Nuclear architecture and ultrastructural distribution of poly(ADP

Journal of Cell Science 109, 409-418 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS4069
409
Nuclear architecture and ultrastructural distribution of poly(ADPribosyl)transferase, a multifunctional enzyme
Wilhelm Mosgoeller1, Marianne Steiner1, Pavel Hozák2, Edward Penner3 and Józefa We˛sierska-Ga˛dek4,*
1Institute of Histology and Embryology, University of Vienna, Schwarzspanierstrasse 17, A-1090
2Academy of Sciences, Institute of Experimental Medicine, Prague, Czech Republic
3University Clinic (AKH), Department of Gastroenterology and Hepatology, Vienna, Austria
4Institute of Tumorbiology-Cancer Research, Borschkegasse 8a, A-1090 Vienna, Austria
Wien, Austria
*Author for correspondence
SUMMARY
A monospecific autoimmune serum for poly(ADPribosyl)transferase (pADPRT) was used to localise the
enzyme in ultrastructural cellular compartments. We
detected enzyme in mitochondria of HeLa and Sertoli cells.
Within the nucleoplasm the enzyme concentration was positively correlated with the degree of chromatin condensation, with interchromatin spaces being virtually free of
pADPRT. During spermatogenesis we observed a gradual
increase of the chromatin associated pADPRT that parallelled chromatin condensation. The highest concentration
was seen in the late stages of sperm differentiation, indicating the existence of a storage form in transcriptionally
inactive nuclei.
In nucleoli pADPRT is accumulated in foci within the
dense fibrillar component. Such foci are seen in close
spatial relationship to sites of nucleolar transcription as
revealed by high resolution immunodetection of bromouri-
dine uptake sites. It is suggested that nucleolar pADPRT
plays a role in preribosome processing via the modification
of nucleolus specific proteins that bind to nascent transcripts and hence indirectly regulates polymerase I activity.
The persisting binding of pADPRT to ribonucleoproteins
may explain the observed disperse enzyme distribution at
lower concentrations in the granular component. The
fibrillar centres seem to contain no pADPRT. We conclude
that known compounds of fibrillar centres like polymerase
I are unlikely candidates for modification via direct
covalent ADP-ribosylation.
INTRODUCTION
which breaking and rejoining of DNA strands may occur.
There is evidence from different experimental systems that
pADPRT plays an important role in cell differentiation
(Althaus et al., 1982; Farzaneh et al., 1982; Ueda et al., 1982).
Recently pADPRT was found to modify specific nucleolar
proteins in the nucleolus (Leitinger and We˛sierska-Ga˛dek,
1993). The specific activity was comparable to that in whole
nuclei thereby indicating that the nucleolar activity was not due
to chromatin contamination.
Since in the nucleolus at least three morphologically distinct
components can be defined and attributed to steps of ribosomal
biogenesis, we were interested in the distribution of pADPRT
within the ultrastructural components of nucleoli in different cell
types. Using human testes for high resolution immunohistochemical studies, we were also able to trace the physical
rearrangements of pADPRT during sperm differentiation in situ.
Poly(ADP-ribosyl)transferase (pADPRT) catalyses the transfer
of ADP-ribose moieties to covalent linkages with various
acceptor proteins utilizing NAD+ as a substrate (for review see:
Berger, 1985; Ueda and Hayaishi, 1985). pADPRT activity has
been found almost ubiquitously among animal tissues, plants
and lower eukaryotic organisms. Enzyme activity is mostly
localized in the nucleus (Boulikas, 1991, 1993; Malanga and
Althaus, 1994; Nishizuka et al., 1967; Shall, 1994). The major
part of nuclear pADPRT is located in chromatin in the internucleosomal space (Mullins et al., 1977; Niedergang et al., 1985).
Enzyme activity has also been found associated with the nuclear
matrix (We˛sierska-Ga˛dek and Sauermann, 1985). Some
extranuclear activity was detected in the ribosomal (Roberts et
al., 1975) and in the mitochondrial fraction (Burzio et al., 1981;
Kun et al., 1975; Richter et al., 1983).
This enzyme, strongly inducible by DNA strand breaks has
been suggested to play an important role in cellular events such
as DNA replication or DNA repair (Malanga and Althaus,
1994; Muller et al., 1994; Yoshida and Simbulan, 1994) in
Key words: Poly(ADP-ribosyl)transferase, ADP-ribose,
Autoimmunity, Immunelectron microscopy, Immunfluorescence
microscopy, Nucleolus, Ribosomal transcription, Human
spermatogenesis
MATERIALS AND METHODS
Anti-pADPRT antibodies
Serum was obtained from a patient who, following an allogenic bone
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W. Mosgoeller and others
marrow transplantation (BMT), developed clinical complications
defined as graft versus host disease. He had been conditioned by total
body irradiation (single dose of 3 Gy). While sera obtained pre-BMT
and one and two months after transplantation were negative for antinuclear antibodies, specimens obtained at day 100 and thereafter
revealed a strong nuclear and nucleolar staining pattern. In this serum,
which displayed homogenous nuclear and strong nucleolar staining in
indirect immunofluorescence, anti-pADPRT and anti-histone H1
autoantibodies were detected (data not shown). To purify specific antipADPRT we applied immunoaffinity chromatography.
Purification of human pADPRT
The full-length human pADPRT was overproduced in insect cells
using a baculovirus expression vector system (Giner et al., 1992).
Infected insect Sf9 cells were lysed twice in a hypotonic buffer and
combined lysates were chromatographically fractionated in two consecutive steps: on DNA-agarose (Sigma) and on Blue-Sepharose.
Proteins eluted from DNA-agarose with buffer containing 1 M NaCl
were diluted to a final concentration of 200 mM NaCl and loaded on
a Blue-Trisacryl column (Pharmacia). pADPRT was eluted at a concentration of 400 mM NaCl. To check the purity of isolated pADPRT,
eluates were analysed on 10% SDS-polyacrylamide gels. Proteins
were visualised by Coomassie Blue and silver staining.
Covalent immobilization of pADPRT
Purified to homogeneity human pADPRT was immobilized on Mini
Leak Medium beads (Kem-En-Tec, Copenhagen, Denmark)
according to the manufacturer’s instructions. Mini Leak is a divinyl
sulfone activated matrix of spherical agarose especially designed for
gentle immobilization of biomolecules. pADPRT diluted with
phosphate buffered saline (PBS) to a concentration of 0.5 mg/ml was
added to the activated matrix and diluted with coupling buffer containing 30% polyethylene glycol 20,000 (PEG) to a final concentration of 10% PEG and was incubated overnight. The supernatant was
discarded, the matrix was then washed and incubated with 0.2 M
ethanolamine-HCl, pH 9.0, for 4 hours to block excess active groups.
Coupling yield was estimated by measurement of optical density at
280 nm before and after coupling reaction.
Immunoaffinity chromatography
A 500 µl sample of patient’s serum was 5-fold diluted with PBS and
loaded on a preequilibrated pADPRT affinity column. The column
was washed with PBS until the basic level of optical density was
reached. Then autoantibodies were eluted with 150 mM NaCl in
phosphate buffer (pH 3.0) and immediately neutralized with 2 M TrisHCl, pH 9.0.
Purified antibodies were examined by immunoblotting using total
nuclear proteins from mycoplasma free HeLa cells and isolated
human pADPRT as antigen source. For control, affinity-purified antipADPRT antibodies were incubated for 1 hour at room temperature
with purified pADPRT. The samples were centrifuged and clear
supernatant was used for immunoblotting.
Immunostaining
For light microscopic (LM) investigations cells were grown on
coverslips, rinsed with PBS, fixed in 100% acetone for 10 minutes at
−20°C, air dried and extracted in 0.2% Triton X-100 in PBS, pH 7.4,
for 2.5 minutes at RT (room temperature) washed twice and kept in
PBST (PBS, 0.025% Tween-20, pH 7.4) with 1% (w/v) BSA (bovine
serum albumin Fraction V, Boehringer) added. Subsequently the
monolayer was incubated in the purified anti-pADPRT serum for 60
minutes at 37°C diluted 1:10 to a final antibody concentration of 0.03
µg/µl in 1% BSA-PBST. After 3 washes in PBST and incubation with
goat anti-human FITC-labelled antibody (Sigma) diluted 1:100 in 1%
BSA-PBST for one hour at room temperature, the coverslips were
washed again and mounted with anti-fade medium (Citiflour).
Immunfluorescence signal was detected either by conventional flu-
orescence-microscopy (Leitz-Dialux) or by optical sectioning in a
confocal laser scanning microscope followed by digital image
enhancement (LSM-Zeiss).
For electron microscopic (EM) preparations HeLa cells grown on
coverslips were fixed with 2% freshly depolymerized paraformaldehyde, with 0.5% glutaraldehyde added in 0.1 M sodium phosphate
buffer, pH 7.4, washed in buffer, postfixed in 1% OsO4, washed,
dehydrated and flat embedded according to the method of Steiner at
al. (1994). Testis material was obtained, fixed in aldehydes only and
processed as earlier (Mosgoeller et al., 1993).
Thin sections on gold grids of the osmified material were deosmified in 2% INaO4 in distilled water for 40 minutes at RT and washed
extensively in distilled water. Following incubation with 5% BSA
(w/v) in PBST the grids were kept in the anti-pADPRT antibodies
overnight in a moist chamber. Secondary antibodies were sheep antihuman Fab fragments labelled with TRITC (Boehringer) diluted 1:40
in 1% BSA-PBST. The third antibody incubation was in rabbit antisheep IgG coupled with 5 nm gold (Chemicon) diluted 1:40 in 3%
BSA-PBS (pH 8.2). The grids were washed 4 times in PBS (pH 8.2)
followed by 6 washes in distilled water, which was pH adjusted to 8.0
by the addition of a few grains of Tris (Merck). Following air drying
the sections were contrasted in 1% (w/v) aqueous uranylacetate for 3
minutes at room temperature, air dried again and investigated in a
transmission electron microscope (Jeoul, EM 1200).
Controls were performed by omitting the first or second antibody
and absorption of the primary antibody by addition of excess purified
enzyme. In all cases no significant label was observed.
During the evaluation of the ultrastructural images it became
evident that the grain distribution over some microscopic structures
was not random but tended to cluster. For the sake of objective data
evaluation we defined a signal cluster as an accumulation of a certain
number of grains within a field of 50 nm in diameter. The number of
grains was chosen for each experiment so that in corresponding
controls using non immunogenic incubation less than 2.5% of the
investigated cellular substructures would be scored as labelled by
clusters. This procedure inherently controls also the quality of
secondary gold coupled antibodies that tend to cluster when aged.
Double labelling with nascent RNA
HeLa cells were incubated with BrUTP fixed with aldehydes and
embedded as described by Hozak et al. (1994). Thin sections on gold
grids were incubated in a mixture of mouse anti-BrdUTP IgG
(Boehringer) diluted 1:5 in PBST with 5% (w/v) BSA and the antipADPRT serum, overnight in a moist chamber at room temperature.
After three washes in PBS and preincubation for unspecific protein
binding the grids were incubated with sheep IgG-Fab fragments
specific for the human immunglobulins (diluted 1:30 in 1% BSAPBST, pH 7.4) for two hours. A third incubation was in polyclonal
immunoabsorption purified donkey anti-sheep IgG diluted 1:20 in 3%
BSA-PBST, pH 8.2, coupled with 5 nm gold. The detection for the
primary anti-BrdUTP antibody was performed consecutively.
Following 3 washes in PBS (pH 8.2) and blocking of unspecific
binding sites with 5% (v/v) normal rabbit serum in 0.2 M sodium
phosphate buffer (pH 7.5) the next incubation was with rabbit antimouse IgG coupled to 10 nm gold (Dako) diluted 1:60 in the blocking
buffer. All incubation times with secondary antibodies varied from
one to three hours and were performed at room temperature. The grids
were finally washed 6 times in 0.2 M phosphate buffer (pH 7.5) and
6 times in double distilled boiled water (pH 8.2) and processed as
above.
For the double labelling additional controls were performed: (1) to
evaluate the interaction and colocalization of the secondary antibodies
one or both of the primary antibodies were exchanged by non-immune
serum. In this case we could not observe interaction of the secondary
antibodies. (2) To rule out unspecific signal due to interaction of the
first antibodies, both staining protocols were performed separately on
consecutive sections with one of them being turned upside down to
Immunological pADPRT detection
411
enable a one sided ‘face en face’ staining. After superimposing the
two corresponding face en face images of the same cells, the label
distribution of both protocols gave the same result as the double
stained sections.
densities were computed over a representative number of cytoplasmic
sections. The Student’s t-test (Sokal and Rohlf, 1981) was used to
estimate the significance of label differences between mitochondria
and the surrounding cytoplasm.
Statistics
At the EM level it was difficult to judge the intensity of the colloidal
gold stain in mitochondria due to very low signal. Similar to previously described statistical techniques (Mosgoeller et al., 1993) grain
RESULTS
kDa
Fig. 1. Purification of human pADPRT and examination of affinitypurified anti-pADPRT antibodies. Proteins were separated on 10%
SDS-polyacrylamide gels. M, marker proteins: 200 kDa myosin; 116
kDa β-galactosidase; 94 kDa phosphorylase b; 68 kDa bovine serum
albumin; 55 kDa glutamic dehydrogenase; 36 kDa lactate
dehydrogenase; 31 kDa carbonic anhydrase. Lane 1, lysate of control
insect cells; lane 2, first lysate of transfected insects cells; lane 3,
second lysate of transfected cells; lane 4, 1 M NaCl eluate from
DNA agarose column; lane 5, 400 mM NaCl eluate from BlueTrisacryl column; lanes 6 and 7, proteins transferred onto membrane
and Ponceau red stained; lanes 8 and 9, immunoblotting using
affinity-purified anti-pADPRT antibodies in a dilution of 1:500;
lanes 6 and 8, nuclear proteins of HeLa cells; lanes 7 and 9,
chromatographically purified human pADPRT.
Anti-pADPRT serum purification
Fig. 1, lane 5 shows the human pADPRT after expression in
the baculovirus system and chromatographical purification to
homogeneity, immobilized on activated agarose beads. This
activated matrix is especially designed for gentle coupling of
biomolecules under mild conditions. The affinity-purified antipADPRT antibodies were then tested in immunoblotting using
total nuclear proteins of HeLa cells and purified enzyme (Fig.
1, lanes 8 and 9).
Cytoplasmic immuno-pADPRT-staining in HeLa
cells and Sertoli cells
Fig. 2 shows a HeLa cell after immunofluorescent pADPRT
staining. The cytoplasm reveals regional differences in label
density, particularly well seen in the confocal scanning optical
section (Fig. 2C). Bright fluorescence appears around the
nuclear envelope. Within the nuclear centre there are confined
regions with signal of a rather homogeneous fluorescence.
Most of these homogeneously stained regions have a
nucleolus-like shape. Occasionally in some nuclei some fluorescent spots can be recognized in addition to nucleolar signal
(Fig. 2C).
After EM immunostaining of LR-White thin sections and
colloidal gold detection at the ultrastructural level, the signal distribution was similar in both HeLa cells and Sertoli cells. In the
cytoplasm a few dispersed grains were observed only occasionally. Although many mitochondrial sections were free of label,
the signal density was, on average, significantly higher
compared to the cytoplasmic staining (Fig. 3). We measured the
average grain density over more than one hundred mitochon-
Fig. 2. HeLa cells after fluorescent staining of pADPRT (A) and DNA (B), and optical sectioning of the same preparation using a confocal
laser microscope and digital contrast enhancement (C). In the standard fluorescent image (A) the nucleoli appear almost homogeneously
stained. The nucleoplasm, in particular the peripheral nuclear region and the adjacent cytoplasm reveal fluorescence. In the DAPI image (B) the
nucleoli are recognized as dark regions surrounded by bright fluorescence. In the optical section (C) arrows point out bright fluorescent spots in
the cytoplasm, arrowheads mark the signal along the nuclear membrane. Bar, 10 µm.
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Fig. 3. Cytoplasm with two mitochondria of a human Sertoli cell
after EM immunostaining and 5 nm colloidal gold detection of
pADPRT. One mitochondrium is labeled in the central compartment.
Bar, 0.2 µm.
drial and extramitochondrial regions. By means of the Student’s
t-test the average mitochondrial labelling (30.1±23.0 s.d.
grains/µm) was found to be significantly higher (P<0.001) as
compared with surrounding cytoplasm (5.2±8.0 s.d. grains/µm).
Chromatin staining
LM optical sections showed brightly fluorescing dots at the
peripheral nucleus (Fig. 2C). At the EM level all of the
chromatin was labelled with increasing signal density over the
chromatin associated with the nuclear membrane (Fig. 4) and
nucleolus associated chromatin (Figs 5-7). The interchromatin
space is more or less devoid of signal. Occasional chromatin
inclusions in nucleoli were also labelled.
In spermatogonia the labelling distribution was similar to
HeLa cells. The gold grains decorated chromatin all over the
nucleus (Fig. 7). In the course of meiosis and in later stages of
sperm differentiation, the grain density increases as the condensation of chromatin advances to form chromatids and later
on the highly compacted nucleus of the spermatid (Fig. 8A-C).
The synaptonemal complex remains free of label (Fig. 8A).
The highest grain density in our material was observed over
the compacted chromatin of spermatids (Fig. 8C).
Nucleolar staining
The standard light microscopic fluorescent images reveal a
homogeneous signal emitted from the nucleolus (Fig. 2A). The
nucleolar signal density after confocal scanning image
recording and digital contrast enhancement shows regional
intensity differences (Fig. 2C). At the EM level we observed
grain cluster over the DF and occasionally some grains over
the GC. We did not see label clearly attributable to fibrillar
centres (FCs). However, the nucleolus of HeLa cells is a very
dynamic structure which is reflected in a rather complex
arrangement of components. In a major part of the sections
signal allocation to a particular component can be difficult and
in some cases not possible. For a better ultrastructural resolution of nucleolar components the human Sertoli cell provides
a good model due to a very large FC and a distinct segregation
of the other components (Cataldo et al., 1988). Fig. 6 shows a
section of a Sertoli cell revealing all three nucleolar components. The FC and the major part of the strands of DF are free
Fig. 4. HeLa cell after EM-pADPRT staining. Arrowheads indicate
label over the entire nucleoplasm (np) which is more intense close to
the nuclear envelope (ne). Bar, 0.5 µm.
of label. However, within the DF there are confined regions
decorated by gold clusters. No such clusters were observed
over the granular component, which typically revealed few
single grains dispersed over the section. A similar label distribution was also seen in nucleoli of spermatogonia (Fig. 7).
Colocalization of pADPRT and nucleolar
transcription
In HeLa cells that were incubated with a short pulse of BrUTP
prior to fixation and embedding, we were able to perform
double labelling for nascent transcription sites and pADPRT
simultaneously using different gold grain sizes as markers.
Within the nucleoli we found many more foci revealing incorporation of BrUTP as compared with the labelling sites for
pADPRT. To investigate the relation of transcription foci to
focal accumulations of pADPRT we scored more than a
hundred nucleoli. Not in a single case were the two grain sizes
from the different detection systems found intermingled as
would be expected in a mixed cluster. Table 1 gives a summary
of the evaluation. Typically, labelling clusters were observed
over the DF but independent from each other. In 11% of all
cluster events the two different grain sizes were arranged in a
side by side manner as shown in Fig. 9.
DISCUSSION
pADPRT antibodies
Interestingly, the native serum did not react with B23, C23,
Immunological pADPRT detection
413
Fig. 5. HeLa cell nucleolus after EMpADPRT staining. Arrowheads indicate
gold grains in chromatin and dense
fibrillar component (df). Bar, 0.6 µm.
Fig. 6. Sertoli cell nucleolus after EMpADPRT staining. Arrowheads indicate
label in the chromatin, the dense
fibrillar component (df) and a few
single grains in the granular component
(gc). The fibrillar centre (fc) is not
labelled. Bar, 0.5 µm.
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W. Mosgoeller and others
Fig. 7. pADPRT EM-immunostaining
of a spermatogonium nucleolus
attached to the nuclear envelope (ne).
Arrowheads indicate signal in the
chromatin and in the dense fibrillar
component (df). Bar, 0.5 µm.
Table 1. Colocalization of BrUTP uptake and pADPRT
Independent pADPRT cluster
Independent BrUTP cluster
Side by side (2 clusters within 100 nm)
pADPRT +
BrUTP
n=127
Negative control +
BrUTP
n=111
18.9%
70.1%
11.0%
1.8%
98.2%
0%
RNA polymerase I and fibrillarin (data not shown), common
nucleolar antigens in human pathology (We˛sierska-Ga˛dek et
al., 1992), but revealed strong reactivity with nucleolar
pADPRT.
The immunoabsorbed serum reacted solely with the enzyme
thereby demonstrating its purity and specificity. Due to the
origin of the anti-pADPRT serum we consider them of high
specificity and reactivity with naturally occurring epitopes and
hence for cytobiological studies superior to monoclonal antibodies.
Cytoplasmic stain
Our LM data (Fig. 2A,C) show that a considerable amount of
pADPRT is contained in the cytoplasm. Using optical sectioning (Fig. 2C) it becomes apparent that the enzyme is concentrated in some small structure. A statistical test at the EM level
suggests that the major portion of cytoplasmic pADPRT
resides in the mitochondria (Fig. 3). The label distribution on
cross sections suggests that the enzyme resides in central compartments and not at the outer membrane. The fixation and
embedding of our material was designed to preserve antigenicity which naturally compromises on structure preservation. Although membranes are difficult to distinguish, the outer
mitochondrial membrane can be clearly located. It seems that
we visualized pADPRT which has been found in the inner
membrane by biochemical means (Burzio et al., 1981; Richter
and Schlegel, 1993), where it binds to the mitochondrial
protein-DNA complex (Masmoudi et al., 1993).
Nucleolar localization of pADPRT
The fluorescent pattern in the nucleoli and the size of labelled
regions indicates that the enzyme resides in more than one
nucleolar component (Fig. 2A). When superimposing both
images the pADPRT-fluorescence covers the entire nucleolus
(as recognized in the DAPI staining) and some adjacent DNA
rich chromatin (see Fig. 2A and B). The comparison of signal
intensity of the pADPRT staining with the corresponding
DAPI-DNA stain (Fig. 2A,B) suggests that within the
nucleolus the enzyme exists in a form not associated with DNA
and/or in a concentration highly in excess of DNA.
At the ultrastructural level we were able to observe two
different labelling patterns in DF and GC of the nucleolus. The
typical cluster-like arrangement of gold grains suggests a focal
accumulation of pADPRT within confined regions of the DF
(Figs 5-7, 9). The major portion of DF was devoid of label; we
estimate that in Sertoli cells less than 10% of the entire DF
contains the enzyme. Wachtler et al. (1989) could show that
the DF is inhomogeneous by means of nucleolus specific silver
staining. In agreement with this, a previous observation on the
distribution of DNA in nucleoli (Mosgoeller et al., 1993) and
the present study clearly show that the DF as recognized in
routine electron micrographs consists of domains with
different molecular compositions. This kind of heterogeneity
within the nucleolus seems to occur in the DF only; in our data
we have no evidence for a similar molecular focality in the
other components, i.e. FCs or GCs.
Fakan et al. (1988) succeeded in locating pADPRT on
nascent transcripts as visualized on chromatin spread preparations. With the technique available at that time it was not
possible to investigate intranucleolar transcription. We
combined the recently developed non-autoradiographic high
resolution detection of rDNA transcription (Hozak et al., 1994;
Schöfer et al., 1993) with EM pADPRT immunostaining. This
approach enabled us to directly visualize nucleolar transcription and pADPRT simultaneously in situ, without spreading
induced artifacts. We found a close relationship between
Immunological pADPRT detection
415
Fig. 8. Stages of chromatin
condensation during spermiogenesis.
In spermatocytes I (A) when DNA
condenses around the synaptonemal
complex (sc) the concentration of
immunogold-detected pADPRT also
increases in the surrounding region. In
later stages (B) when chromosomes
take shape the condensed chromatin is
heavily labelled by gold particles.
Little or no label is seen in the
nucleoplasm (np). In final stages of
sperm differentiation (C) the highest
signal density is seen over the most
tightly packed chromatin of the
spermatid. Bar, 0.5 µm.
pADPRT foci and nascent intranucleolar RNA in the DF (Fig.
9). Our data do not allow us to suggest a strict colocalization
of pADPRT sites and transcription foci in one point, since the
grain cluster of the different labelling systems did not overlap.
Our interpretation of the label distribution in double labelling
experiments would rather be in favour of a side by side
arrangement of individual foci, which theoretically may reflect
an involvement of pADPRT sites either before or directly after
transcription.
The nascent rRNA has several binding sites for nucleolin
and B23 (Dumbar et al., 1989; Ghisolfi et al., 1990). Since
some binding sites of nucleolin depend on a native hair pin
loop structure of ribosomal RNA and most binding sites are
localized in the 18 S and 28 S sequence, an important role of
the protein in the preribosome assembly was suggested
(Ghisolfi et al., 1990). Interestingly enough, both proteins have
been shown to be poly(ADP-ribose) acceptors (Leitinger and
We˛sierska-Ga˛dek, 1993) despite their acid nature. Since
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W. Mosgoeller and others
Fig. 9. Section of a HeLa cell nucleolus after
BrUTP incorporation and EM double immunostaining of BrUTP incorporation sites (large
grains) and pADPRT (small grains). Focal
accumulations of pADPRT-label (arrowheads)
and BrUTP-label (arrows) may occur completely
independent of each other within the dense
fibrillar component (df) or may be arranged side
by side. The fibrillar centre (fc) is not labelled.
Bar, 0.5 µm.
intranucleolar transcription almost exclusively accounts for
ribosomal RNA production (Hozak et al., 1993) we suggest
that most if not all of the pADPRT, which we detected in the
DF, might be associated with nascent ribosomal RNA. The
association may be mediated by proteins like B23 or nucleolin.
In eukaryotic cells it was not possible to show a direct modification of polymerase I by pADPRT (Momii and Koide, 1982)
although some kind of mutual dependence between the two
enzymes has been established (Taniguchi et al., 1982).
However, the time course given for the pADPRT induced RNA
polymerase I downregulation by Taniguchi et al. (1982) is
highly suggestive of an indirect mechanism.
The binding of pADPRT to ribonucleoproteins (RNPs)
seems to persist throughout later stages of ribosome biogenesis in the GC (Figs 6, 7) and even to the stage of cytoplasmic
ribosomes where pADPRT activity has been described
(Roberts et al., 1975). We did find pADPRT in the GC, where
it appears to be distributed at random over the entire
component (Figs 6, 7). However, the concentrations as
revealed by immunochemisty are low.
We used cells with nucleoli that have clearly distinct
fibrillar centres. However, after evaluating a large number of
sections in the different cell types used, we were not able to
detect significant amounts of pADPRT in the FCs. The
function of FCs in nucleoli is still a matter of controversial
discussion (for review see Schwarzacher and Wachtler, 1993).
FCs are a highly dynamic structure that change shape, number
and size during nucleolar activation (Hozak et al., 1989).
Although they contain many different proteins (including transcription enzymes) they do not necessarily participate in transcription or transcript processing (Hozak, 1995). Since there
is also no nucleosomal DNA present in this component
(Derenzini et al., 1985), this might explain the low concentration or absence of pADPRT in FCs. Furthermore, our data
suggest that no other proteins localized in FCs (e.g. polymerase I, topoisomerase I) are typical candidates for pADPRT
dependent modification.
Chromatin associated pADPRT
Our results concerning the distribution of pADPRT in
chromatin are in agreement with those of Fakan et al. (1988).
It is distributed at random in the condensed parts (Figs 4, 6).
The enzyme may be located in the internucleosomal space
(Mullins et al., 1977; Niedergang et al., 1985) where it may be
involved in a histone shuttling mechanism (Althaus et al.,
1994; Wesierska-Gadek and Sauermann, 1988) or in chromatin
relaxation (de Murcia et al., 1988). Furthermore, there are indications that pADPRT can be associated with nuclear matrix
proteins (Wesierska-Gadek and Sauermann, 1985). During
meiosis the pADPRT label density gives a good reflection of
the grade of chromatin condensation (Fig. 8A-C). This characteristic may well account for the relatively high signal in
nucleolus associated condensed chromatin (Fig. 6) and in
chromatin near the nuclear envelope (Fig. 4).
There is evidence from different cell systems that pADPRT
plays an important role in cell differentiation (Althaus et al.,
1982; Farzaneh et al., 1982) without being dependent on cell proliferation (Ueda et al., 1982). Interestingly pADPRT remains
chromatin associated throughout human sperm differentiation.
The highest enzyme concentrations we observed occurred in the
differentiated sperm head (Fig. 8C). Transcription processes
(Simbulan et al., 1993; Yoshida and Simbulan, 1994) or involvement in gene expression regulation (Qu et al., 1994; Yamagoe et
al., 1991) are unlikely to occur at this late stage of spermatogenesis in inactive cells. During spermiogenesis the nucleolus
decreases in activity and size, while the chromatin condenses as
sperm maturation advances (Kierszenbaum and Tres, 1978; Stahl
et al., 1991). The function of the enzyme at this stage remains
unclear. It may be a ‘dormant, stand-by form’ awaiting involvement in chromatin relaxation (Aubin et al., 1983; Niedergang et
al., 1985). More likely it may bind to DNA breaks (Gradwohl et
al., 1990; Ikejima et al., 1990) that decrease constraints on the
supercoiled DNA. In this case it would be crucially involved in
DNA repair and strand ligation (Creissen and Shall, 1982) before
the chromatin can be decondensed and the genes reactivated.
Immunological pADPRT detection
This work was supported by: the Oesterreichische Nationalbank,
grant no. 4478; the Welcome Trust; the Grant Agency of the Academy
of Sciences of the Czech Republic (no. 539402); and the Grant
Agency of the Czech Republic (no. 304/94/0148). We are grateful to
Dr G. de Murcia for the most generous gift of the baculovirus
construct, expressing the full length human pADPRT. We thank Dr
P. Kier for the serum and Dr H. G. Schwarzacher and Dr F. Wachtler
for helpful discussion and comments.
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(Received 4 September 1995 - Accepted 13 November 1995)