1 Unité MéDIAN, CNRS UMR 6142, UFR de Pharmacie, 51 rue
Cognacq-Jay, 51096 Reims Cedex, France
2 DTI, UMR 6107, UFR de Sciences, BP 1039, 51687 Reims Cedex, France
3 Laboratoire de Biologie Cellulaire et Tissulaire, Université de
Liège, 20 rue de Pitteurs, 4020 Liège, Belgium
4 IFR53, 51 rue Cognacq-Jay, 51096 Reims Cedex, France
* Author for correspondence (e-mail: dominique.ploton{at}univ-reims.fr )
Accepted 23 May 2002
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Summary |
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Key words: Cell nucleolus, RNA polymerase I, Pre-ribosomal RNA, BrUTP incorporation, Electron tomography
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Introduction |
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Our present knowledge of the molecular organization of transcribed rRNA
genes was mainly established using chromatin spreads
(Miller and Beatty, 1969).
After hypotonic treatment, each transcribing gene decondenses and appears as a
so-called `Christmas tree', 4.5 to 6 µm in length
(Trendelenburg and Puvion-Dutilleul,
1987
). This `Christmas tree' consists of 135 to 180 fibrils of
growing pre-rRNAs, each of which is connected to the rDNA axis by a RPI
molecule (Reeder and Lang,
1997
). By contrast, localization of these `Christmas trees'
relative to the different compartments within the nucleolus has proved to be a
complex problem. There is a general consensus that transcription occurs in the
fibrillar regions of the nucleolus, but whether it is within the DFC or at the
border of the DFC and the FC, or even within the FC, remains a matter of much
debate (Goessens, 1976
;
Fakan, 1978
;
Scheer and Rose, 1984
;
Thiry et al., 1985
;
Dupuy-Coin et al., 1986
;
Derenzini et al., 1987
;
Raska et al., 1989
;
Thiry and Goessens, 1991
;
Dundr and Raska, 1993
;
Schöfer et al., 1993
;
Bréchard et al., 1994
;
Hozak et al., 1994
;
Cmarko et al., 1999
;
Biggiogera et al., 2001
).
Recently, we reported a new method for BrUTP incorporation within optimally
preserved cells that allowed us to follow the kinetics of rRNA synthesis and
maturation (Thiry et al.,
2000). The results of this work showed that BrUTP-labelled rRNAs
are initially localized both within the FC and the inner part of the DFC.
However, the speed of rRNA synthesis in vivo is so high [25-50
nucleotides/second (Grummt,
1978
)] that the localization of incorporated BrUTP (or
[3H]-UTP) could be representative of both incorporation and
accumulation sites. In order to resolve this problem, a model in which the
molecular events are slowed down is a prerequisite. In addition, conventional
microscopy gives two-dimensional pictures that only account for a partial view
of the labelling and do not provide a description of the in situ volumic
organization of the rDNA genes. Clearly, alternative approaches providing
three-dimensional information, such as electron tomography, must be performed
to answer this biological question
(Crowther et al., 1970
;
Héliot et al., 1997
;
Baumeister et al., 1999
).
In this work, we have studied the precise localization of initial BrUTP
incorporation sites at the ultrastructural level. To address this point, we
used isolated nucleoli, which (when compared with intact cells) exhibit
decreased transcriptional activity
(Grummt, 1978;
Hadjiolov, 1985
). Using a
pulse-chase procedure and an elongation inhibitor, we demonstrated that the
ribosomal transcripts elongate in the cortex of the FC and then enter into the
surrounding DFC. Furthermore, we have immunolabelled RPI molecules prior to
embedding in order to study their three-dimensional organization in whole
cells. Electron tomography was performed on sections whose thickness (500 nm)
is sufficient to visualize entire FCs. This approach revealed that RPI
molecules are located within the cortex of the FC, where they are organized
within three to five curved coils constituted with bent twines. These results
led us to propose a model for the volumic organization of the `Christmas
trees' that reconciles all the previous findings by placing the rDNA genes in
the FC and the growing rRNA molecules both in the FC and within the DFC.
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Materials and Methods |
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In vitro transcription measured by [3H]-GTP
incorporation
Isolated nucleoli from ELT cells were suspended in TS buffer (25% glycerol,
10 mM KCl, 50 mM Tris-HCl, pH 7.0, 5 mM MgCl2, 0.5 mM EGTA, 0.2 mM
spermidine, 0.4 mM spermine, 25 U/ml RNase inhibitor) (Roche, Meylan, France),
and a set of protease inhibitors (0.75 mM PMSF, 30 µg/ml antipain, 2
µg/ml aprotinin, 100 µg/ml EDTA, 0.5 µg/ml leupeptin, 200 µg/ml
Pefabloc®, 30 µg/ml phophoramidon, 5 µg/ml bestatin, 0.7 µg/ml
pepstatin, 10 µg/ml E-64 and 10 µg/ml chymostatin) (Roche). Reactions
were performed in TS buffer to which 0.2 mM ATP, CTP, UTP or BrUTP (Roche) and
500 dpm/pmol [3H]-GTP (Amersham Pharmacia Biotech, Little Chalfont,
UK) were added. After incubation for 1 to 30 minutes at 37°C, the
transcription process was stopped by adding 1.6 mg/ml bovine serum albumin
(BSA) and 5% trichloroacetic acid (TCA). After 30 minutes on ice and a 6
minute centrifugation at 8000 g, the pellet was washed three
times in 5% TCA and centrifuged for 6 minutes at 8000 g. The
remaining precipitate was diluted in Ecoscint A (National Diagnostics,
Atlanta, GA) and counted in triplicate in a Beckman LS3801 scintillation
counter (Beckman Coulter, High Wycombe, UK).
In vitro transcription measured by BrUTP incorporation
Isolated nucleoli from ELT cells were suspended in 500 µl TS buffer
containing 0.2 mM ATP, GTP and CTP and 0.4 mM BrUTP (TSB buffer). After
incubation for 1 to 120 minutes at 37°C, the transcription process was
stopped by adding a fixative solution composed of 4% formaldehyde and 0.1%
glutaraldehyde in 0.1 M Sorensen's buffer (pH 7.4). After 60 minutes on ice,
the pellet was washed in the same buffer before being dehydrated and embedded
in Epikote 812. In other experiments, the nucleoli were suspended in TSB
buffer containing either 0.2 µg/ml actinomycin D (Sigma) or 100 µg/ml
-amanitin (Roche). For pulse-chase experiments, the nucleoli were
pulse-labelled for 5 minutes or 10 minutes, then centrifuged for 1 minute at
665 g and washed in 8 ml TS buffer containing 0.4 mM UTP.
After centrifugation at 665 g for 1 minute, they were
suspended for 15 or 20 minutes in 500 µl TSB buffer containing 0.4 mM UTP
instead of BrUTP before being fixed as described above. Finally, to perform
inhibition studies, the nucleoli were suspended at 37°C in TSB buffer
containing UTP instead of BrUTP and either 0.4 mM
cordycepin-5'triphosphate (Sigma) or 0.2 mM ATP, then incubated for 15
minutes. After, a chase was carried out by incubating the nucleoli in TSB
buffer at 37°C for 15 minutes in the presence or absence of cordycepin.
Fixation and embedding were performed as described above.
Immunogold labelling of BrUTP nucleotides
Ultrathin sections of nucleoli that had incorporated BrUTP were incubated
for 30 minutes in PBS (0.14 M NaCl, 6 mM Na2HPO4, 4 mM
KH2PO4, pH 7.2) containing diluted (1:30) normal goat
serum and 1% BSA and then rinsed with PBS containing 1% BSA (PBS-BSA). The
sections were then incubated for 4 hours at room temperature with a monoclonal
anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ) diluted 1:50 in PBS
and containing 0.2% BSA and diluted (1:50) normal goat serum. After washing
with PBS-BSA, the sections were incubated at room temperature for 1 hour with
goat anti-mouse IgG coupled to colloidal gold particles, 10 nm in diameter
(Janssen Life Sciences, Beerse, Belgium), diluted 1:40 with PBS (pH 8.2)
containing 0.2% BSA. After washing with PBS-BSA, the sections were rinsed in
deionized water. Finally, the ultrathin sections were mounted on nickel grids
and stained with uranyl acetate and lead citrate before examination in a Jeol
CX 100 electron microscope at 60 kV (Jeol, Brussels, Belgium). Three kinds of
control experiments were carried out: (i) the primary antibody was omitted;
(ii) the sections were incubated with antibody-free gold particles; and (iii)
BrUTP was replaced by UTP in the transcription buffer.
Quantitative evaluations of colloidal gold particles on ultrathin
sections
To evaluate the labelling density, the area of each compartment was first
estimated morphometrically by the point-counting method
(Weibel et al., 1969). Gold
particles were counted from 7 to 15 random micrographs (for the time-course of
BrUTP incorporation and effect of actinomycin D) or from 13 to 25 random
micrographs (for pulse-chase experiments and studies performed in the presence
of cordycepin). After evaluating the areas (Sa) occupied by the various
compartments, the number of gold particles (Ni) over each compartment was
determined and the labelling density (Ns=Ni/Sa) was calculated. The numerical
data obtained do not reflect the exact amounts of BrUTP molecules in the
compartments but rather their relative density. For this reason, all specimens
in comparative studies were submitted to identical fixation and embedding
conditions, and incubation of the different sections was performed
simultaneously.
Immunostaining of RPI before embedding
A549 cells were fixed for 10 minutes in 3% paraformaldehyde diluted in PBS,
then permeabilized with 0.3% Triton X-100 in PBS for 2 minutes. Cells were
soaked for 30 minutes in 3% BSA in PBS, then incubated for 4 hours with a
rabbit polyclonal antibody directed against the large subunit of RPI (a gift
from K. Rose, Houston, TX) diluted 1:400. After a 30 minute incubation with a
biotinylated goat anti-rabbit antibody (Sigma), the secondary antibody was
revealed with fluoronanogold streptavidin (Nanoprobes, New York, USA). The
cells were fixed again with 1.6% glutaraldehyde in PBS, washed in deionised
water and amplified with HQ silver (Nanoprobes) in order to obtain 10 nm
particles. Cells were then harvested by scraping, dehydrated in graded
alcohols and embedded in Epikote 812. Ultrathin sections (80 nm) mounted on
copper grids were stained with uranyl acetate and lead citrate before
examination using a Jeol 200 CX microscope at 80 kV (Jeol).
Electron tomography
500 nm thick sections prepared from A549 cells labelled with anti-RPI
antibodies were observed in a medium-voltage CM30 electron microscope working
at 250 kV in the STEM mode (Philips, Eindhoven, The Netherlands)
(Beorchia et al., 1992). In
order to get a high contrast, these sections were not counterstained. Before
initiating a tilt series, the section was stabilized under the electron beam
at a dose of 100 e-/(Å2xseconds) for 10
minutes to limit anisotropic thinning of the specimen during data collection.
Each section was tilted every 2° from -50° to +50°. The 51
corresponding images (512x512 pixels) were recorded directly on a
disk-type scintillator-photomultiplier detector system and digitized on-line
using Orion hardware (ELI sprl, Belgium). Images were then aligned
individually by using a sinogram technique
(Bahr et al., 1979
). In order
to study individual clusters in more detail, the corresponding part of the
image was isolated from the original image, resampled and finally realigned.
Tomographic reconstruction was performed by using an extended field-additive
algorithm-reconstruction technique on aligned images, as previously described
(Héliot et al., 1997
).
Extensive investigations were performed by using the Analyze Software package
(Mayo BIR, CN Software, Southwater, UK) and the Amira software (TGS Europe,
Mérignac, France) on the reconstructed volumes of 16 clusters taken
from different cells. Three-dimensional visualizations with a high depth of
field were calculated to obtain pertinent images at fixed angles of view and
rotations around any chosen axis. Complex movements around the volumes were
also performed, as shown in the movies (available at
jcs.biologists.org/supplemental
). Finally, the inner parts of the volumes were investigated by presenting
sections performed in any chosen plane. For
Fig. 8, sections, stereo-pairs
and movies were performed after a 2x2x2 downsampling.
|
Quantification of the labelling density within thick sections
Images of seven 500 nm thick sections obtained from A549 cells labelled
with anti-RPI antibodies before embedding were used to quantify the density of
the labelling within different parts of the nucleus. By using Metamorph
software (Roper Scientific, France), a circle 270 nm in diameter was
positioned on 90 different zones of the image (30 outside of the cell, 30 on
the nucleoplasm and 30 on clusters), and we measured the density of the
labelling for each case. After subtracting the value of the density measured
outside of the cell from that of the nucleoplasm and the clusters, the ratio
of the labelling density within clusters was calculated relatively to the
density within nucleoplasm.
Quantification of the number of active rDNA genes per fibrillar
center in A549 cells
Control and DRB-treated A549 cells (100 µg/ml for 6 hours) were fixed
for 10 minutes in 3% paraformaldehyde diluted in PBS, then permeabilized with
0.3% Triton X-100 in PBS for 2 minutes. Cells were then immunolabelled with
UBF antibody, and a series of optical sections were obtained by confocal
microscopy as previously described (Klein
et al., 1998). Processing was performed using the Analyze computer
software (CNSoftware, Southwater, UK) working on a Sun Sparc20 (Sun
Microsystems, Mountain View, CA) workstation. Volumes were isotropically
restored to obtain the same pixel size in x, y, z directions. A
3x3x3 cubic median filter was applied to decrease noise, and local
enhanced contrast was achieved using Bengali-Le Negrate's method
(Le Negrate et al., 1992
).
After, volumes were binarised. Finally, the number of objects contained within
a volume were estimated using three-dimensional quantification software
[Analyze package; Qt software (Klein et
al., 1998
)].
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Results |
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Sites of BrUTP incorporation in isolated nucleoli
Isolated nucleoli from ELT cells were incubated in the presence of BrUTP
for 1, 5, 10 and 25 minutes. The distribution of BrRNAs among the different
nucleolar compartments was subsequently visualized and quantified after
immunogold labelling with 10 nm gold particles
(Fig. 1). The three main
nucleolar components (FC, DFC and GC) were easily identified, attesting that
the nucleolar ultrastructure was preserved. BrUTP incorporation was rapid (62%
and 82% of nucleoli were labelled after 1 and 5 minutes, respectively), with
92-97% of nucleoli being labelled after 10 minutes. Within nucleoli, labelling
was observed on the two fibrillar components (FC and DFC) but almost never on
the GC or on nucleolus-associated condensed chromatin. Closer examination
revealed that on the FC, gold particles were preferentially localized in the
cortical regions near to the surrounding DFC
(Fig. 1A,B). Quantification of
labelling over a 1 to 25 minute period revealed that labelling density was
progressively increased on both the FC and the DFC as a function of time
(Fig. 2). In addition,
labelling density was significantly higher on the FC than on the DFC (with the
particle density on the FC and DFC being significantly higher than that in
resin). In the presence of a concentration of actinomycin D that inhibits
nucleolar transcription, labelling was strongly reduced on all the nucleolar
components (Fig. 3). By
contrast, in the presence of -amanitin (an extranucleolar
transcriptional inhibitor), no reduction of the nucleolar labelling was
observed (data not shown).
|
|
|
Localization of elongating rRNAs by pulse-chase
In order to determine whether pre-rRNAs enter new compartments during the
elongation process, the distribution of labelling was analysed in isolated
nucleoli submitted to a 10 minute pulse with BrUTP, followed by an optional 20
minute chase with UTP. In both cases, labelling was consistently present on
the fibrillar components (data not shown). Gold particles were mainly observed
on the cortical part of the FC, whereas its central part was almost devoid of
labelling. However, relative to the FC, labelling density was higher on the
DFC after the chase. A quantitative evaluation revealed that the labelling
density on the FC was significantly higher than that on the DFC in the pulse
experiment (Fig. 4). A 5 minute
pulse followed by a 15 minute chase led to similar results (data not
shown).
|
Identification of the initial sites of rRNA elongation
In this experiment, isolated nucleoli were incubated with BrUTP after a
transient inhibition of elongation by cordycepin, which leads to premature
transcription termination and release of incomplete transcripts from their
templates (Siev et al., 1969;
Suhadolnik, 1979
). After the
release of inhibition, RPI molecules are immediately re-engaged in
transcription, and this allows us to identify the initial sites of BrUTP
incorporation. When cordycepin was present throughout the experiment
(Fig. 5), BrUTP incorporation
was significantly reduced, and only 20% of nucleoli were labelled. The
labelling density on both the FC and the DFC, although still significant, was
very low. After a cordycepin pretreatment, 82% of nucleoli incorporated BrUTP
(relative to 89% in control experiments without cordycepin). In these
experimental conditions, the FC displayed high labelling density
(Fig. 5B,C), which was
significantly higher than that of the FC of control nucleoli labelled in the
absence of cordycepin (Fig.
5A,C).
|
Three-dimensional distribution of RPI
In order to study the volumic organization of RPI, immunolabelling was
performed in A549 cells before embedding. First, the localization of the whole
labelling relatively to the ultrastructure was checked by producing ultrathin
sections counterstained with lead and uranyl. This revealed a preserved
nucleolar ultrastructure (Fig.
6) in which several FCs (270 nm in diameter) were identified
as the sites where most silver particles were found.
|
From similar preparations, 500 nm thick sections were produced. Under
observation at 250 kV, RPI labelling always appeared as several discrete
clusters of particles in which the density of the labelling is 10 times higher
than that of the nucleoplasm (data not shown). The clusters are organized as
spheroids 270 nm in diameter (Fig.
7A). They contained
150 individualized silver particles and
displayed a similar volumic organization. The cluster framed in
Fig. 7A was taken as a
representative example. By rotating the tomogram at different angles, it was
possible to reveal all the details of the structure and choose the most
pertinent and demonstrative views. The particles presented a highly organized
structure within a cluster, as evidenced by a rotation at +15°
(Fig. 7D). This view revealed
that the cluster is composed of several coils, 60 nm in diameter, as also seen
on a stereopair displayed with a surfacic rendering mode
(Fig. 7E). Rotations and
complex spatial moving around this reconstructed object were performed to
determine the number of coils, to measure their length and to investigate
their spatial organization (movie abailable at
jcs.biologists.org/supplemental). Three curved coils (150-200 nm in length,
#1-3) and two shorter ones (30-50 nm in length, numbers 4 and 5) were observed
(Fig. 7D). These coils share a
common origin (Fig. 7D,I,
circles) but display separate extremities (brackets).
|
In order to investigate the internal arrangement of RPI within the FC, 30 nm thick coronal sections were computed from the tomogram displayed in Fig. 7D (Fig. 7F-I). Notably, the central region is devoid of particles (asterisks). The coronal sections revealed the internal structure of the coils, in which RPI particles were frequently aligned to form 20 nm thick twines with a free terminal end (Fig. 7G-I, arrows). To further investigate their orientation, the volume was rotated to place the coils in a vertical position, and a sectioning plane parallel to the axis of coil number 2 was applied (Fig. 8A,B). In these conditions, two successive, 1.5 nm thick sections revealed seven twines, which were orthogonally disposed relatively to the axis of the coil and were parallel to each other [Fig. 8A,B, arrows; Movie 2 (available at jcs.biologists.org/supplemental )]. When 1.5 nm thick sections were produced perpendicularly to the axis of the coil, it appeared that the twines formed full or partially open circles, 60 nm in diameter [Fig. 8C,D, white circles; Movie 3 (available at jcs.biologists.org/supplemental )]. These features were also evidenced on a three-dimensional visualization performed on one half of the volume [Fig. 8E; Movie 4 (available at jcs.biologists.org/supplemental )].
Number of active rDNA genes per fibrillar center
DRB treatment was used to estimate the number of active rDNA genes per
transcription site. DRB unravels nucleolar components and the fibrillar
components (FC and DFC) form separate beads, each of which containing a single
rDNA gene (Haaf et al., 1991).
After UBF immunostaining, a marker of both FC and DFC, a series of optical
sections in control and DRB-treated cells were produced and analyzed by
confocal microscopy. In control cells, UBF staining appeared as frequently
superimposed dots within the whole nucleolar volume, whereas in treated cells,
UBF was found within discrete dots, arranged as extended necklaces. Whole
projections were then calculated and a quantification led to 89 (±16)
and 180 (±25) dots for 12 control and 12 treated cells, respectively.
The value obtained for control cells was probably underestimated because of
the relatively low resolution of the confocal microscope on the z-axis (0.4
µm) compared with the size of the dots (0.3 µm). These results indicate
that in control cells, most transcription sites contain either a single active
rDNA copy or two rDNA copies seen as two distinct clusters arranged in close
proximity.
![]() |
Discussion |
---|
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With this in vitro system, nascent transcripts are mainly detected in the
FC and to a lesser extent in the DFC. This observation correlates with
previous in vivo studies showing that the first sites to be labelled by BrUTP
in lipofected cells are the FC and the DFC
(Thiry et al., 2000), but it
does not agree with experiments using BrUTP incorporation within permeabilized
cells (Hozak et al., 1994
) or
BrUTP introduced by microinjection (Cmarko
et al., 1999
). To explain this discrepancy, the dynamics of
nascent rRNAs must be taken into account. In order to analyze their migration,
we performed pulse-chase experiments (Fig.
4). Our data showed a significant decrease of the labelling in the
FC and a simultaneous increase in the DFC. This demonstrates that the FC is
the site of primary BrUTP incorporation and that the pre-rRNAs enter within
the surrounding DFC during the elongation process. Moreover, the primary sites
of BrUTP incorporation were identified after a transient inhibition of
elongation by cordycepin, whose action leads to premature transcriptional
termination (Siev et al.,
1969
; Suhadolnik,
1979
), thus allowing new RPI molecules to be engaged in
transcription immediately after the release of inhibition. Upon transcription
reactivation, a subsequent BrUTP pulse initiated preferential labelling of the
FCs, whereas DFC-specific labelling was very low
(Fig. 5). This finding
demonstrates that the FCs are the sites of rDNA transcription within the
nucleolus. Taken together, these experiments demonstrate that transcription of
rDNA genes takes place in the cortex of FC and that growing rRNA rapidly enter
the DFC. This is consistent with the results of previous work in which RPI
have been located exclusively or preferentially in the FCs
(Scheer and Rose, 1984
;
Reimer et al., 1987
;
Raska et al., 1989
). Moreover,
other studies have found DNA, including rDNA genes, in the FCs, preferentially
in their cortical part (Thiry and
Thiry-Blaise, 1989
;
Puvion-Dutilleul et al., 1991
;
Thiry and Thiry-Blaise, 1991
;
Thiry et al., 1993
) and also
small amounts of nascent RNAs over the FCs themselves
(Goessens, 1976
;
Thiry et al., 1985
;
Dupuy-Coin et al., 1986
;
Derenzini et al., 1987
;
Thiry and Goessens, 1991
;
Thiry et al., 2000
).
We also addressed the question of the volumic organization of the
transcription machinery by performing electron tomography on cells
immunolabelled with anti-RPI antibodies prior to embedding. This technique is
the only one that can reveal the distribution of labelled particles within a
cellular volume at the electronic level
(Koster et al., 1997;
McEwen and Marko, 2001
). A549
cells were chosen because they contain numerous FCs, whose small size
(
0.27 µm in diameter) allows them to be fully contained within the
section thickness (0.5 µm). In addition, FCs of A549 cells are homogenous
in size, in contrast to ELT cells. This factor was essential to allow
structural comparisons between the different FCs analyzed in the present
study. Since antigen detection represents a critical parameter for tomographic
studies, we performed a fixation with paraformaldehyde and a mild
permeabilization with Triton X-100 to achieve the best compromise between
antigen accessibility and ultrastucture preservation
(Humbel et al., 1998
).
Similarly, fluoronanogold was chosen for its reduced size, which facilitates
its penetration within the specimen and reveals antigens, unlike 5 or 10 nm
gold particles, which are unable to penetrate
(Robinson et al., 2000
).
Moreover, the use of fluoronanogold before embedding increases significantly
the sensitivity of antigen detection compared with classic methods using 5 to
10 nm gold particles on the surface of ultrathin sections (Hainfeld and
Furaya, 1992; Hainfeld and Powell,
2000
). Finally, silver amplification was controlled by using a
protective colloid that favoured the growth of silver particles centred on the
gold clusters in order to reveal the exact location of antigens
(Baschong and Stierhof, 1998
).
The relevance of this detection procedure has been demonstrated previously
(Robinson and Vandré,
1997
), using pre-embedding immunolabelling of tubulin in
leukocytes. Robinson and Vandré observed 25 nm thick fibres that
correspond almost exactly to the size of microtubules, which indicates that
fluoronanogold reveals the exact size of structures containing antigens.
As shown previously, the observation of counterstained ultrathin sections
demonstrated that the RPI molecules are mainly localized within the FC
(Scheer and Rose, 1984;
Reimer et al., 1987
;
Raska et al., 1989
). The
detection of RPI before embedding led to a labelling density that was
significantly higher than that observed classically on ultrathin sections
(Fig. 6). This can be explained
by both the sensitivity of the detection (see above) and the fact that the
antigens were observed throughout the thickness of the section (80 to 100 nm),
unlike conventional detection protocols that only reveal antigens located at
its surface. In addition to a strong labelling in the FCs, a weak RPI
labelling was also seen in the GC. Interestingly, a similar distribution of
RPI was obtained by STEM analyses performed on sections containing whole FCs
(i.e. 0.5 µm in thickness), followed by three-dimensional reconstructions
(Fig. 7A). Indeed, the main
sites of RPI labelling are organized into discrete clusters, clearly separated
from one another and surrounded by a much weaker labelling. The coincidence of
ultrastructural analyses and three-dimensional data allows us to conclude that
RPI clusters are located in the FCs. Quantification showed that the labelling
density of RPI was 90% in the clusters and 10% in their environment. This
view, although seen at a much higher resolution, is in agreement with the
picture of GFP-tagged nucleolar proteins observed by confocal microscopy,
showing main nucleolar sites surrounded by a weaker labelling
(Phair and Misteli, 2000
;
Chen and Huang, 2001
;
Savino et al., 2001
). FRAP
experiments have proved that this pattern reflects the rapid exchange of
nucleolar proteins between their nucleolar binding sites and the nucleoplasm
(Phair and Misteli, 2000
;
Chen and Huang, 2001
).
Several lines of evidence indicate that the three-dimensionally organized
clusters are the sites at which transcribing RPI molecules are located.
However, the question of whether the clusters are composed of
transcriptionally active or inactive molecules cannot be answered, as at the
present time no antiserum or modification can discriminate between these two
states. First, the localization of RPI relative to the ultrastructure shows
that RPI is predominantly found in the FCs. In the present study, the FCs were
further identified as the initial sites where BrUTP incorporation takes place.
These findings point to the FCs as the sites where transcribing RPI are
located. It corroborates numerous other studies, showing that the other
counterparts of RPI transcription, rDNA genes and nascent pre-rRNAs, are also
found in the FCs (Goessens,
1976; Thiry et al.,
1985
; Dupuy-Coin et al.,
1986
; Derenzini et al.,
1987
; Thiry and Thiry-Blaise,
1989
; Thiry and Thiry-Blaise,
1991
; Thiry et al.,
1993
; Thiry et al.,
2000
). From these data, we conclude that the FCs are the sites
where RPI transcription occurs. We addressed the question of the number of
rDNA genes that are present in each FC. Our experiments using DRB treatment on
A549 cells revealed that most FCs contain a single active rDNA gene, which is
in agreement with previous results obtained from highly proliferating cells
(Ochs and Smetana, 1989
).
From the tomographic analyses of 16 different tomograms, the structure of
each cluster was further described, and it revealed two levels of
organization. First, several coils, displaying a common origin, showed a
spatial arrangement recalling that of a corolla, as confirmed by the presence
of a central cavity. Second, the internal organization of the coils revealed
bent twines, approximately 60 nm long and 20 nm thick. We hypothesize that the
coils are constituted with RPI molecules engaged on the rDNA gene and that
their spatial distribution can be used to propose a model for the
three-dimensional organization of the rDNA gene in situ
(Fig. 9). Using images of
spreads, the transcribed region of a mammalian rDNA gene (6000 nm in
length) was considered to be covered with 30 RPI molecules/µm
(Trendelenburg and Puvion-Dutilleul,
1987
). The size of a RPI molecule was estimated to 16 nm, and the
length of fully elongated rRNAs to be 350 nm (these pre-rRNAs molecules being
3 nm in diameter and ending with a 20 nm wide particle)
(Fig. 9A). Our data, which
revealed four to five coils emanating from a common origin, can be correlated
with previous results showing the presence of matrix-associated regions both
in the 5'NTS and in the 3'NTS of rDNA genes
(Stephanova et al., 1993
).
Moreover, the termination transcription factor TTF1, which can oligomerize and
form aggregates, binds both to the initiation and the termination sites of
rDNA genes, thus organizing each rDNA gene as a loop
(Reeder and Lang, 1997
). These
results were schematized by folding the rDNA gene into four loops
(Fig. 9B), each of which
undergoes another level of folding, thus forming two parallel rows
(Fig. 9C). These rows (
200
nm in length) consist of successive small loops (brackets),
60 nm in
length, each of which contains three to four RPI molecules. The bending of all
the loops on the matrix of a cylinder produces a coil, 60 nm in diameter and
200 nm in length (Fig. 9D,E),
similar to those observed after RPI immunolabelling. Consequently, a coil
could be obtained by stacking about six open rings (brackets on D), each of
which are constituted as two twines (Fig.
9F,G). These views are in agreement with the transversal and
longitudinal sections of the coils (Fig.
8). The high level of bending and looping needed in this model
could be performed by UBF, a very abundant nucleolar protein. Indeed, UBF has
the ability to bend rDNA (Putnam et al.,
1994
), and its binding is not restricted to the regulatory
sequences of the ribosomal DNA repeats, but it can occur all along these
sequences (O'Sullivan et al.,
2002
).
|
Finally, a transcribed rDNA gene can be spatially organized within a
limited volume as four similarly organized coils
(Fig. 9H,I), with a compaction
factor of approximately seven. This model still allows enough spacing between
RPI molecules and rDNA for pre-rRNAs molecules to be elongated without steric
hindrance. The openings of the rings (arrows on
Fig. 9F) give rise to two
longitudinal grooves along each coil, which can be observed in the rotation of
the whole model (Movie 5, available at jcs.biologists.org/supplemental). A
transverse section of the figure presented in
Fig. 9H (270 nm in diameter)
fits within a FC, as represented at the same magnification on an ultrathin
section (Fig. 9J). Our BrUTP
incorporation data, which demonstrate that growing pre-rRNAs molecules are
synthesized in the FC and enter the surrounding DFC, have been schematized on
Fig. 9J as polarized threads.
It illustrates, by using in vivo BrUTP incorporation and classic
post-embedding detection methods, why the probability of finding BrUTP over
the DFC is very high and why a slowed-down system is a prerequisite to observe
BrUTP on the FC. Our model supposes the positioning of the growing rRNA
extremities in two different compartments, which is supported by the finding
that pre-rRNAs are in a extended conformation during transcription
(Stanek et al., 2001)
Our model correlates with the fact that the presence of the DFC in the
nucleolus is critically dependent on ongoing transcription of the rRNA genes.
Microinjection of anti-RPI antibodies reduces rRNA synthesis
(Schlegel et al., 1985). It
also modifies the nucleolar morphology: the DFC is rapidly disorganized after
microinjection and forms extra-nucleolar bodies that are devoid of RPI
(Benavente et al., 1988
).
Moreover, after hypotonic shock leading to a complete reorganization of the
nucleolar structures, the FCs and the DFCs are totally separated and rDNA is
only found in the remains of the FCs
(Zatsepina et al., 1997
). The
intimate association of the FCs with the DFC might provide a structural
framework that maintains transcriptional products in a specific topological
order, which is necessary for the subsequent cascade of maturation and
processing steps.
Finally, this model allows the juxtaposition of several active genes, whose association would then form larger FCs. This could account the difference in sizes of FCs from different cellular models. Further tomographic studies performed on FCs from various cell lines will be necessary to assess this hypothesis.
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Acknowledgments |
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Footnotes |
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References |
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Bahr, G. F., Boccia, J. A. and Engler, W. F. (1979). Reconstruction of a chromosome model from its projections. Ultramicroscopy 4, 45-53.[Medline]
Baschong, W. and Stierhof, Y. D. (1998). Preparation, use and enlargement of ultrasmall gold particles in immunoelectron microscopy. Microsc. Res. Tech. 42, 66-79.[Medline]
Baumeister, W., Grimm, R. and Walz, J. (1999). Electron tomography of molecules and cells. Trends Cell Biol. 9,81 -85.[Medline]
Benavente, R., Reimer, G., Rose, K. M., Hugle-Dorr, B. and Scheer, U. (1988). Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells. Chromosoma 97,115 -123.[Medline]
Beorchia, A., Héliot, L., Ménager, M., Kaplan, H. and Ploton, D. (1992). Applications of medium-voltage STEM for the 3-D study of organelles within thick sections. J. Microsc. 170,247 -258.
Biggiogera, M., Malatesta, M., Abolhassani-Dadras, S., Amalric,
F., Rothblum, L., and Fakan, S. (2001). Revealing the unseen:
the organizer region of the nucleolus. J. Cell Sci.
114,3199
-3205.
Bréchard, M. P., Hartung, M., de Lanversin, A., Cau, P. and Stahl, A. (1994). Localization of rDNA transcription sites in nucleoli of human Sertoli cells by EM quantitative autoradiographic study using 3H-uridine. Biol. Cell 81,247 -256.[Medline]
Chen, D. and Huang, S. (2001). Nucleolar
components involved in ribosome biogenesis cycle between the nucleolus and
nucleoplasm in interphase cells. J. Cell Biol.
153,169
-176.
Cmarko, D., Verschure, P. J., Martin, T. E., Dahmus, M. E.,
Krause, S., Fu, X. D., van Driel, R. and Fakan, S. (1999).
Ultrastructural analysis of transcription and splicing in the cell nucleus
after bromo-UTP microinjection. Mol. Biol. Cell
10,211
-223.
Crowther, R. A., de Rosier, D. J. and Klug, A. (1970). The reconstruction of a three-dimensional structure from projections and its application to electron microscopy. Proc. R. Soc. Lond. Ser. A 317,319 -340.
Derenzini, M., Hernandez-Verdun, D., Farabegoli, F., Pession, A. and Novello, F. (1987). Structure of ribosomal genes of mammalian cells in situ. Chromosoma 95, 63-70.[Medline]
Derenzini, M., Thiry, M. and Goessens, G. (1990). Ultrastructural cytochemistry of the mammalian cell nucleus. J. Histochem. Cytochem. 38,1237 -1256.[Abstract]
Dundr, M. and Raska, I. (1993). Nonisotopic ultrastructural mapping of transcription sites within the nucleolus. Exp. Cell Res. 208,275 -281.[Medline]
Dupuy-Coin, A. M., Pebusque, M. J., Seite, R. and Bouteille, M. (1986). Localization of transcription in nucleoli of rat sympathetic neurons. A quantitative ultrastructural autoradiography study. J. Submicrosc. Cytol. 18, 21-27.[Medline]
Fakan, S. (1978). High resolution autoradiography studies on chromatin functions. In The Cell Nucleus (ed. H. Busch), pp. 3-53. New York: Academic Press.
Goessens, G. (1976). High resolution autoradiographic studies of Ehrlich tumour cell nucleoli. Exp. Cell Res. 100,88 -94.[Medline]
Grummt, I. (1978). In vitro synthesis of pre-rRNA in isolated nucleoli. In The Cell Nucleus (ed. H. Busch), pp. 373-414. New York: Academic Press.
Haaf, T., Hayman, D. L. and Schmid, M. (1991). Quantitative determination of rDNA transcription units in vertebrate cells. Exp. Cell Res. 193,78 -86.[Medline]
Hadjiolov, A. A. (1985). Ribosomal genes. In The Nucleolus and Ribosome Biogenesis (ed. A. A. Hadjiolov), pp. 5-51. Wien, Austria: Springler Verlag.
Hainfeld, J. F. and Furuya, F. R. (1992). A
1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.
J. Histochem. Cytochem.
40,177
-184.
Hainfeld, J. F. and Powell, R. D. (2000). New
frontiers in gold labeling. J. Histochem. Cytochem.
48,471
-480.
Héliot, L., Kaplan, H., Lucas, L., Klein, C., Beorchia,
A., Doco-Fenzy, M., Ménager, M., Thiry, M., O'Donohue, M. F. and
Ploton, D. (1997). Electron tomography of metaphase nucleolar
organizer regions: evidence for a twisted-loop organization. Mol.
Biol. Cell 8,2199
-2216.
Hozak, P., Cook, P. R., Schöfer, C., Mosgöller, W. and
Wachtler, F. (1994). Site of transcription of ribosomal RNA
and intranucleolar structure in HeLa cells. J. Cell
Sci. 107,639
-648.
Humbel, B. M., de Jong, M. D. M., Müller, W. H. and Verkleij, A. J. (1998) Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers. Microsc. Res. Tech. 42,43 -58.[Medline]
Jackson, D. A., Hassan, A. B., Errington, R. J. and Cook, P. R. (1993). Visualization of focal sites of transcription within human nuclei. EMBO J. 12,1059 -1065.[Abstract]
Klein, C., Cheutin, T., O'Donohue, M. F., Rothblum, L., Kaplan,
H., Beorchia, A., Lucas, L., Héliot, L. and Ploton, D.
(1998). The three-dimensional study of chromosomes and of
UBF-immunolabelled NORs demonstrates their non-random spatial arrangement
during mitosis. Mol. Biol. Cell
9,3147
-3159.
Koster, A. J., Grimm, R., Typke, D., Hegerl, R., Stoschek, A., Walz, J. and Baumeister, W. (1997). Perspectives of molecular and cellular electron tomography. J. Struct. Biol. 120,276 -308.[Medline]
Le Negrate, A., Beghdadi, A. and Dupoisot, H. (1992). An image enhancement technique and its evaluation through bimodality. Comp. Vis. Graph. 54, 13-22.
Lepoint, A. and Bassleer, R. (1978). Number of nucleoli in Ehrlich tumor cells during interphase. Virchows Arch. B. Cell Path. 26,267 -273.
McEwen, B. F. and Marko, M. (2001). The
emergence of electron tomography as an important tool for investigating
cellular ultrastructure. J. Hist. Cytochem.
49,553
-563.
Mélèse, T. and Xue, Z. (1995). The nucleolus: an organelle formed by the act of building a ribosome. Curr. Biol. 7,319 -324.
Miller, O. L. and Beatty, B. R. (1969). Visualization of nucleolar genes. Science 164,955 -957.[Medline]
Moss, T. and Stefanovsky, V. (1995). Promotion and regulation of ribosomal transcription in eukaryotes by RNA polymerase I. Prog. Nucl. Acid Res. Mol. Biol. 50, 25-66.[Medline]
Ochs, R. L. and Smetana, K. (1989). Fibrillar center distribution in nucleoli of PHA-stimulated human lymphocytes. Exp. Cell Res. 184,552 -557.[Medline]
Olson, M. O., Dundr, M. and Szebeni, A. (2000). The nucleolus: an old factory with unexpected capabilities. Trends Cell Biol. 10,189 -196.[Medline]
O'Sullivan, A., Sullivan, G. and McStay, B.
(2002). UBF binding in vivo is not restricted to regulatory
sequences within the vertebrate ribosomal DNA repeat. Mol. Cell.
Biol. 22,657
-668.
Phair, R. D. and Misteli, T. (2000). High mobility of proteins in the mammalian cell nucleus. Nature 404,604 -609.[Medline]
Putnam, C. D., Copenhaver, G. P., Denton, M. L. and Pikaard, C. S. (1994). The RNA polymerase I transactivator upstream binding factor requires its dimerization and high-mobility-group (HMG) box 1 to bend, wrap, and positively supercoil enhancer DNA. Mol. Cell. Biol. 14,6476 -6488.[Abstract]
Puvion-Dutilleul, F., Mazan, S., Nicoloso, M., Christensen, M. E. and Bachellerie, J. P. (1991). Localization of U3 RNA molecules in nucleoli of HeLa and mouse 3T3 cells by high resolution in situ hybridization. Eur. J. Cell Biol. 56,178 -186.[Medline]
Raska, I., Reimer, G., Jarnik, M., Kostrouch, Z. and Raska, K. Jr (1989). Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components. Biol. Cell 65,79 -82.[Medline]
Reeder, R. H. and Lang, W. H. (1997). Terminating transcription in eukaryotes: lessons learned from RNA polymerase I. Trends Biochem. Sci. 22,473 -477.[Medline]
Reimer, G., Rose, K., Scheer, U. and Tan, E. (1987). Autoantibody to RNA polymerase I in scleroderma sera.J. Clin. Invest. 79,65 -72.[Medline]
Robinson, J. M. and Vandré, D. D.
(1997). Efficient immunocytochemical labeling of eukocyte
microtubules with fluoroNanogold: an important tool for correlative
microscopy. J. Hist. Cytochem.
45,631
-642.
Robinson, J. M., Takizawa, T. and Vandré, D. D.
(2000). Enhanced labeling efficiency using ultrasmal immunogold
probes: immunocytochemistry. J. Hist. Cytochem.
48,631
-642.
Savino, T. M., Gébrane-Younès, J., de Mey, J.,
Sibarita, J. B. and Hernandez-Verdun, D. (2001). Nucleolar
assembly of the rRNA processing machinery in living cells. J. Cell
Biol. 153,1097
-1110.
Scheer, U. and Rose, K. M. (1984). Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc. Natl. Acad. Sci. USA 81,1431 -1435.[Abstract]
Scheer, U. and Hock, R. (1999). Structure and function of the nucleolus. Curr. Opin. Cell Biol. 11,385 -390.[Medline]
Schlegel, R. A., Miller, L. S. and Rose, K. M. (1985). Reduction in RNA synthesis following red cell-mediated microinjection of antibodies to RNA polymerase I. Cell. Biol. Int. Rep. 9,341 -350.[Medline]
Schöfer, C., Müller, M., Leitner, M. D. and Wachtler, F. (1993). The uptake of uridine in the nucleolus occurs in the dense fibrillar component. Immunogold localization of incorporated digoxigenin-UTP at the electron microscopic level. Cytogenet. Cell. Genet. 64,27 -30.[Medline]
Shaw, P. and Jordan, E. G. (1995). The nucleolus. Annu. Rev. Cell Dev. Biol. 11, 93-121.[Medline]
Siev, M., Weinberg, R. and Penman, S. (1969).
The selective interruption of nucleolar RNA synthesis in HeLa cells by
cordycepin. J. Cell Biol.
41,510
-520.
Stanek, D., Koberna, K., Pliss, A., Malinsky, J., Masata, M., Vecerova, J., Risueno, M. C. and Raska, I. (2001). Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus. Chromosoma 110,460 -470.[Medline]
Stephanova, E., Stancheva, R. and Avramova, Z. (1993). Binding of sequences from the 5'-and 3'-nontranscribed spacers of the rat rDNA locus to the nucleolar matrix. Chromosoma 102,287 -295.[Medline]
Suhadolnik, R. J. (1979). Naturally occuring nucleoside and nucleotide antibodies. Progr. Nucl. Acids Res. Mol. Biol. 22,193 -291.[Medline]
Thiry, M. and Goessens, G. (1991). Distinguishing the sites of pre-rRNA synthesis and accumulation in Ehrlich tumor cell nucleoli. J. Cell Sci. 99,759 -767.[Abstract]
Thiry, M. and Goessens, G. (1996). The Nucleolus during the Cell Cycle. In Molecular Biology Intelligence Unit (ed. R. G. Landes), pp. 1-144. Heidelberg, Germany: Springler-Verlag.
Thiry, M. and Thiry-Blaise, L. (1989). In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA. Eur. J. Cell Biol. 50,235 -243.[Medline]
Thiry, M. and Thiry-Blaise, L. (1991). Locating transcribed and non-transcribed rDNA spacer sequences within the nucleolus by in situ hybridization and immunoelectron microscopy. Nucleic Acids Res. 19,11 -15.[Abstract]
Thiry, M., Lepoint, A. and Goessens, G. (1985). Re-evaluation of the site of transcription in Ehrlich tumour cell nucleoli. Biol. Cell 54,57 -64.[Medline]
Thiry, M., Ploton, D., Ménager, M. and Goessens, G. (1993). Ultrastructural distribution of DNA within the nucleolus of various animal cell lines or tissues revealed by terminal deoxynucleotidyl transferase. Cell Tissue Res. 271, 33-45.[Medline]
Thiry, M., Cheutin, T., O'Donohue, M. F., Kaplan, H. and Ploton,
D. (2000). Dynamics and three-dimensional localization of
ribosomal RNA within the nucleolus. RNA
6,1750
-1761.
Trendelenburg, M. and Puvion-Dutilleul, F. (1987). Electron microscopy in molecular biology, a practical approach (eds J. Sommerville and U. Scheer), pp.101 -146. Oxford, IRL Press.
Vandelaer, M., Thiry, M. and Goessens, G. (1996). Isolation of nucleoli from ELT cells: a quick new method that preserves morphological integrity and high transcriptional activity. Exp. Cell Res. 228,125 -131.[Medline]
Wansink, D.G., Schul, W., van Der Kraan, I., van Steensel, B., van Driel, R. and de Jong, L. (1993). Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J. Cell Biol. 122,283 -293.[Abstract]
Weibel, E. R., Staubli, W., Gnagi, H. R. and Hess, F. A.
(1969). Correlated morphometric and biochemical studies on the
liver cell. I. Morphometric model, stereologic methods, and normal
morphometric data for rat liver. J. Cell Biol.
42, 68-91.
Zatsepina, O. V., Dudnic, O. A., Chentsov, Y. S., Thiry, M., Spring, H. and Trendelenburg, M. F. (1997). Reassembly of functional nucleoli following in situ unravelling by low-ionic-strength treatment of cultured mammalian cells. Exp. Cell Res. 233,155 -168.[Medline]
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