J. Cell Set. 84, 53-67 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
53
STRUCTURAL INTEGRITY OF THE NUCLEAR
MATRIX: DIFFERENTIAL EFFECTS OF THIOL
AGENTS AND METAL CHELATORS
PETER A. DIJKWEL* AND PAUL W. WENINK
Department of Chemical Cytology, University of Nijmegen, Toernooiveld, 6525 ED
Nijmegen, The Netherlands
SUMMARY
Nuclear matrices, associated with over 80% of the chromosomal DNA, could be isolated from
BHK nuclei by extraction with 2M-NaCl. The matrices were found to impose at least two levels of
structural order upon nuclear DNA.
From sedimentation studies it was inferred that metal depletion of the salt-extracted nuclei
generated matrix structures, which sedimented at significantly lower rates than control matrices.
Fluorescence microscopy revealed that the reduced sedimentation rate is a consequence of the
increase in the radius of the DNA halo, i.e. the DNA loops emanating from the residual nucleus.
Addition of Cu ions to nuclei prior to salt extraction was found to induce contraction of this DNA
halo. These results indicate that Cu ions may play an important role in stabilizing one level of DNA
folding.
When metal depletion had been brought about by thiol agents, a second effect was observed to
occur. Within 15min, salt-extracted nuclei disintegrated, generating irregularly shaped, slowly
sedimenting structures. Disintegration only occurred when the full complement of DNA was still
attached to the nuclear matrices.
Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that treatment
with thiols did not detectably alter the polypeptide composition of DNA-depleted residual nuclei.
Results of these experiments suggest that both metal-protein interactions and disulphide bonds
are important in maintaining higher-order structure in the nucleus. A model to account for these
observations is discussed.
INTRODUCTION
In both interphase nuclei and metaphase chromosomes higher-order structure of
chromatin is mediated by non-histone chromosomal proteins. For the packing of the
chromatin fibre in chromosomes a 'radial loop' model has been proposed (Laemmli
et al. 1978; Marsden & Laemmli, 1979), i.e. chromatin fibres are attached to an axial
proteinaceous scaffolding in a loop-wise fashion. Comparable structural order is
thought to exist in interphase, the framework being provided by the nuclear matrix
(Berezney & Coffey, 1974; Cook & Brazell, 1976; Wankaef al. 1977).
Since the nuclear matrix has been shown to be involved in a variety of nuclear
functions, such as DNA replication (Berezney & Coffey, 1975; Dijkwel et al. 1979;
Pardoll et al. 1980), RNA transcription and processing (Jackson et al. 1981;
•Author for correspondence.
Key words: nuclear matrix, thiol agents, metal chelators.
54
P. A. Dijkwel and P. W. Wenink
Mariman et al. 1982), hormone-receptor binding (Barrack & Coffey, 1980) and
binding of carcinogens (Hemminki & Vainio, 1979; Tew et al. 1980), considerable
effort has been invested in order to elucidate nuclear matrix structure and establish
its protein composition. In this approach, the nuclear matrix has been operationally
denned as the residual structure obtained after treatment of nuclei with non-ionic
detergent and extraction with high concentrations of NaCl. Generally, the residual
structure consists of approximately 10 % of total nuclear protein (Wanka et al. 1977;
Lebkowski & Laemmli, 1982a), of which the lamins are the most prominent class
(Mullenders et al. 1982; KaufmanneJ al. 1981; Lebkowski & Laemmli, 19826), and
varying amounts of chromosomal DNA, depending on whether or not nuclease
treatment is included in the isolation procedure.
Though some insight into the three-dimensional architecture of the nuclear matrix
has been provided (Brasch, 1982; Capco & Penman, 1983), the precise way in which
matrix proteins are ordered to form a nuclear scaffolding remains to be established.
This greatly complicates the attribution of functions to specific nuclear constituents.
A novel attempt to establish the positioning of subnuclear structures, to which DNA
is attached, was recently reported (Lebkowski & Laemmli, 1982a,b). It was reported
that bivalent cations, notably Cu 2+ , are required for one level of DNA organization
in the nucleus. Removal of this ion resulted in expansion of the DNA halo and loss of
several proteins (Lebkowski & Laemmli, 19826). The effect could be observed both
when metal chelators such as orthophenantroline, and thiol agents such as mercaptoethanol and dithiothreitol, were added. However, a study reported previously (Cook
et al. 1976), in which HeLa nucleoids were found to be unstable in dithiothreitolcontaining media, seems to question the results of the study mentioned earlier as far
as the thiol effect is concerned. The aim of the present study was to evaluate the
relevance of the data presented to date concerning the effects of thiol agents on the
nuclear matrix.
MATERIALS AND METHODS
Cell culture and preparation of nuclear matrices
BHK-A3 cells (Cupido & Simons, 1984), obtained from the Department of Radiation Genetics
and Chemical Mutagenesis at Leiden University, were maintained in monolayer culture in Minimal
Essential Medium (MEM; Flow Labs) supplemented with 8% (v/v) foetal calf serum (Gibco).
Cells were labelled for three to five generations with 1/iCiml" 1 L-[4,5-3H]leucine (sp.
act. 4 6 C i m m o r ' ; Amersham) and 0-02/iCiml" 1 [2-14C]thymidine (sp. act. 5 2 - 8 C i m o r ' ;
Amersham).
Nuclei were isolated essentially as described (Wanka et al. 1977). Monolayers were rinsed
sequentially with 0-9 % NaCl in 5 mM-Tris • HC1 (pH 8-0) and with 0' 1 % Triton X-100 in 5 mMTris-HCl (pH8-0) ( T T buffer). Cells were then washed from the glass surface with T T buffer.
Nuclei were isolated by forcing the suspension twice through a hypodermic needle (0-8 mm
diameter) and centrifuging the suspension subsequently for 3 min at SOOg. The nuclear pellet was
resuspended in 50mM-Tris-HC1 (pH8 - 0) and checked for contamination by phase-contract
microscopy. Finally, an equal volume of 4M-NaCl in SO mM-Tris • HC1 (pH 8-0) was added to the
nuclear suspension.
All steps were performed on ice.
Structural integrity of the nuclear matrix
55
Centrifugation and determination of radioactivity
After appropriate treatment with thiol agents or metal chelators, 5-ml samples of nuclear lysate
in 2M-NaCl were carefully layered on 5 % to 2 5 % sucrose gradients containing 2M-NaCl and
50mM-Tris-HCl (pH8-0). The gradients were prepared on 65% sucrose shelves containing
0-4gml~' CsCl. Centrifugation was performed at 4°C in Beckman SW 2711 rotors at 5000
rev. min" 1 for different periods of time.
After centrifugation, fractions were collected starting from the bottom of the tube. Trichloroacetic acid was added and the acid-insoluble material was pelleted by centrifugation, washed once
with 70% ethanol and dried. Subsequently, the pellet was allowed to dissolve in 0 - 2M-NaOH at
80°C. After 30 min an equal volume of 1 M-perchloric acid was added and hydrolysis was allowed to
proceed for another 30 min at 80°C. After addition of scintillation mix (Packard) the radioactivity
in the samples was determined in a Philips LSA.
Fluorescence microscopy
From the nuclear lysates prepared for sedimentation analysis samples were taken to which
4/igml~' ethidium bromide was added. The sample was then viewed with a Zeiss Photomikroskop
III, using a 50 W HBO-mercury lamp. Photographs were taken with high-speed films (Kodak VR
1000) to minimize illumination time.
Poly aery lamide gel electrophoresis
For analysis of the polypeptide composition of nuclear matrices, nuclei were DNA-depleted
either by treatment with DNase I in the presence of 1 mM-MgCl2 or by digestion with
staphylococcal nuclease in the presence of 0-1 mM-CaCl2. The nuclei were then collected, suspended in 50 mM-Tris • HC1 (pH 8-0) and extracted with 2 M-NaCl. Protein samples were prepared
as described (Pieck et al. 1985). Protein samples were dissolved in sample buffer (Laemmli, 1970)
supplemented with 6M-urea and analysed on 4 % to 18% polyacrylamide slab gels according to
Laemmli (1970).
When the effect of thiols and the polypeptide composition of nuclear matrices was analysed, all
solutions used were supplemented with either /J-mercaptoethanol or dithiothreitol in the appropriate concentrations. Gels were stained with Coomassie Blue.
RESULTS
Effects of thiol agents
As from a variety of eukaryotic cells, matrices could be prepared from nuclei of
BHK cells by extraction with 2M-NaCl. Fig. 1A shows the sedimentation profile on
neutral sucrose gradients of salt-extracted nuclei, of which DNA and protein were
labelled with [14C]thymidine and [3H]leucine, respectively. Approximately 80% of
the DNA and 20 % of the nuclear proteins are found to sediment to the lower half of
the gradient heterogeneously, as indicated by the broad peak. As expected for
different labels residing in the same structure, the distribution of the protein label
closely follows that of the DNA label. Residual nuclei were observed to sediment
with average values between 6000 and 9000 S. From the top of the gradients, most of
the protein and a small amount of DNA were invariably recovered. Both fractions
were unable to enter the gradient under the conditions chosen.
Previous studies have shown that inclusion of /3-mercaptoethanol and dithiothreitol (DTT) in the isolation procedure resulted in a decrease in the sedimentation
rate of residual nuclei in sucrose gradients. Fluorescence microscopy revealed an
increase of the average radius of the DNA halo by a factor of 2. Most probably, the
56
P. A. Dijkwel and P. W. Wenink
DNA-unfolding occurred as a consequence of the collapse of internal matrix
structure as a result of chelation of either copper or calcium ions (Lebkowski &
Laemmli, 1982a).
We also observed that addition of thiol agents, in concentrations ranging from
0-5M to 50mM, to nuclei suspended in 2M-NaCl, induced significant changes. As
shown in Fig. IB, addition of DTT destroys the rapidly sedimenting component.
Almost all label applied to the gradient is recovered at the uppermost fractions. Only
a small fraction, approximately 15% of the DNA cosedimenting with 5 % of the
protein, was found to have entered the gradient with an s value of 1500. Similar
results were obtained when thiol agents had been present in the isolation procedure
from the nuclear isolation step onwards. Neither addition of MgCl2 in millimolar
amounts, nor the inclusion of 0-5mM-phenylmethylsulphonyl fluoride (PMSF) was
found to affect these observations.
To establish more directly to what extent residual nuclei are morphologically
altered in thiol-containing high-salt buffers, the samples were also subjected to
fluorescence microscopy. Prior to addition of thiols, salt-extracted nuclei were
characterized by a uniformly fluorescing residual nucleus surrounded by a less
intensely fluorescing DNA halo (Fig. 1C). Addition of 10mM-DTT had two
immediate effects. Concomitant with the loss of the uniform appearance of the
residual nuclear core, an expansion of the DNA halo was observed (Fig. ID). These
structures were found to be unstable. Consequently, it is difficult to estimate the
radius of the expanded halo. Nevertheless, our data indicate a 1-5-fold increase in the
10
15
b
Fraction number
10
15
Fig. 1. Disintegration of residual nuclei in high-salt buffer containing dithiothreitol.
Cells were continuously labelled with [3H]leucine and [14C]thymidine. Nuclei were
isolated and extracted with 2M-NaCl. A. Sedimentation of salt-extracted nuclei on a
linear 5 % to 3 5 % sucrose gradient; centrifugation for 60min at 5000rev. min" 1 at 4°C.
(O
O) [3H]leucine, 21 OOOdisintsmin"1; ( •
• ) [14C]thymidine, 57700disints
1
min" . B. Sedimentation of salt-extracted nuclei prepared in the presence of lOmM-DTT
(as for A). (O
O) [3H]leucine, 21 OOOdisintsmin
>) [ u C]thymidine,
60 300 disints min '. Fluorescence visualization of residual nuclei, stained with 4 i*g ml '
ethidium bromide in: C. 2M-NaCl; D, 2M-NaCl, immediately after addition of 10mMD T T ; E, 2M-NaCl, 20min after addition of lOmM-DTT.
Structural integrity of the nuclear matrix
57
58
P. A. Dijkwel and P. W. Wenink
radius (control nuclei, 12 ± 2 fim, n - 11; D T T nuclei, 19 ± 3 fim, n - 9). Finally,
approximately 15 min after addition of thiol agents to the salt-extracted nuclei, only
the structures shown in Fig. IE could be observed. In these structures the nuclear
matrix appears to have disintegrated. Consequently, these structures have no
characteristic shape. They seem to consist of aggregates of material originating from
the former residual nucleus spread out over a background of diffusely fluorescing
DNA. Furthermore, as a consequence of their dramatically increased diameters,
these structures are expected to sediment at rates considerably lower than those of
control salt-extracted nuclei.
Effects of metal chelators and reducing agents
As thiols are bifunctional, i.e. metal chelators as well as reducing agents, we tried
to unravel these effects by using EDTA and 1,10-orthophenantroline (OP) on the
one hand and NaBH 4 on the other.
Addition of lmM or lOmM-EDTA to salt-extracted nuclei had no effect on
sedimentation characteristics or on morphology. However, OP, tested in concentrations between (M mM and 50 mM, did affect residual nuclei. Compared to control
matrices, sedimenting at 9000 S (Fig. 2A), matrices prepared in 1 mM-OP sedimented at a reduced rate of 5000 S (Fig. 2B). The amount of DNA and protein in
these structures was not detectably altered by OP.
To visualize the effect of OP by fluorescence microscopy an alternative method
was adopted. Salt-extracted nuclei were prepared, attached to coverslips (Vogelstein
et al. 1980). This makes gentle removal of the OP-containing high-salt buffer
possible, which was imperative because of the high background fluorescence of OP
under these circumstances. Fig. 2E shows that OP-treated residual nuclei are
surrounded by a more-extensive halo than control matrices (Fig. 2D). Moreover,
and as in the initial stage of thiol-mediated expansion, features within the residual
core can be observed. However, structures prepared in OP were stable. From this we
conclude that the decrease of the sedimentation rate is a consequence of the
expansion of the DNA halo.
Therefore, a role for metal ions in nuclear structure is likely. This conclusion is
supported further by results of experiments in which, immediately prior to saltextraction, 1 mM-CuCl2 was added to nuclei. Nuclear matrices obtained from these
nuclei were found to sediment at higher rates (Fig. 2C) than the corresponding
controls (Fig. 2A). It should be noted, however, that the residual structures prepared in the presence of copper ions, contained significantly more protein than the
control matrices.
Results of fluorescence microscopy were consistent with the sedimentation data.
Fig. 2F shows a residual nucleus prepared as a control. Salt-extraction of nuclei
prepared in the presence of 1 rnM-CuC^, yielded structures with a significantly
contracted halo (Fig. 2G). Moreover, copper ions were found to counteract the
effect of OP on residual nuclei (data not shown).
From the data presented to date it can be concluded that in addition to metal
chelation, thiol agents have another effect on nuclear matrices. This effect could arise
Structural integrity of the nuclear matrix
59
as a consequence of reductive disruption of disulphide bonds, which might stabilize
the structure. This possibility was assessed by treating salt-extracted nuclei with
NaBH4. However, 1 mM-NaBH4 did not convert salt-extracted nuclei to a slowly
sedimenting form. On the contrary, a slight increase in sedimentation rate was
observed (data not shown), consistent with data presented by Lebkowski & Laemmli
(1982a). As expected, morphological changes were not found to be induced by
NaBH4, even when the concentration was raised to lOmM (data not shown).
Since neither metal chelators nor reducing agents, when used separately, can
induce the thiol-induced disintegration of the nuclear matrix, it must be concluded
that the bifunctional character of thiols is a prerequisite for their action.
The role ofDNA in thiol-mediated disintegration
In contrast to nuclear matrices, nuclei appeared to be stable in thiol-containing
buffers (data not shown). The most obvious effect of addition of 2M-NaCl to nuclei
is removal of histones from the DNA. Consequently, the now naked DNA will adopt
a configuration minimizing repulsion. As DNA is anchored to the nuclear matrix at
more or less regular intervals, the well-known halo surrounding the proteinaceous
matrix will be the endpoint of this process. In this configuration, though minimized,
repulsion is not reduced to zero. Consequently, destabilization of the residual
nucleus, for instance by the action of the bifunctional thiols, might result in further
expansion and, eventually, disintegration.
To assess whether this is the case, salt-extracted nuclei were prepared; some of
these were subsequently treated with DNase I to remove most of the DNA attached.
Sedimentation analysis revealed that the DNA-rich matrices disintegrate in the
presence of DTT (namely, Fig. 1). However, DTT was found not to affect the
sedimentation behaviour of DNA-depleted matrices (Fig. 3A,B). Fluorescence
microscopy showed that, in contrast to residual nuclei not treated with DNase I,
DNA-depleted matrices (Fig. 3C) were stable in DTT-containing high-salt buffer
(Fig. 3D).
These results indicate that, though thiols have an effect on salt-extracted nuclei,
this by itself is not sufficient to destabilize the structures to such an extent that
disintegration occurs.
The effect of thiols on the polypeptide composition of residual nuclei
Destabilization of salt-extracted nuclei by thiol agents might be related to
dissociation of certain proteins from these structures. Consequently, nuclei were
isolated both in the absence and in the presence of DTT. Chromatin was then
removed by nuclease digestion after which the nuclei were salt-extracted.
Lane b of Fig. 4 shows the polypeptide composition of nuclear matrices isolated
in the absence of thiols. The prominent bands in the 60-70 (XlO3)Mr range
represent the lamin proteins. Comparison with lane c shows that the presence of
D T T during the isolation procedure does not change the protein composition,
except for a protein of high molecular weight. The relative amounts of this protein
were found to vary considerably from one matrix preparation to the other, indicating
60
P. A. Dijkwel and P. W. Wenink
5
10
Fraction number
15
Structural integrity of the nuclear matrix
Fig. 2. Reversible unfolding of DNA induced by 1,10-orthophenantroline and Cu 2 + .
Residual nuclei were prepared as described. A. Sedimentation of salt-extracted nuclei on
a linear 5 % to 25 % sucrose gradient; centrifugation for 60 min at 5000 rev. min"' and at
4°C. (O
O) [3H]leucine, 53 200 disints min" 1 ; ( •
• ) [14C]thymidine, 16300
disints min"'. B. Sedimentation of salt-extracted nuclei prepared in the presence of 1 mM• ) [ 14 C]thymidine,
OP (as for A). (O
O) [3H]leucine, 67000disintsmin~^; ( •
22 500 disints min" 1 . C. Sedimentation of salt-extracted nuclei prepared in the presence
of lmM-CaCl 2 (as for A). (O
O) [3H]leucine, 43 100 disints min" 1 ; ( •
•)
[ 14 C]thymidine, 17 800 disints min" 1 . For fluorescence visualization cells, grown on
coverslips, were treated with 0-5% NP-40, 0-0, 0-4, 0-8, 1-2, 1-6 and 2-OM-NaCl in
5 mM-Tris • HCl (pH 8-0) containing 5 mM-MgCl2 for 30 s sequentially. Then the residual
cells were stained with 4 ^ g m l " ' ethidium bromide, either in the absence (C) or presence
(D) of 1 mM-OP. For analysis of the effect of Cu z + by fluorescence microscopy, residual
nuclei were prepared in the usual way: F, a residual nucleus in 2M-NaCl; G, a residual
nucleus, to which 1 mM-CuCl2 had been added prior to extraction.
61
62
P. A. Dijkwel and P. W. Wentnk
10
15
b
Fraction number
10
15
Structural integrity of the nuclear matrix
63
it might consist of a complex of matrix proteins. Disulphide bonding most probably
generates this complex, as indicated by its greatly reduced abundance in matrices
prepared in the presence of DTT.
Furthermore, from the banding pattern depicted in lane c it can be inferred that
salt-extracted nuclei are essentially free of proteolytic activity which is activated by
sulphydryl groups. Consequently, the disintegration observed to occur in thiolcontaining media cannot be ascribed to protein degradation by enzymes of this type.
a
x10
-94
_
-68
-43
*-
-29
Fig. 4. Effect of thiols on the polypeptide composition of DNA-depleted residual nuclei.
Nuclei were isolated, digested with nuclease and extracted with 2M-NaCl. Nuclear
matrix proteins were subsequently prepared for electrophoresis on 4 % to 18% polyacrylamide slab gels. The Coomassie Blue staining patterns are depicted: lanes A,D,
marker proteins; lane B, nuclear matrix proteins obtained from matrices isolated in the
absence of thiols; lane C, nuclear matrix proteins obtained from matrices isolated in the
presence of lOmM-DTT.
Fig. 3. Effect of dithiothreitol on DNA-rich and DNA-depleted residual nuclei.
Residual nuclei were prepared as described for Fig. 1. A. Sedimentation of salt-extracted
nuclei incubated for lOmin with 50 units ml" DNase I prior to salt extraction, on a
linear 5 % to 2 5 % sucrose gradient. Centrifugation was for 45 min at 5500 rev. min" 1 ,
at 4°C. ( O — O ) [3H]leucine, 23 lSOdisintsmin" 1 ; ( •
• ) [ 14 C]thymidine,
11800disintsmin~'. B. As for A; sedimentation in the presence of lOmM-DTT.
• ) [14C]thymidine, 13000disints
(O
O) [3H]leucine, 25 4O0disintsmin~ 1 ; ( •
1
min" . Fluorescence visualization of a residual nucleus in: C, 2M-NaCl, after removal of
DNA with DNase I; D, 2M-NaCl, containing lOmM-DTT, after removal of DNA with
DNase I.
64
P. A. Dijkwel and P. W. Wenink
DISCUSSION
In this paper the nature of the protein-protein interactions stabilizing nuclear
matrix structure was probed with thiol agents, metal chelators and reducing agents.
Effects of these substances on salt-extracted nuclei were analysed by sedimentation
analysis and fluorescence microscopy.
Nuclear matrices were found to sediment rather heterogeneously on sucrose
gradients. The close resemblance of the sedimentation profiles of DNA label and
protein label indicate that this is a consequence of the heterogeneity of the matrix
population. Fluorescence microscopy revealed residual nuclei to be surrounded by
the characteristic DNA halo.
Addition of the metal chelator OP to salt-extracted nuclei led to a decrease in the
average sedimentation rate of the structures. As neither the relative amount of DNA
nor the proportion of protein residing in the residual nuclei was affected by OP, the
decrease in the sedimentation rate was considered to be a consequence of DNA
unfolding. This assumption was proved to be correct by fluorescence microscopy.
Concomitant with DNA decompaction, internal features of the salt-extracted nuclei
became visible.
Our results confirm data presented by Lebkowski & Laemmli (1982a), indicating
involvement of divalent cations, presumably copper, in long-range order of DNA in
matrix structures. Our study also indicates that copper is important for the maintenance of matrix structure. Salt extraction of nuclei, suspended in CuCl2-containing
buffers, gave rise to matrices sedimenting at increased rates. These matrices were
found to be surrounded by contracted halos. Furthermore, our observations suggest
that the heterogeneous sedimentation behaviour of control matrices might be
accounted for by the fact that nuclei lose their divalent ions to different extents
during isolation.
Though copper ions appear to be involved in maintaining long-range DNA order
in nuclei, some care has to be taken in interpretating the results along these lines.
Copper-treated residual nuclei were found to contain significantly more protein than
control matrices, while the amount of DNA was found unchanged. Increase in mass
could therefore contribute to the increase in the sedimentation rate. Moreover,
merely as a consequence of twice the regular amount of protein in the matrix,
unfolding of DNA might be hampered physically. Consequently, the radius of the
DNA halo would be reduced.
Addition of thiol agents to nuclei suspended in 2M-NaCl initially had effects
comparable to those of OP. The radius of the DNA halo was found to increase by a
factor 1-5. However, in contrast to experiments in which the metal chelator had been
used, this situation did not represent an endpoint. In the course of 15 min all residual
nuclei expanded greatly losing the shape characteristic of the original nucleus. Since
the nuclear lamina, a prominent feature of the nuclear matrix, appeared disrupted in
the expanded structures, loss of the original morphological appearance might be a
consequence of this disruption.
Structural integrity of the nuclear matrix
65
The lamina as a target for thiols has been made plausible by several studies
(Shelton & Cochran, 1978; Cobbs & Shelton, 1978; Lamm & Kasper, 1979), which
indicate that nuclear envelope proteins can be reversibly crosslinked. Whether or not
internal matrix structure is in some way affected by disruption of disulphide bonds
could not be established (Kaufmann et al. 1981).
Since Lebkowski & Laemmli (1982a) did not observe the disintegrating effect of
thiol agents on HeLa nuclear matrices, we investigated whether our observations
were specific for BHK cells. It was found, however, that salt-extracted nuclei
obtained from CHO, bovine liver and HeLa cells all responded to thiols similarly.
The disintegration observed to occur in thiol-containing high-salt media might
therefore be a universal phenomenon. Our conclusion is supported by an observation
of Cook et al. (1976). They reported destruction of HeLa nucleoid integrity in media
containing 50mM-DTT.
DNA-depleted residual nuclei were stable in thiol-containing 2M-NaCl. It is
therefore concluded that, after metal depletion and disruption of disulphide bonds,
nuclear matrix proteins are still able to interact to such an extent that a structure
reminiscent of that of the original nucleus is preserved. This is consistent with
previous results, which indicate that after removal of DNA, residual nuclei could be
obtained from /8-mercaptoethanol-containing high-salt buffers (Kaufmann et al.
1981). However, when forces, such as are generated by the dehistonized DNA
complement, are exerted on these structures the matrices fall apart.
Assuming that chromosomal DNA is attached to the nuclear matrix both
internally and peripherally, our results suggest that the nuclear lamina might be
stabilized by disulphide bonding while divalent cations stabilize the internal matrix.
Metal depletion leads to a residual structure, in which the lamina remains intact, but
as suggested by Lebkowski & Laemmli (1982a) the internal matrix has collapsed.
Consequently, the radius of the DNA halo increases and the nuclear interior acquires
an empty appearance. When both metal depletion and disruption of disulphide
bonds have been induced, the lamina is also destabilized. Disruption follows
subsequently if a considerable amount of DNA is still attached to the residual
nucleus. The model further suggests that mere disruption of disulphide bonds by
reducing agents will have no observable effect. The lamina might be disrupted, but
as the internal matrix is thought to be unaffected, indicated by the absence of a
change in the radius of the DNA halo, the residual nucleus will be prevented from
disintegrating. Alternatively, our observations might also fit the dimercaptide model
that was proposed recently (Jeppesen & Morten, 1985). In agreement with this study
it was also found that for disintegration to occur, thiol-mediated elimination of
specific proteins from the residual nuclei does not seem to be required.
We are grateful to J. Eijgensteijn and J. Poddighe for performing the protein analyses and to Dr
F. Wanka for his advice and discussions during the preparation of this manuscript. This study was
supported by the Dutch Cancer Foundation (KWF), grant SNUKC 81-10 to Dr F. Wanka.
66
P. A. Dijkwel and P. W. Wenink
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(Received 27 September 1985 -Accepted, in revised form, 5 February 1986)