine serum albumin in PBS were incubated with the rabbit anti-lamin A/C antibody at RT for 2 hr. After washing in PBS, the sections were incubated with 10 nm colloidal gold-conjugated goat anti-rabbit IgG antibodies (Amersham Biosciences; Tokyo, Japan) at room temperature for 1 hr. The sections were stained with uranyl acetate and lead citrate and examined with the electron microscope. Control sections were incubated with non-immune rabbit serum in place of the antibody.
For quickly frozen and freeze-substituted samples, the quickly frozen anterior pituitaries were transferred to 0.3% glutaraldehyde-containing acetone at 80C for 2 days, and then warmed stepwise to 20C for 2 hr, to 4C for 2 hr, and finally to RT. The specimens were washed in 100% acetone, then in 100% ethanol, and embedded in Lowicryl K4M. Immunostaining for lamin A/C and labeling of the gold-conjugated second antibody was performed as described above and examined with the electron microscope.
For quantification of the immunolabeling, each electron microphotograph was taken at the direct magnification of x50,000 and enlarged in printing to x71,000. The distance between each gold particle and the inner nuclear membrane was measured. In total, 1149 gold particles in and around the nuclei of 24 anterior pituitary cells were counted.
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Results |
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At higher magnification, remarkable differences in ultrastructural preservation were recognizable depending on the depth from the slammed surface (Figure 2). In the first cell layer (within 15 µm from the surface), there existed an electron-lucent layer as wide as 1020 nm just inside the inner nuclear membrane (Figure 2B). The layer was homogeneous only with less-electron-dense amorphous materials. The boundary between the layer and the adjoining heterochromatin was quite definitive. According to the immunogold electron microscopy, this layer was heavily labeled with the gold particles for lamin A/C (Figure 5B and Figure 6), thus suggesting the nuclear lamina. On the contrary, such an electron-lucent layer was never visualized just beneath the inner nuclear membrane by conventionally glutaraldehyde-fixed anterior pituitary cells (Figure 2A). In this kind of preparation, it seemed that the heterochromatin was attached directly to the inner nuclear membrane.
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The nuclear lamina structure was observed by quick-freeze deep-etch electron microscopy. Quick-freeze deep-etch electron microscopy is a useful technique for visualization of fibrous components at high resolution (Senda and Fujita 1987; Senda et al. 1994
; Senda 1998
; Senda and Umemoto 1998
; Senda and Yoshinaga-Hirabayashi 1998
). As the nuclear lamina is a meshwork of lamin intermediate filaments (Dwyer and Blobel 1976
; Aebi et al. 1986
; Akey 1989
; Ris 1997
), we expected that this technique might provide some aspects for differences in the ultrastructure of the nuclear lamina with and without chemical fixation.
In quickly frozen and deeply etched mouse anterior pituitary cells, the nuclear envelope consisting of outer and inner nuclear membranes and the nuclear pore can be identified (Figure 3). The nucleoplasm was filled with a large amount of globular material of various sizes and with some fibrous components among them. These globular materials are likely to consist of components forming the chromatin loops and other soluble nuclear proteins (Senda and Umemoto 1998). We noticed that the globular materials localized just inside the inner nuclear membrane were smaller than those in other regions of the nucleoplasm. Nevertheless, we could not definitively distinguish the layer corresponding to the nuclear lamina in either chemically fixed or unfixed cells. Although a tangentially fractured plane of the nuclear lamina could have visualized the lamina structure, we did not encounter such a fractured plane. When compared carefully, the globular material was more tightly packed in the nucleoplasm of the fresh cell (Figure 3B) than of the glutaraldehyde-fixed cell (Figure 3A).
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When frozen sections of the mouse anterior pituitary glands fixed with 3% paraformaldehyde for 2 hr were immunostained with anti-lamin A/C antibody, no significant immunofluorescence was detected in the cells (Figures 4A4C). Next, when frozen sections of the pituitary glands fixed with 3% paraformaldehyde for 10 min were immunostained for lamin A/C, the nuclear margin of the pituitary cells was slightly immunopositive (Figures 4D4F). Finally, when frozen sections of the unfixed pituitary glands were fixed with pure acetone for 10 min, the strong immunostaining for lamin A/C was detected at the periphery of almost all the nuclei in the tissue (Figures 4G4I). Moreover, immunopositive dots were observed in the nuclei of some cells. In the control sections of the three kinds of samples, which were incubated with normal rabbit IgG, no fluorescence against the rabbit serum was visible (Figures 4J4L).
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Discussion |
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The question of why the quick-freeze freeze-substitution technique is effective for the ultrastructural and immunocytochemical preservation of the nuclear lamina is an intriguing issue to be addressed. One possible reason is that exposure to aldehyde fixatives could be avoided or minimized in the quick-freeze freeze-substitution procedures. No aldehyde fixative was contained in the solutions used in the process for quick-freeze freeze-substitution electron microscopy. In immunoelectron microscopy of the quickly frozen samples, only 0.3% glutaraldehyde was contained in acetone as a fixative. This concentration of glutaraldehyde seemed to be enough for preservation of the subcellular constituents of the quickly frozen cells (Figure 5B) although it could not maintain the fine structure of the anterior pituitary cells by the conventional fixation (data not shown).
Aldehyde fixatives are not likely to ruin the ultrastructure of the nuclear lamina because quick-freeze deep-etch electron microscopy showed no significant difference in the appearance of the nuclear lamina-corresponding layer between fresh unfixed cells and glutaraldehyde-fixed ones (see Figure 3). Also, in previous investigations, the nuclear lamina fixed with glutaraldehyde-containing fixatives was successfully visualized by electron microscopy (Fawcett 1966; Aebi et al. 1986
; Ris 1997
). The nuclear porenuclear lamina complexes in Necturus were observed using cryo-electron microscopy of unfixed and frozen specimens (Akey 1989
). A meshwork of the lamina observed by this technique without fixation was fundamentally identical to that with aldehyde fixation.
In the immunofluorescence study, it was found that the immunoreactivities for lamin A/C were higher in the tissues less exposed to paraformaldehyde (see Figures 4A4F). Furthermore, in the tissues fixed only with acetone, most of the nuclei showed a strong immunoreactivity for lamin A/C (see Figures 4G4I). Minimization of the aldehyde fixation definitely has a beneficial effect on the immunodetection of lamin A/C. Consistently, the quick-freeze freeze-substituted samples with minimum aldehyde fixation (0.3% glutaraldehyde) showed intense labeling of the gold particles for lamin A/C (see Figure 5B). However, in some previous reports, lamins in the paraformaldehyde-fixed tissues could be detected by immunoelectron microscopy (Stick et al. 1988; Hozák et al. 1995
; Goldman et al. 1998
). Diversity of the lamin antibodies employed and the cell types investigated might provide different results.
Another possible reason why quick-freeze freeze-substitution could provide good preservation of the nuclear lamina is that quick freezing might not allow nuclear materials to move around. Though immobilization of antigens is one of the purposes of fixation, it cannot always be fulfilled. The usual chemical fixation by perfusion of and/or immersion in chemical fixatives requires a certain amount of time for them to penetrate into the tissues and cells. Thus, cellular components may move around by the time chemical fixation is completed. In the present study, during chemical fixation, some extrinsic components might have moved into the nuclear lamina layer and have been stained with heavy metals, leading to the obscuring of the proper lamina structure. Quick freezing of the biological samples using liquid helium can complete fixation at millisecond order (Fernández-Morán 1960; Heuser and Reese 1976
). This physical fixation technique would never allow the wandering of cellular constituents. Therefore, the homogeneous electron-lucent layer with less electron-dense amorphous materials must be the real in situ state of the nuclear lamina. In other words, at least in mouse anterior pituitary cells, the nuclear lamina can be preserved ultrastructurally by quick-freezing fixation and visualized as naturally as it exists in situ by quick-freeze freeze-substitution electron microscopy.
The cells located near the slammed surface were less stained with both toluidine blue in semi-thin sections (see Figure 1A) and uranium/copper in thin sections (see Figure 1B). This superficial layer within 15 µm from the surface was ultrastructurally well preserved (see Figure 2B). The layer was surely so quickly frozen (frozen in vitrification) that it would be expected to consist homogeneously of tissue components in in situ nature. On the contrary, in the tissue located deeper than the superficial layer, numerous ice crystals formed by insufficient quick freezing should push the tissue components away and press them among the ice crystals. Such aggregated tissue components would be stained intensely while the regions where the ice crystals occupied would not (see Figure 2D). The ice-crystal formation by insufficient freezing thus causes heterogeneity in both toluidine blue and uranium/copper staining. As a result, the deeper tissue was well stained, and the superficial layer was relatively less stained.
The nuclear lamina of mouse anterior pituitary cells was found in this study to be 1020-nm thick. Most of the cells that were observed also had a 1020-nm-thick nuclear lamina. However, some cell types contain a thicker than 50-nm nuclear lamina (Fawcett 1966). As the nuclear lamina is a meshwork of intermediate-type filaments (Aebi et al. 1986
), a thicker lamina may contain many layers of the filaments.
Even though lamin A/C was localized to the nuclear periphery, it was also found in the inner nucleoplasm of the mouse anterior pituitary cells. This was also found during the G1 and S phases of the cell cycle or in some pathological conditions (Moir et al. 1994; Hozák et al. 1995
; Machiels et al. 1995
). In HeLa cells, lamin A was found to form part of the nucleoskeleton that ramifies throughout the nucleoplasm (Hozák et al. 1995
). This possibility was also suggested in human erythroleukemia cells (Neri et al. 1999
). In fact, we have noticed the occurrence of <10-nm strands in the nucleoplasm of the anterior pituitary cells, which seemed to be associated with chromatin loops (Senda and Umemoto 1998
).
The nuclear lamins were initially confined to the nuclear lamina just inside the inner nuclear membrane and thus provided a framework for nuclear envelope organization and an anchoring site for interphase chromatin (Gerace et al. 1978; Hancock 1982
; Lebkowski and Laemmli 1982
). Increasing evidence from recent investigations suggests that nuclear lamins are involved in other functions, such as DNA synthesis, transcription, and even apoptosis (Goldman et al. 2002
). Wider distribution of nuclear lamins other than in the nuclear lamina layer may reflect a multiplicity of their function.
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Acknowledgments |
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Footnotes |
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