Visualization of the Nuclear Lamina in Mouse Anterior Pituitary Cells and Immunocytochemical Detection of Lamin A/C by Quick-freeze Freeze-substitution Electron Microscopy

Abstract

We examined the nuclear lamina in the quickly frozen anterior pituitary cells by electron microscopic techniques combined with freeze substitution, deep etching, and immunocytochemistry and compared it with that in the chemically fixed cells. By quick-freeze freeze-substitution electron microscopy, an electron-lucent layer, as thick as 20 nm, was revealed just inside the inner nuclear membrane, whereas in the conventionally glutaraldehyde-fixed cells the layer was not seen. By quick-freeze deep-etch electron microscopy, we could not distinguish definitively the layer corresponding to the nuclear lamina in either fresh unfixed or glutaraldehyde-fixed cells. Immunofluorescence microscopy showed that lamin A/C in the nucleus was detected in the acetone-fixed cells and briefly in paraformaldehyde-fixed cells but not in the cells with prolonged paraformaldehyde fixation. Nuclear localization of lamin A/C was revealed by immunogold electron microscopy also in the quickly frozen and freeze-substituted cells, but not in the paraformaldehyde-fixed cells. Lamin A/C was localized mainly in the peripheral nucleoplasm within 60 nm from the inner nuclear membrane, which corresponded to the nuclear lamina. These results suggest that the nuclear lamina can be preserved both ultrastructurally and immunocytochemically by quick-freezing fixation, rather than by conventional chemical fixation.

Keywords

nuclear lamina lamin A/C quick freezing freeze substitution deep etching anterior pituitary cell immunogold electron microscopy

The nuclear lamina is a fibrous layer localized at the nucleoplasmic surface of the inner nuclear membrane (Fawcett 1966; Gerace et al. 1978; Krohne and Benavente 1986). The major components of the nuclear lamina are three lamins (A, B, and C), which make up a filamentous meshwork just beneath the inner nuclear membrane (Gerace et al. 1978; Krohne and Benavente 1986; Stuurman et al. 1998; Goldman et al. 2002). The biochemical properties in addition to the close association of the lamina to the inner nuclear membrane suggest that the nuclear lamina provides mechanical support for the nuclear envelope (Aaronson and Blobel 1975; Aebi et al. 1986; Fisher et al. 1986; Goldman et al. 1986; McKeon et al. 1986; Moir et al. 1995). Also, involvement of the nuclear lamina in the anchorage and organization of chromatin has been postulated (Coggeshall and Fawcett 1964; Paddy et al. 1990).

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.

Materials and Methods

Animals

Male ICR mice aged 8-10 weeks were used in this study according to the guidelines of Fujita Health University on the use and care of laboratory animals. The animals were kept in air-conditioned animal quarters and given food and water ad libitum.

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 nonreactive 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 Alexa-Fluor 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 nonimmune 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.

Results

Visualization of the Nuclear Lamina by Quick-freeze Freeze-substitution Electron Microscopy

Unfixed mice anterior pituitary glands were quickly frozen, processed for freeze substitution, and finally embedded in epoxy resin. Semi-thin sections were cut perpendicular to the slammed surface, stained with toluidine blue, and observed with a light microscope (Figure 1A). At the light microscopic level, it was judged that the whole pituitary tissue and each pituitary cell were both well preserved. The pituitary cells located near the slammed surface seemed to be less stained with toluidine blue.

Thin sections cut perpendicular to the surface were stained with uranyl acetate and lead citrate and observed under an electron microscope. At lower magnification, each pituitary cell exhibited the natural cell contour and normal profiles of the intracellular cytoorganelles (Figure 1B), as compared with those in conventionally glutaraldehyde-fixed anterior pituitary cells (data not shown). The pituitary cells located near the slammed surface were less electron dense.

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 10-20 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.

In the second cell layer (between 15 and 30 μm from the surface), the electron-lucent layer was not continuous and was often interrupted by the heterochromatin (Figure 2C). Outer and inner nuclear membranes and other cytoorganelle membranes did not clearly show the trilamellar structure of the membrane lipid bilayer. Therefore, the subcellular ultrastructure in this cell layer appeared not to be well preserved. In the third cell layer (between 30 and 45 μm from the surface), most of the membranes, including the nuclear membranes, were not identified, and all other cellular components were severely distorted and destroyed due to obvious formation of ice crystals (Figure 2D). The nuclear lamina corresponding layer could not be recognized at all under the nuclear envelope.

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).

Figure 1

Light (A) and electron (B) microphotographs of quickly frozen and freeze-substituted mouse anterior pituitary tissues. (A) Semi-thin sections were cut perpendicular to the slammed surface (arrows) and stained with toluidine blue. The whole pituitary tissue and each pituitary cell were both well preserved. The pituitary cells located near the surface seemed to be less stained with toluidine blue. (B) Ultrathin sections were cut perpendicular to the slammed surface (arrows) and examined with the electron microscope at low magnification. Each pituitary cell exhibited the natural cell contour and normal profiles of the intracellular cytoorganelles. The pituitary cells located near the slammed surface were less electron dense as compared with those in the deeper region. Bar: A = 50 μm; B = 2 μm.

Detection of Lamin A/C by Immunofluorescence Microscopy

Because lamin proteins have been shown to be major components of the nuclear lamina (Gerace et al. 1978; Krohne and Benavente 1986; Stuurman et al. 1998; Goldman et al. 2002), we next performed immunohistochemical staining for lamin A/C to assess preservative condition of the nuclear lamina.

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 4A-4C). 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 4D-4F). 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 4G-4I). 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 4J-4L).

Figure 2

Higher magnification view of the cytoplasm-nucleus (N) border region in a conventionally glutaraldehyde-fixed anterior pituitary cell (A) and quickly frozen and freeze-substituted cells (B-D). (A) In the glutaraldehyde-fixed cell, there did not seem to be a gap between the inner nuclear membrane and heterochromatin. (B) In the freeze-substituted cells located in the first cell layer (within 15 μm from the surface), an electron-lucent layer existed as wide as 10-20 nm just inside the inner nuclear membrane (asterisks). (C) In the second cell layer (between 15 and 30 μm from the surface), the electron-lucent layer was not continuous and was often interrupted by the heterochromatin. (D) In the third cell layer (between 30 and 45 μm from the surface), the electron-lucent layer was not recognized at all under the nuclear envelope. Bar = 50 nm.

Detection of Lamin A/C by Immunoelectron Microscopy

To assess whether the nuclear lamina is well preserved in the quickly frozen and freeze-substituted anterior pituitary cells, we next employed immunoelectron microscopy using colloidal gold-conjugated secondary antibody. In the conventionally paraformaldehyde-fixed anterior pituitary cells, the gold particles for lamin A/C were hardly seen around the nucleus (Figure 5A). On the other hand, in the quick-freeze freeze-substituted cells, most of the nuclei were heavily labeled with gold particles for lamin A/C (Figure 5B). The gold particles were frequently localized just inside the nuclear envelope, where an electron-lucent thin layer was discernible even in Lowicryl ultrathin sections. In addition to gold particles along the nuclear membrane, they were also found deeper in the nucleoplasm. In order to determine their tendency for distribution, the distance between each gold particle and the inner nuclear membrane was measured and summarized in a histogram (Figure 6). The peak of the gold particle number was just beneath the inner nuclear membrane within 60 nm from it. Moreover, a considerable number of gold particles were scattered deeper in the nucleoplasm. No gold particle was seen in the sections of the quickly frozen and freeze-substituted tissues incubated with non-immune rabbit serum (Figure 5C). This result confirmed that the electron-lucent layer just under the inner nuclear membrane revealed by quick-freeze freeze-substitution electron microscopy (Figure 2B) was the nuclear lamina where lamin A/C was concentrated.

Figure 3

Observation of the nuclear architecture in glutaraldehyde-fixed (A) and fresh unfixed (B) anterior pituitary cells by quick-freeze deep-etch electron microscopy. The nucleoplasm (N) of both kinds of cells was filled with a large amount of globular material of various sizes and with some fibrous components (arrows) among them. In neither glutaraldehyde-fixed (A) nor fresh unfixed (B) cells, the layer corresponding to the nuclear lamina could be distinguished. Asterisks indicate nuclear pores. Bar = 100 nm.

Discussion

In the present study, a distinct electron-lucent layer as thick as 10-20 nm was visualized just inside the inner nuclear membrane of the anterior pituitary cells processed by the quick-freeze freeze-substitution technique followed by electron microscopic observation. Because the cells fixed by perfusion and immersion with glutaraldehyde never exhibited such a layer, the quick-freeze freeze-substitution procedure was certain to be effective for visualization of this specialized layer, which was later confirmed to be the nuclear lamina with the results from the immunoelectron microscopy for lamin A/C. Furthermore, we could detect lamin A/C, the major components of the nuclear lamina, only in the quick-freeze freeze-substituted samples, but we failed to detect it in conventionally paraformaldehyde-fixed samples by immunoelectron microscopy.

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).

Figure 4

(A-L) Immunofluorescence microscopy for lamin A/C in the mouse anterior pituitary glands. The nucleus was stained with TOPRO-3. (A-C) No immunofluorescence for lamin A/C was detected in the pituitary cells fixed with 3% paraformaldehyde for 2 hr. (D-F) The nuclear margin of the pituitary cells fixed with 3% paraformaldehyde for 10 min was slightly immunopositive for lamin A/C. (G-I) In the sections fixed with pure acetone for 10 min, intense immunostaining for lamin A/C was detected at the nuclear margin of the pituitary cells. In addition, immunopositive dots were observed in the nuclei. (J-L) In the control sections of these three kinds of samples that were incubated with normal rabbit IgG, no fluorescence was detected. Bar = 10 μm.

Figure 5

Immunoelectron microscopy for lamin A/C in conventionally paraformaldehyde-fixed (A) and quickly frozen and freeze-substituted (B,C) mouse anterior pituitary cells. (A) Gold particles were barely seen in the nucleus (N) of the paraformaldehyde-fixed cells. (B) In the quickly frozen and freeze-substituted cells, the nucleus (N) was heavily labeled with gold particles for lamin A/C. The gold particles were frequently localized just inside the nuclear envelope (arrowheads), where an electron-lucent thin layer was discernible even in Lowicryl ultrathin sections. Gold particles were also found deeper in the nucleoplasm. (C) In non-immune rabbit serum-incubated control sections of the quickly frozen and freeze-substituted cells, no gold particle was seen. Arrows in A-C indicate the outer and inner nuclear membranes. Bar = 100 nm.

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 pore-nuclear lamina complexes in Necturus were observed using cryoelectron 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.

Figure 6

A histogram showing the number of gold particles for lamin A/C and their distance from the inner nuclear membrane in the quick-freeze-substituted cells.

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 4A-4F). Furthermore, in the tissues fixed only with acetone, most of the nuclei showed a strong immunoreactivity for lamin A/C (see Figures 4G-4I). 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 10-20-nm thick. Most of the cells that were observed also had a 10-20-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.

Footnotes

Acknowledgments

We thank Emiko Kodera for technical assistance in electron microscopy.

References

Aaronson

Blobel

(1975) Isolation of nuclear pore complexes in association with a lamina. Proc Natl Acad Sci USA 72: 1007–1011

Aebi

Cohn

Buhle

Gerace

(1986) The nuclear lamina is a meshwork of intermediate type filaments. Nature 323: 560–564

Akey

(1989) Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy. J Cell Biol 109: 955–970

Beams

Tahmisian

Devine

Anderson

(1957) Ultrastructure of the nuclear membrane of a gregarine parasitic in grasshoppers. Exp Cell Res 13: 200–204

Coggeshall

Fawcett

(1964) The fine structure of the central nervous system of the leech Hirudo Medicinalis . J Neurophysiol 27: 229–289

Dwyer

Blobel

(1976) A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J Cell Biol 70: 581–591

Ely

D'Arcy

Jost

(1978) Interaction of antibodies against nuclear envelope-associated proteins from rat liver nuclei with rodent and human cells. Exp Cell Res 116: 325–331

Fawcett

(1966) On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates. Am J Anat 119: 129–146

Fernández-Morán

(1960) Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid helium II. Ann NY Acad Sci 85: 689–713

10.

Fisher

Chaudhary

Blobel

(1986) cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci USA 83: 6450–6454

11.

Gerace

Blum

Blobel

(1978) Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution. J Cell Biol 79: 546–566

12.

Goldman

Maul

Steinert

Yang

Goldman

(1986) Keratin-like proteins that coisolate with intermediate filaments of BHK-21 cells are nuclear lamins. Proc Natl Acad Sci USA 83: 3839–3843

13.

Goldman

Baccetti

Collodel

Gambera

Moretti

Piomboni

(1998) Localization of lamins in mammalian spermatozoa. J Submicrosc Cytol Pathol 30: 573–580

14.

Goldman

Gruenbaum

Moir

Shumaker

Spann

(2002) Nuclear lamins: building blocks of nuclear architecture. Genes Dev 16: 533–547

15.

Gray

Guillery

(1963) On nuclear structure in the ventral nerve cord of the leech, Hirudo medicinalis. Z Zellforsch Mikrosk Anat 59: 738–745

16.

Hancock

(1982) Topological organization of interphase DNA: the nuclear matrix and other skeletal structures. Biol Cell 46: 105–122

17.

Heuser

Reese

(1976) Freeze-substitution applied to the study of quick-frozen synapses. J Cell Biol 70:357a

18.

Heuser

Reese

Dennis

Jan

Evans

(1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol 81: 275–300

19.

Hirokawa

Kirino

(1980) An ultrastructural study of nerve and glial cells by freeze-substitution. J Neurocytol 9: 2243–2254

20.

Hirokawa

Heuser

(1981) Structural evidence that Botulinum toxin blocks neuromuscular transmission by impairing the calcium influx that normally accompanies nerve depolarization. J Cell Biol 88: 160–171

21.

Hozák

Sasseville

Raymond

Cook

(1995) Lamin proteins form an internal nucleoskeleton as well as a peripheral lamina in human cells. J Cell Sci 108: 635–644

22.

Krohne

Franke

Ely

D'Arcy

Jost

(1978) Localization of a nuclear envelope-associated protein by indirect immunofluorescence microscopy using antibodies against a major polypeptide from rat liver fractions enriched in nuclear envelope-associated material. Cytobiologie 18: 22–38

23.

Krohne

Benavente

(1986) The nuclear lamins. A multigene family of proteins in evolution and differentiation. Exp Cell Res 162: 1–10

24.

Lebkowski

Laemmli

(1982) Non-histone proteins and long-range organization of HeLa interphase DNA. J Mol Biol 156: 325–344

25.

Machiels

Broers

JLV

Raymond

de Lay

Kuijpers

HJH

Caberg

NEH

Ramaekers

FCS

(1995) Abnormal A-type lamin organization in a human lung carcinoma cell line. Eur J Cell Biol 67: 328–335

26.

McKeon

Kirschner

Caput

(1986) Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature 319: 463–468

27.

Mercer

(1959) An electron microscopic study of Amoeba proteus. Proc R Soc B 150: 216–232

28.

Moir

Montag-Lowy

Goldman

(1994) Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication. J Cell Biol 125: 1201–1212

29.

Moir

Spann

Goldman

(1995) The dynamic properties and possible functions of nuclear lamins. Int Rev Cytol 162B: 141–182

30.

Neri

Raymond

Giordano

Capitani

Martelli

(1999) Lamin A is part of the internal nucleoskeleton of human erythroleukemia cells. J Cell Physiol 178: 284–295

31.

Paddy

Belmont

Saumweber

Agard

Sedat

(1990) Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery. Cell 62: 89–106

32.

Pappas

(1956) The fine structure of the nuclear envelope of Amoeba proteus. J Biophys Biochem Cytol 2(suppl):431–434

33.

Ris

(1997) High-resolution field-emission scanning electron microscopy of nuclear pore complex. Scanning 19: 368–375

34.

Senda

(1998) Cytoskeletal organization in the neurohypophysial axons of rats. Med Electron Microsc 31: 94–99

35.

Senda

Fujita

(1987) Ultrastructural aspects of quick-freezing deep-etching replica images of the cytoskeletal system in anterior pituitary secretory cells of rats and mice. Arch Histol Jpn 50: 49–60

36.

Senda

Nishii

Fujita

(1991) Immunocytochemical localization of synapsin I in the adrenal medulla of rats. Histochemistry 96: 25–30

37.

Senda

Okabe

Matsuda

Fujita

(1994) Quick-freeze, deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II. Cell Tissue Res 277: 51–60

38.

Senda

Umemoto

(1998) Electron microscopic demonstration of chromatin-nuclear matrix association in the nuclei of Triton-W-extracted rat anterior pituitary cells. Biomed Res 19: 83–91

39.

Senda

Yoshinaga-Hirabayashi

(1998) Intermembrane bridges within membrane organelles revealed by quick-freeze deep-etch electron microscopy. Anat Rec 251: 339–345

40.

Stick

Angres

Lehner

Nigg

(1988) The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum. J Cell Biol 107: 397–406

41.

Stuurman

Heins

Aebi

(1998) Nuclear lamins: their structure, assembly, and interactions. J Struct Biol 122: 42–66