Three-dimensional Organization of pKi-67 : A Comparative Fluorescence and Electron Tomography Study Using Fluoronanogold
Unité MéDian, CNRS UMR 6142, UFR de Pharmacie (TC,M-FO,DP), Reims, France; DTI, UMR 6107, UFR de Sciences (AB), Reims, France; Service Commun d'Imagerie Cellulaire et de Cytométrie, INSERM IFR58, Institut Biomédical des Cordeliers, Paris, France (CK); and IFR53, Reims, France (HK)
Correspondence to: Dominique Ploton, Unité MéDian, CNRS UMR 6142, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France. E-mail: dominique.ploton{at}univ-reims.fr
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Summary |
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Key Words: confocal microscopy electron tomography heterochromatin Ki-67 antigen nucleolus
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Introduction |
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As revealed by Western blotting, pKi-67 is a large protein consisting of two main variants. These isoforms (with theoretical molecular masses of 320 and 359 kD) are obtained by alternative splicing of a mRNA precursor encoded by a unique gene (Gerdes et al. 1991; Duchrow et al. 1994
). Analysis of the pKi-67 primary sequence has not revealed any significant homology to other known sequences. However, several putative nuclear targeting sequences have been identified, as well as more than a hundred potential phosphorylation sites (Schlüter et al. 1993
). In addition, several striking features have been determined. Both variants of the protein contain sixteen repetitive elements ("Ki repeats"), each of which includes a 66-bp motif, the Ki motif, which is highly conserved (Schlüter et al. 1993
). Moreover, a forkhead-associated (FHA) domain has been found in the N-terminal portion of pKi-67 (Sueishi et al. 2000
). This domain, believed to be a modular phosphopeptide recognition motif that might mediate proteinprotein interactions (Henckel et al. 1999
; Li et al. 2000
), is shared by several proteins involved in cell cycle regulation (Hofmann and Bucher 1995
). This finding can be related to previous data, which revealed the role played by pKi-67 in cell cycle progression. Indeed, it has been reported that Ki-67 specific antisense oligonucleotides prevent incorporation of [3H]-thymidine (Schlüter et al. 1993
) and that microinjection of antibodies directed against the murine homologue of pKi-67 delays cell cycle progression (Starborg et al. 1996
).
Many data suggest that pKi-67 might be involved in the organization of chromatin higher-order structure (Takagi et al. 1999; MacCallum and Hall 2000
). This hypothesis is indirectly supported by other evidence. Ki-67 immunolabeling disappears after digestion with DNase I but not after RNase treatment (Sasaki et al. 1987
). Moreover, Ki-67 antibodies display a stronger affinity when pKi-67 is bound to DNA (Lopez et al. 1994
). In addition, an increase of pKi-67 follows the increase of DNA during S-phase, whereas the global protein content decreases. Finally, recent biochemical data obtained by subcellular fractionation have confirmed that pKi-67 is a chromatin-associated protein, which probably resides in densely packed regions such as heterochromatin (Kreitz et al. 2000
). Although many data support an involvement of pKi-67 in chromatin organization, some contradictory studies have localized pKi-67 mainly within the nucleolus, in close association with the nucleolar components that are directly involved in rRNA elongation and maturation (Verheijen et al. 1989
; Kill 1996
; MacCallum and Hall 2000
) or in association with a new nucleolar protein (Takagi et al. 2001
). Because most morphological studies published thus far were mostly bi-dimensional, they only partially revealed the complex distribution of pKi-67 and may have led to ambiguous interpretations. In addition, electron and optical microscopy data are very difficult to compare because they are usually obtained with different labeling protocols.
In this present study we used an electron-dense probe linked to a fluorescent dye, FluoroNanogold (FNG) (Robinson et al. 2000), to examine the precise 3D organization of pKi-67 during interphase at the optical and electronic levels. After acquiring a series of optical sections by confocal microscopy or collecting projections at different angles with a scanning and transmission electron microscope (STEM), volume reconstructions and tomographic analyses were performed (Beorchia et al. 1992
; Lucas et al. 1996
; Héliot et al. 1997
; Perkins et al. 1997
; Baumeister et al. 1999
; Cheutin et al. 2002
; Frank et al. 2002
). The results reveal both the fine localization and the different levels of organization of pKi-67. pKi-67 co-localizes with certain heterochromatin domains at the periphery of nucleoli, which strongly suggests that this protein is involved in the folding of perinucleolar chromatin.
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Materials and Methods |
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Immunocytochemistry
Cells were simultaneously fixed and permeabilized for 4 min in 3% paraformaldehyde and 1% Triton X-100 diluted in PBS (140 mM NaCl, 6 mM Na2HPO4, 4 mM KH2PO4, pH 7.2) (Sigma). Then they were saturated for 30 min in PBS containing 3% bovine serum albumin (BSA), 1 mM CaCl2, and 0.5 mM MgCl2. Slides were incubated for 30 minutes in the presence of MM1 antibodies against human pKi-67 (Novocastra; LePerray en Yvelines, France) diluted 1:50 in PBS containing 1% BSA, 1 mM CaCl2, and 0.5 mM MgCl2, and rinsed three times for 5 min in PBS. A goat anti-mouse, biotinylated antibody (Jackson ImmunoResearch; Avondale, PA) was applied for 30 min and revealed for 15 minutes with either a streptavidinFluoroNanogold (diluted 1:20) consisting of streptavidin conjugated both to a fluorescein (FITC) molecule and a 1.4 nm Nanogold particle (Nanoprobes; Yaphank, NY) (Cheutin et al. 2002), or a streptavidin-Texas Red conjugate (diluted 1:50) (Amersham Biosciences; Saclay, France). Staining of DNA was performed for 5 min at room temperature in the presence of 100 µM chromomycin A3 diluted in PBS containing 150 mM MgCl2. For confocal microscopy studies, cells on coverslips were mounted in Citifluor AF1 (Agar Scientific; Saclay, France). For electron microscopy studies, cells immunolabeled with FluoroNanogold were over-fixed for 12 min with 1.6% glutaraldehyde in PBS. After rinsing in de-ionized water, HQ silver enhancement (Nanoprobes) was performed for 8 min. Finally, cells were harvested by scraping, dehydrated in graded alcohols, and embedded in Epikotte 812.
Confocal Microscopy
Observations were made with a Bio-Rad MRC 600 System (Bio-Rad; Hercules, CA), mounted on an Axioplan optical microscope (Carl Zeiss; Oberkochen, Germany), using a planapochromat x63, 1.4 numerical aperture oil immersion objective. For FITC coupled to FluoroNanogold, the 488-nm line of an argon ion laser was used to perform optical sections. Depending on the cell thickness, 2030 sections per cell were recorded with a 0.2 µm z step. For double labeling experiments, dual channel acquisitions were performed by alternatively exciting chromomycin A3 with the 457-nm line of an air-cooled argon ion laser and Texas Red with the 543-nm line of an HeNe laser. To avoid misalignment caused by block filter exchange, a special filter set (DC 570, high-pass 560) was used. This enabled the simultaneous excitation and collection of fluorescence from both Texas Red and chromomycin A3. An accurate picture of the co-localization pattern was obtained by using the Bio-Rad 1024ES software. In this procedure, the intensity in the green channel was plotted against that of the red one for each pixel of both images. The scatterplot thus obtained was used to select the pixels that display the highest levels of both green and red. These pixels, which correspond to a significant labeling of both species, were shown in white relative to all the other pixels.
Electron Microscopy
For classical studies, ultrathin sections (80 nm) were counterstained with lead citrate and uranyl acetate. For the specific staining of DNA, ultrathin sections were mounted on gold grids and stained with the Feulgen-like osmium-ammine reaction as earlier described (Derenzini et al. 1982). All ultrathin sections were observed with a 200 CX electron microscope at 100 kV (JEOL). Sections 0.5 µm or 2 µm in thickness were observed in a medium voltage CM30 electron microscope working at 250 kV in the STEM mode (Philips), as described previously (Beorchia et al. 1992
; Héliot et al. 1997
; Cheutin et al. 2002
). Before starting a tilt series, the whole-mounted cells were irradiated for 10 min at 100 e-/(Å2 x sec). After correcting the axis alignment of the eucentric goniometer stage, each tilt series was collected from -60° to +60° with a pitch of 2°. Images were directly recorded on a disk-type scintillatorphotomultiplier detector system and digitized on line using Orion hardware (ELI; Brussels, Belgium), working on a PC. Images (512 x 512 pixels) with a high signal-to-noise ratio were collected using low-speed scanning, in contrast to the focus, which was realized at high-speed scanning.
Three-dimensional Reconstructions
Files corresponding to z series acquired by confocal microscopy were transferred to a Sun Sparc20 workstation (Sun Microsystems; Tucson, AZ) for processing, which was performed using the Analyze software (CNSoftware; Southwater, UK) (Klein et al. 1998). Volumes were re-sampled to have an identical pixel size in x, y, and z directions and a (3 x 3 x 3)-cubic median filter was applied to decrease the noise within images. For projections acquired by STEM, the contrast of digital images was reversed. Image alignment was achieved by translating images in x and y directions relative to a fiducial marker, using a sinogram technique (Bahr et al. 1979
). Due to the parallel imaging system of the STEM, image translation had no consequence on the 3D reconstruction. Next, the 512 x 512 images were converted to 256 x 256 ones, and tomographic reconstruction was performed by using an extended, field-additive algorithmic reconstruction technique on lined-up images, as described (Crowther et al. 1970
; Gordon et al. 1970
). For each slice of the volume, seven iterations were computed on a Sun Sparc20 workstation.
Three-dimensional Visualization
The same three-dimensional visualization tools were applied to volumes obtained by confocal microscopy or STEM studies. Two visualization processes were used, both based on ray-tracing methods. The first method allows the ray to pass through the whole volume, and the resulting pixel reflects the intensity of the most intense voxel encountered by the ray. By scanning all the volume with the ray, a projection is obtained that allows a volume visualization accounting for differences in signal intensity. This type of 3D visualization was performed with the Analyze Software package. In the second method, the ray is reflected when it meets a voxel that has an intensity superior to a threshold. In contrast to the first method, it leads to a surface visualization that allows a detailed analysis of the surface of the labeling. In this case, Visuvoxel (Lucas et al. 1996), a software package developed in our laboratory, was used on a Sun Sparc20 workstation.
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Results |
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Ultrastructural Localization of pKi-67
The ultrastructural localization of pKi-67 was then investigated. Highly sensitive immunolocalization of pKi-67 was performed before embedding by revealing the primary antibodies with FluoroNanogold complexes further enhanced with silver (Cheutin et al. 2002). Counterstained ultrathin sections allowed clear identification of typical nucleolar components, such as the fibrillar centers (FCs) surrounded by the dense fibrillar component (DFC) (circles associate both components) and the granular component (GC) (Figure 4B). In the nucleolus, FCs, DFC, and GC were almost totally devoid of pKi-67. Most of the labeling was found at the periphery of the nucleoli, where the perinucleolar condensed chromatin is located. Most silvergold particles were organized in small clusters, which in some places seemed to form short threads
50 nm in diameter (arrows and parallel lines). The nucleoplasmic labeling was sparse and was always found in close association with chromatin.
STEM Analysis of pKi-67 in 2-µm-thick Sections
We further investigated the 3D organization of pKi-67 by using a STEM working at 250 kV. Thick sections (2 µm) containing pKi-67-immunolabeled cells before embedding were observed to perform electron tomography on large subvolumes of the nucleolus. In Figures 5A5E
, five projections from a tilt series show the nucleolus from a transverse view (-40°) to a front view (+40°), indicating that its whole thickness is visualized during tilting. Clearly, the nucleolar peripheral regions are more intensely stained and show the presence of a cord 250300 nm in diameter (bracketed by arrowheads, Figures 5A5D). Some labeled areas were in close proximity to the nuclear envelope (arrows, Figure 5E). A weak perinuclear labeling corresponding to small nucleoplasmic heaps was also observed. The combination of all the projections of a tilt series allowed the generation of a digital volume (i.e., a tomogram) after alignment and tomographic reconstruction. To validate this reconstruction, a projection at 0° was calculated (Figure 5F) and compared to its initial projection (Figure 5C). To study the internal distribution of pKi-67, digital sections were performed in the tomogram (Figures 5G5I). In these virtual sections, pKi-67 formed cords (bracketed with arrowheads, Figures 5H and 5I) surrounding an unlabeled central area, as already observed in optical sections (Figure 1). The thickness of the cords was relatively constant (250300 nm) and many protrusions emanating from the cords were in direct contact with the nuclear envelope (arrows). A closer examination of these images revealed that the cords and the protrusions consisted of 3050-nm-thick fibers, (frame, Figures 5G and 5H). In some areas these fibers appeared as straight segments 500 nm in length (frame, Figure 5G), whereas in others they were twisted (frame, Figure 5H).
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To further increase our comprehension of their structural features and to tentatively individualize the goldsilver particles, two series of projections were extracted from the areas framed in Figures 6B and 6C. The corresponding tomograms were reconstructed and used to perform either projections of the whole volume at different angles (Figures 7A7E
and Figures 8A8E)
, digital sections in the (x, y) plane for the projection obtained at 0° (Figures 7F7H and Figures 8F8H), or stereopairs calculated with a surfacic visualization mode (Figures 7I and 8I). Although the protrusions in contact with the nuclear envelope (Figure 7) and the perinucleolar areas (Figure 8) were composed of fibers 3050 nm in diameter, their 3D organizations were different. In the protrusions, pKi-67 was distributed within parallel fibers, which were particularly visible at certain view angles (arrows, Figure 7B). Their orientation was perpendicular to the axis of the cord formed by the labeling. Digital sections revealed their internal organization, in which some of the fibers clearly displayed a free extremity directed towards the inner part of the nucleolus (arrows, Figure 7F7H). By using a surface visualization, the 3D disposition of these fibers was clearly visible (stereopair, Figure 7I). In contrast, the cord located in the perinucleolar area, although still consisting of 3050-nm-thick fibers, was narrower. The fibers were sometimes difficult to see because they were tangentially disposed with respect to the cord axis (semicircles, Figure 8A). They appeared more clearly on transverse sections (circles, Figures 8E8H) and on stereopairs using a surface visualization (Figure 8I). These tomographic studies showed that the fibers were homogeneously localized throughout the whole thickness of the labeling and that they were regularly spaced (
100 nm distance between fibers measured in 10 different nucleoli).
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Discussion |
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Light and electron microscopy visualization of higher-order chromatin structures is complicated by their tightly folded and coiled nature (Kornberg and Lorch 2002). However, different strategies, including cryoelectron microscopy of isolated chromatin fibers (Bednar et al. 1998
), medium-voltage electron microscopy to investigate thick sections of nuclei (Belmont et al. 1999
), and approaches based on both optical sectioning and immunoelectron microscopy (Tumbar et al. 1999
), proved to be extremely useful in this field.
In the present study the precise 3D distribution of pKi-67 and its different levels of organization have enabled us to indirectly obtain a detailed structure of a specific heterochromatin domain, as performed previously with other proteins binding these regions (Woodcock and Dimitrov 2001).
To achieve our goal, we used an electron-dense probe linked to a fluorescent dye, FluoroNanogold (FNG). By use of this probe, the same sample can be observed at different resolutions by confocal microscopy (200-nm resolution), electron tomography (10-nm resolution), and conventional electron microscopy (1-nm resolution).The use of streptavidinFNG before embedding greatly increases the sensitivity of antigen detection compared to classical methods using 510-nm gold particles on the surface of ultrathin sections (Hainfeld and Furuya 1992; Hainfeld and Powell 2000
), and reveals very precisely the location and size of the structures containing antigens without altering their organization (Robinson and Vandré 1997
; Baschong and Stierhof 1998
; Robinson et al. 2000
). This detection process preserves the nuclear structure (Humbel et al. 1998
) and more particularly the nucleolar ultrastructure, as seen on ultrathin sections in our present and previous studies (Cheutin et al. 2002
).
Previous optical studies have reported that pKi-67 is found either in the cortex of the nucleoli (Kill 1996) or at the periphery of the nucleolus (O'Donohue et al. 1998
; Endl and Gerdes 2000
). Moreover, several ultrastructural studies have localized pKi-67 in small nucleoplasmic spots, in the cortex of the nucleoli, in the granular component (GC), or in the dense fibrillar component (DFC) of the nucleolus (van Dierendonck et al. 1989
; Verheijen et al. 1989
; Isola et al. 1990
). In the present study the comparison of double labeling of pKi-67 and DNA observed by confocal microscopy and of pKi-67 observed on ultrathin sections demonstrates that pKi-67 co-localizes with perinucleolar-located DNA and, to a lesser extent, with DNA near the nuclear envelope. These results unequivocally exclude the possibility of pKi-67 localization within the nucleolar components FC, DFC, and GC, which are known as the sites for rRNA synthesis and for early and late processing, respectively (Thiry and Goessens 1996
). By using the same experimental conditions, we were recently able to localize GFP-tagged proteins within the FC and DFC (unpublished results). These latter results indicate that the absence of pKI-67 in these nucleolar components is due to the absence of pKi-67 and not to a problem of penetration of reagents.
By using electron tomography on the same specimens, i.e., tilting of thick sections, volume reconstruction, virtual sectioning, and 3D visualization, our study clearly revealed two levels of organization of pKi-67, i.e., 3050-nm-thick fibers organized as a cord 250300 nm in diameter. These fibers seem to be polarized because the side of the cord directed toward the nucleolus is less densely organized than the nucleoplasmic side. This polarization is also suggested by the presence of fibers with free extremities in the inner part of the cord and by their parallel alignment in some places, which correlates with various levels of compaction within the cord itself.
These data strongly suggest that pKi-67 is involved in the higher-order organization of perinucleolar chromatin. Indeed, the 3050-nm thick fibers are reminiscent of the first level of chromatin packaging, in which adjacent nucleosomes fold into a 30-nm structure that has been described both as a solenoid (Belmont et al. 1999) and as a zigzag conformation (Bednar et al. 1998
). On the other hand, the 250300-nm cord is similar to a level of chromatin folding found in both late-replicating heterochromatic homogeneously staining regions (HSRs), which are several hundreds of mega-base pairs in size (Belmont et al. 1999
), and in living cells (Sadoni et al. 2001
).
In addition, we found some fibers in close proximity to the nuclear envelope, suggesting an intimate association with pKi-67, which is also supported by structural data. Indeed, the Ki-67 consensus motif is believed to adopt an -helical structure with a typical amphophilic arrangement, which may favor its association with membranes such as the nuclear envelope (Duchrow et al. 1994
; Kourmouli et al. 2000
). To specify the link between pKi-67 and HP1, Scholzen et al. (2002)
have shown that overexpression of HP1 leads to a relocation of endogenous pKi-67 from the periphery of the nucleolus to sites containing HP1. However, this had no consequence on the nucleolar organization. Interestingly, this result confirms that pKi-67 is not directly involved in the synthesis and processing of rRNAs, because G0 cells do not express pKi-67 although the nucleoli are active (Bridger et al. 1998
; Endl and Gerdes 2000
). However, when cells re-engaged within the cell cycle, nucleoli and heterochromatin undergo a complete reorganization (Derenzini et al. 1990
), which is concomitant with the reactivation of pKi-67 expression. In addition, it has been demonstrated that in S and G2 cells pKi-67 co-localizes only with centromeres of acrocentric chromosomes, i.e., chromosomes bearing potentially active rRNA genes segregated in the nucleolar organizer regions (NORs) and actively transcribed within the nucleolus after mitosis (Bridger et al. 1998
). Taken together, these findings suggest that pKi-67 may be involved in the organization of heterochromatin surrounding rRNA genes that are actively transcribed within the nucleolus after G0 exit (Lopez et al. 1991
; Landberg and Roos 1993
), or more generally when the cell is cycling (Endl and Gerdes 2000
; Traut et al. 2002
).
Here we demonstrated that by using the same marker, FluoroNanogold, it is possible to perform a comparative 3D confocal and electron tomography study of a very abundant nuclear protein such as pKi-67. This work confirmed the usefulness of such an approach to study the volume organization of the nucleus and more particularly of the nucleolus (Cheutin et al. 2002). Finally, preliminary studies (unpublished results) demonstrated that electron tomography of GFP-tagged nucleolar proteins is possible and could be correlated to spatiotemporal studies of proteins in living cells.
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Acknowledgments |
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We are grateful to Dr Tom Misteli and Dr Mike O'Donohue for their critical reading of the manuscript.
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Footnotes |
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Received for publication March 3, 2003; accepted June 12, 2003
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