Download perinuclear dense bodies: characterization as dna

J. Cell Set. 80, 1-11 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
PERINUCLEAR DENSE BODIES: CHARACTERIZATION
AS DNA-CONTAINING STRUCTURES USING
ENZYME-LINKED GOLD GRANULES
SIGRID BERGER AND HANS-GEORG SCHWEIGER
Max-Planck-Institut fur Zellbiologie, D-6802 Ladenburg bet Heidelberg, West Germany
SUMMARY
Cytoplasmic structures of the Acetabularia cell that appear close to the nucleus during the
vegetative phase of the life cycle have been characterized cytochemically. The results obtained by
using RNase and DNase linked to gold granules indicate that RNA and also DNA occur in these
cytoplasmic structures.
INTRODUCTION
Interactions between the nucleus and the cytoplasm play a major role in gene
expression and have been investigated in particular in the unicellular and uninucleate
green alga Acetabularia (Schweiger & Berger, 1979).
The ultrastructure of the nucleus and the surrounding cytoplasm has been studied
in detail (Frankeef al. 1974; Berger & Schweiger, 1975; Liddle, Berger & Schweiger,
1976). Preliminary evidence suggested that the perinuclear region of Acetabularia,
although it is extranuclear, contains DNA. The most striking features of this region
are: (1) a 100nm thick perinuclear intermediate zone filled with filamentous material
from which the characteristic cytoplasmic organelles and nuclear particles are
excluded. (2) An adjacent lacunar labyrinth interlaced by many plasmatic junction
channels connecting the intermediate zone and the cytoplasm. (3) Perinuclear dense
bodies (PB), which are only found in close proximity to the intermediate zone and in
particular to the junction channels (Fig. 1). The number of perinuclear dense bodies
(PB) surrounding an Acetabularia primary nucleus is more than 20000 (Franke
et al. 1974). Their structure is composed of fibrillar and granular material (Fig. 2).
They are formed during the early vegetative phase of the life cycle and they disappear
before the onset of nuclear division (Berger et al. 1975).
It has been suggested that the PB contain DNA since a characteristic bleaching of
these bodies was observed with Bernhard's staining technique (Bernhard, 1969;
Franke et al. 1974; Spring, Franke, Falk & Berger, 1975; Liddle et al. 1976).
Although this staining method cannot be considered as final proof of the extranuclear
presence of DNA, it was nonetheless an interesting observation in that it was in
contrast to the generally accepted dogma that DNA is located exclusively in nuclei
Key words: Acetabularia, cytochemistry, enzyme—gold granules, nucleic acids, perinuclear dense
bodies.
S. Berger and H.-G. Schweiger
Fig. 1. Section through nuclear region of A. mediterranea. The nucleus, which usually
has a diameter of 100/an, has just been grazed to demonstrate the surrounding area.
Numerous perinuclear dense bodies are observed in the close neighbourhood of the
nucleus; n, nucleus. Bar, 4/*m.
Fig. 2. Section through nuclear periphery. The nucleus is covered by a —100 nm thick
layer of cytoplasm, surrounded by highly vacuolized cytoplasm. The perinuclear dense
bodies are composed of fibrillar and granular regions with attached granules and some
ribosomes in their vicinity; n, nucleoplasm;/>6, perinuclear dense body. Bar, 1 /im.
DNA-containing cytoplasmic structures
and in cytoplasmic organelles. Non-organelle cytoplasmic DNA has been described
in different organisms by several authors (Kraszweska & Buchowicz, 1983; for
references, see also Reid & Charlson, 1979) but it still represents an unorthodox view
that needs further investigation.
Recently, specific enzyme-gold complexes have been used to localize substrates
cytochemically (Bendayan, 1984). It was shown that nucleases attached to small
colloidal gold beads are still active, undergo enzyme-substrate interactions and
adhere to the substrate (Bendayan, 1981). This cytochemical approach has been
applied to sections through the nuclear and perinuclear regions of Acetabularia cells.
MATERIALS AND METHODS
Cells of Acetabularia mediterranea were grown in a 12h dark/l2h light cycle in an artificial sea
water as previously described (Schweiger, Dehm & Berger, 1977).
Cells in stages before cap formation were selected and fixed by immersion in 1 % glutaraldehyde
buffered with O'l M-phosphate buffer (pH7-4) at room temperature. After 15min the cells were cut
into 2-mm long pieces. Fixation was stopped after two more hours by rinsing the cell fragments six
times in 0-1 M-phosphate buffer for lOmin each. The samples were not subjected to post-fixation
with osmium tetroxide. They were dehydrated in graded series of ethanol and embedded in Epon.
For labelling with antibodies the cells were fixed with 0-17 % glutaraldehyde and 2 % formaldehyde
in 0-1 M-cacodylate buffer (pH7-4) for 90min. The cells were cut into 2-mm long pieces after
15min fixation time and washed six times for lOmin in 0-1 % cacodylate buffer to remove the
fixative. They were dehydrated and embedded in glycol methacrylate (GMA). Thin sections were
mounted on 300-mesh nickel grids covered with carbon-coated collodion film.
For all steps where gold was involved plastic or siliconized glassware was used. The method for
preparing gold granules and for loading the granules with protein followed that described by Frens
(1973) and Horisberger (1981) in general.
For the preparation of colloidal gold 100 ml of 0-01% tetrachloroauric acid (HAuCU) was
heated. As soon as the solution started to boil 2ml 1 % Na3citrate was added. The solution was
boiled for 5 min. During this period the gold chloride was reduced. The formation of colloidal gold
was indicated by the appearance of a light red colour. Immediately after cooling down to room
temperature the solution was adjusted to pH6 - 0 (for loading with DNase) or pH9-0 (for loading
with RNase) or pH6-9 (for loading with protein A) with 0 - 2 M - K 2 C O 3 .
For the preparation of the enzyme-gold complexes either 200/d DNase solution ( l O m g m F 1
DNase I) or RNase solution (Smgml" 1 bovine pancreas RNase A) was added to l m l doubledistilled water and 50 ml of the pH-adjusted gold suspension was added dropwise with constant
stirring. After 2 min 600/il of the suspension was mixed with 100/il 2-5M-NaCl. If the colour of the
enzyme-gold complex did not change from red to violet it was presumed that no gold particles were
flocculated since all of them were coated with enzyme. After three more min of incubation 2-5 ml
1 % polyethylene glycol (Carbowax 20 M) was added to stabilize the complexes.
Preparation of the protein A-gold complexes in general followed the same recipe. The minimal
amount of protein A for loading of the gold granules was determined (Roth, Bendayan & Orci,
1978). A 10 % excess of protein A was used in the preparation of the protein A-gold complexes.
The suspension containing the protein-gold complexes was centrifuged for 50 min at 40 000 £
and 4°C to remove non-bound protein. The supernatant was carefully aspirated. The sediment was
resuspended in 4ml PBS (phosphate-buffered saline; 0-02M-phosphate buffer, 0-13M-NaCl;
pH 6-0 for the DNase-gold complex; pH 7-5 for the RNase-gold complex) or 0-01 M-Tris buffer;
pH 8-0 (for the protein A-gold complex) containing 0-5 mgml" 1 polyethylene glycol. The suspension was gently shaken for 20 min at room temperature and centrifuged for 10 min at 1000 g to
remove clumped granules. The centrifugation steps were repeated once more in order to wash off
all non-bound molecules. The supernatant of the last 1000 £ centrifugation step was used for the
labelling experiments. The enzyme-gold complexes were stored at 4°C. They were freshly
prepared every week.
3
4
5. Berger and H.-G. Schweiger
For control experiments DNase inactivated by heating at 100°C for lOmin was bound to gold
granules. Pilot and control experiments were performed on mouse liver sections that had been fixed
and embedded under the same conditions as thtAcetabularia cells.
For cytochemical labelling with enzymes the grids with the sections were floated for 5 min on
PBS (pH6'0 for DNase labelling, pH7-5 for RNase labelling) containing O-Smgml" 1 polyethylene glycol. The grids were transferred to the corresponding enzyme-gold complex and floated on
the suspension for 45-55 min at either room temperature, 30°C, 34°C or 37CC. The grids were
floated six times for 5 min on PBS plus polyethylene glycol, rinsed briefly with double-distilled
water and stained with 5 % uranyl acetate for 15 min.
For cytochemical labelling with antibodies the loaded grids were floated for 2 min on a solution
containing inactivated (30 min 56°C) anti-rabbit immunoglobulin G (IgG) that had been diluted
1:10 with 0-01 M-Tris buffer (pH8-0). The grids were then washed twice for 10 min with Tris
buffer. Thereafter the grids were floated on a solution of anti-actin antibodies from rabbit that had
been diluted 1:5 with Tris buffer for 2h at either room temperature or 34°C. Non-bound
antibodies were removed by washing the grids five times for 10 min with Tris buffer. The grids
were then transferred to gold-labelled protein A that had been diluted 1:5 with Tris buffer
containing O-Smgml" 1 polyethylene glycol. They were incubated for 60 min at room temperature.
Non-bound protein A was removed by washing six times for 10 min with Tris buffer and polyethylene glycol. They were rinsed briefly with double-distilled water and stained with 5 % uranyl
acetate for 15 min.
The sections were examined with a Siemens I or Philips 400 T electron microscope.
RESULTS
Low concentrations of glutaraldehyde, short times for fixation and omitting
osmification, as used in this study, affect the preservation of subcellular structures.
But even under these conditions the major cellular structures of thtAcetabularia cell
can be identified unequivocally. No significant difference in labelling intensity was
observed at different temperatures if RNase-coated gold granules were used.
In the case of DNase-gold particles the labelling was weak at room temperature
and much more pronounced at higher temperatures. Optimum labelling with a low
background was achieved at 34°C. At 37°C the labelling was as good but the
background was increased.
Incubation with RNase-gold granules resulted in a distinct labelling of the
nucleus (Fig. 3). Especially intense labelling was observed in the granular component of the nucleolus. The PB were also labelled but to a lesser extent (Fig. 4).
After incubation of sections with DNase—gold complexes labelling was observed
over the whole nucleus. The labelling was denser in the vicinity of the nuclear
membrane. It was rather weak in the nucleolus (Figs 5, 6). No labelling was found in
the nucleolar vacuoles. The most striking feature of sections incubated with DNasegold complexes was the labelling of the PB (Figs 5, 6, 7). The label was not evenly
distributed but was preferentially observed close to or in the vicinity of the fibrillar
component.
In a control experiment sections were incubated with gold granules that had been
loaded with inactivated DNase. In this case the DNase-gold complexes had lost
their labelling capability and no labelling was found throughout the sections (Fig. 8).
Using the same RNase-gold or DNase-gold complexes on mouse liver sections
the labelling occurred at the expected sites. Since actin is known to be a naturally
occurring inhibitor of DNase I (Lazarides & Lindberg, 1974; Mannherz, Barrington
DNA-containing cytoplasmic structures
n
•**
.** :
I
Fig. 3. Section through the nucleus, which has been incubated with gold-labelled
RNase. RNase attached to the nucleolus and the nucleoplasm; n, nucleoplasm; no,
nucleolus. Bar, 2 fan.
Fig. 4. Section through one pennuclear dense body, which has been incubated with
gold-labelled RNase. The dense body shows a* pronounced labelling indicative of a high
RNA content. Bar, 0-3 /an.
S. Berger and H.-G. Schiveiger
' -i r «'
pb
Fig. 5. Section through part of the nucleus and attached perinuclear dense bodies, which
have been incubated with gold-labelled DNase. The labelling is found in the nuclepplasm
but not in the granular region of the nucleolus. The perinuclear dense bodies are clearly
labelled indicating DNA content, n, nucleoplasm; no, nucleolus; pb, perinuclear dense
body. Bar, O-S/im.
DNA-containing cytoplasmic structures
•31
• .
n '
-' «>
/7
• ,
**r
Figs 6, 7. Section through part of the nucleus and attached perinuclear dense bodies,
which have been incubated with gold-labelled DNase. The labelling, which shows
attachment of DNase, is observed in the nucleus and perinuclear dense bodies, n,
nucleoplasm;/>£>, perinuclear dense body. Bars, 0-5 [tin.
S. Berger and H.-G. Schiveiger
^
tf K «•/7
8
Fig; 8. Section through part of the nucleus and attached perinuclear dense bodies, which
have been incubated with gold-labelled inactivated DNase. No labelling is observed on
the section, n, nucleoplasm;/>6, perinuclear dense body. Bar, 1 ftm.
Leigh, Leberman & Pfrang, 1975; Hitchcock, Carlsson & Lindberg, 1976; Zechel,
1980) it had to be determined whether gold-labelling of the sections after incubation
with DNase is due to complexes formed between DNase and actin. Therefore,
sections through the nuclear region were incubated with anti-actin antibodies and
thereafter with gold-labelled protein A. The labelling of the sections was rather
high whether the incubation was performed at room temperature or at 34 °C.
DNA-containing cytoplasmic structures
A comparison of the labelling of different areas in the cell showed that there is no
preferential binding of anti-actin antibodies in the PB (Table 1).
DISCUSSION
The technique of incubating sections with enzyme-gold complexes has been used
to localize nucleic acids in the nuclear region of Acetabularia cells. Nucleases lose
their capability of digesting nucleic acids after fixation with aldehydes and embedding in Epon (Monneron & Bernhard, 1966). However, the enzymes retain their
ability to establish stable binding to their substrates, presumably at their active sites
(Bendayan, 1981, 1982).
Studies on mouse liver sections by Bendayan (1981) and in our laboratory have
shown that the enzyme-gold technique gives reliable results. The activity of the
enzyme—gold complex strongly depends on the kind and duration of fixation as well
as on the pH at which the sections are incubated.
The results presented in this paper indicate the presence of RNA in the PB of
Acetabularia although the labelling is substantially weaker than in the nucleolus and
resembles that of the nucleoplasm. This observation suggests that there is RNA in
the PB but that the packing of the RNA is not very dense. On the other hand it could
mean that the RNA in the PB is highly protected against the attachment of RNase.
Attachment of DNase-coated gold particles is mainly observed in the nucleoplasm
and in the PB. The nucleoplasm is more or less evenly labelled with a slight preference for the vicinity of the nuclear membrane. This might mean that in the primary
nucleus the DNA is evenly distributed throughout the nucleus and that its location is
not restricted to a certain part of the giant primary nucleus.
The distinct labelling of the PB by the DNase-coated gold particles represents
additional evidence for the occurrence of DNA in the perinuclear region, i.e. outside
the nuclear membrane. Although it cannot be completely ruled out that the binding
of the DNase—gold particles is non-specific, the failure of heat-inactivated DNase—
gold granules to label indicates that the binding sites are specific for active DNase.
Similar conclusions can be drawn from studies on mouse liver sections. Furthermore, the results obtained with DNase-gold particles are in good agreement with
studies using Bernhard's (1969) cytochemical method, which indicated the presence
of DNA in the PB.
The question arises as to what is the role and the function of DNA in the PB.
Incubation of sections with antibodies against the small subunit of cytoplasmic
ribosomes (60 S) resulted in intense labelling of the granular region of the nucleolus
and distinct labelling of the PB (unpublished results). These results indicate that
amplified rRNA genes occur outside the primary nucleus of Acetabularia.
If in Acetabularia nuclear DNA occurs outside the nucleus the question may be
asked whether the morphogenetic capability of anucleate cells might be due to extranuclear DNA. This, however, is highly improbable since the PB are found exclusively in close proximity to the nucleus. They are removed together with the nucleus
when the rhizoid is removed.
9
10
5. Berger and H.-G. Schweiger
Table 1. Density of gold granules over different areas in an actin antibody control
experiment
Structure
Cytoplasm
Perinuclear dense bodies
Nucleus
Analysed area
(mm2)
6-IS
1-69
5-18
Gold granules
counted (Xl(T 3 )
14-6
2-5
10-0
Gold granules
(mm~ z Xl0~ 3 )
2-4
1-5
1-9
The characterization of the structure and the function of the perinuclear dense
bodies demands detailed studies on isolated preparations of these particles.
Unfortunately, to date attempts to isolate the PB have been unsuccessful.
The authors thank Mrs Ingrid B6nig and Miss Inge Pfrang for their skilful technical assistance.
The photographic assistance of Mrs Renate Fischer as well as the secretarial assistance of Miss
Christiane Schardt are highly appreciated. The authors acknowledge the English revision of the
manuscript by Dr Mark Wiser.
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{Received 17June 1985 -Accepted 4 July 1985)