* Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611; and Institut fuer
Biochemie und Molekulare Zellbiologie der Universität Goettingen, D-37073 Goettingen, Germany
The nuclear lamina is a fibrous structure that
lies at the interface between the nuclear envelope and the
nucleoplasm. The major proteins comprising the lamina,
the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear
lamins have been associated with a number of processes in the nucleus, including DNA replication. To
further characterize the specific role of lamins in DNA
replication, we have used a truncated human lamin as a
dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also
disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and
the mutant lamin protein are found in nucleoplasmic
aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC
complex, proteins required for the elongation phase of
DNA replication, are altered such that they are found
within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA
polymerase , proteins required for the initiation stage
of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.
The nuclear lamin proteins form a fibrous structure,
termed the nuclear lamina, which is concentrated at
the nucleoplasmic face of the nuclear envelope (40).
The lamins are also found in nucleoplasmic foci, the distribution of which is related to the cell cycle (7, 18, 32, 39, 48).
The lamins are highly conserved proteins that are closely
related to cytoplasmic intermediate filament (IF)1 proteins
and, as such, are classified as type V IF (51). In vertebrates, as many as five lamin proteins have been reported. These are divided into two types, A and B, based on criteria such as expression patterns and exon positions (53).
B-type lamins are expressed in all cells, while the A-type
lamins are expressed in differentiated cells (40). In Xenopus laevis, there are five or more lamins, also showing cell
type-specific expression patterns (3, 40, 54). As is the case
with cytoplasmic IF, the lamins have a central rod domain
that forms an In addition to providing mechanical support to the nucleus and influencing its shape and volume, the lamins appear to interact with other nuclear components and thereby
may influence a number of nuclear processes (40). For example, the lamins interact with chromatin in vitro and
probably in vivo (2, 16, 22, 47). Through this interaction,
the lamins may be involved in DNA replication. For example, lamin B is associated with replicating chromatin in
mammalian cells (39). During S phase, lamin B appears not only at the nuclear periphery, but also in nucleoplasmic foci that frequently coincide with sites of bromodeoxyuridine incorporation and the location of the DNA replication factor, PCNA (39). It has also been suggested on
morphological grounds that a filamentous network of lamins
acts as a scaffold for "DNA replication factories" (24, 25).
Further evidence for a role of the nuclear lamins in
DNA replication comes from the nuclear assembly system
prepared from Xenopus laevis eggs (3, 4, 30). In this system, nuclei rapidly assemble when DNA or chromatin is
added to interphase egg extracts. These nuclei carry out
processes such as nuclear import, lamin assembly, and
DNA replication. The presence of highly concentrated,
soluble, nuclear components in the extract makes the Xenopus system particularly useful for biochemical manipulations. Proteins from a wide range of species, including
yeast and humans, have been added to interphase and mitotic extracts to examine their function in the regulation of
the cell cycle (41). In addition, immunodepletion of specific proteins from these extracts has been used to determine their involvement in nuclear functions. For example,
when the major endogenous lamin (lamin B3) is immunodepleted from interphase extracts, nuclei form, but they
cannot replicate their DNA (34, 45). Furthermore, when
the eluted lamin B3 is added back to depleted extracts, nuclear DNA replication is restored (17). These results suggest that nuclear lamins play a role in DNA replication, although it is unclear how or at what stage DNA synthesis is
blocked under these experimental conditions.
The Xenopus nuclear assembly system has also been
useful in characterizing other proteins involved in regulating DNA replication. Immunodepletion experiments involving the removal of XMCM3 and XORC2, as well as a
number of other factors, have demonstrated that they are
essential for DNA replication. XMCM3, for example, is a
putative component of the licensing factor that is thought to limit replication to one round for each cell cycle. It binds to chromatin early in the process of nuclear assembly, before the nuclear membrane forms (10, 29, 33). XORC2 is
the Xenopus homologue of the yeast protein, ORC2 (9).
In yeast, this protein is required to initiate DNA synthesis
and is part of a complex that binds to origins of replication
(55). In Xenopus, XORC2 binds to chromatin before nuclear envelope formation and appears to be involved in the
initiation of DNA synthesis (9). Other proteins involved in
replication have also been identified as constituents of the
Xenopus system, including PCNA (26), a required cofactor of DNA polymerase The immunodepletion approach has been extremely valuable in defining roles for the lamins as well as for other
proteins in DNA synthesis, but this method has several
limitations. For example, in the case of the nuclear lamins, it
is difficult to completely immunodeplete lamin proteins (31).
In addition, any effects seen after immunodepletion may
not be due to the removal of targeted antigenic components,
but rather to the coimmunoprecipitation of bound, associated proteins. Immunodepletions of XMCM3, XORC2, and
lamin B3 all result in the specific removal of other proteins, in addition to the antigen targeted by the antibody
(9, 10, 17, 33).
To define more precisely the functions of nuclear lamins
in nuclear assembly and DNA replication, we have developed a method that avoids some of the pitfalls inherent in
the immunodepletion techniques. Our approach uses a human lamin mutant as a dominant negative disrupter of the
nuclear lamin organization in two experimental systems.
When this mutant protein is microinjected into mammalian cells or added to the Xenopus nuclear assembly extract, the lamin organization is disrupted. The mutant protein as well as the endogenous lamins appear to colocalize
in nucleoplasmic aggregates, with little or no detectable
lamin protein at the nuclear periphery. We have concentrated our efforts on defining the effects of this disruption
in nuclei assembled in Xenopus extracts, since more nuclei
can be studied under conditions permitting coordinated biochemical and morphological assays. As in the case for
Xenopus nuclei assembled after lamin B3 immunodepletion (34, 45), the disrupted nuclei described in this study
cannot complete DNA replication. However, because the
lamins are retained within the nucleus, we are able to examine their distribution relative to other DNA replication
markers. We find that, as a consequence of the disruption,
there is an altered distribution of proteins specifically involved in the elongation phase, but not in the initiation
phase of DNA replication.
Expression of Human Lamins in Escherichia coli
Full-length human lamin A (LA) and
Microinjection of Mammalian Cells and Analysis
by Immunofluorescence
BHK-21 cells were cultured as described elsewhere (18). Single cells were
injected with Xenopus Interphase Extracts and In Vitro Nuclear
Assembly Reactions
Xenopus laevis egg interphase extracts were prepared as described in (43).
The extract was frozen in liquid nitrogen in 70-µl aliquots. Demembranated chromatin from Xenopus sperm was prepared as described in (42).
For all nuclear assembly reactions, aliquots of interphase extract were
thawed rapidly and brought to 15 mM Hepes, pH 7.4. For ATP generation, the extract was made 1 mM ATP (Sigma Chemical Co., St. Louis,
MO), 10 mM phosphocreatine (Sigma Chemical Co.), and 50 µg/ml creatine phosphokinase (Sigma Chemical Co.) (see 43). Stock solutions of
bacterially expressed human LA or In some experiments, assembled nuclei were removed from one assembly reaction and transferred to another. To accomplish this, the assembly
reaction mixture was diluted 50-fold with NWB (200 mM sucrose, 15 mM
Hepes, pH 7.4, 50 mM NaCl, 2.5 mM MgCl2, and 1 mM DTT), and the nuclei
were recovered by centrifugation for 3 min at 1,600 g (5). The resulting
pellets were then resuspended in fresh nuclear assembly reaction mixture.
The effects of In Vitro DNA Replication Assays
DNA replication in the in vitro assembled nuclei was assayed with fluorescence microscopy by adding 1 mM bio-11-dUTP (Enzo Diagnostics,
Farmingdale, NY) to the nuclear assembly reaction to achieve a final concentration of 10 µM (6). The incorporated nucleotides were detected with
fluorochrome-tagged streptavidin as described below. DNA synthesis was
also assayed by adding 1.5 µCi of [32P]dCTP (6,000 Ci/mM; Amersham
Corp., Arlington Heights, IL) to a 15-µl nuclear assembly reaction before
the addition of sperm chromatin. DNA replication was measured as described in (12). Briefly, the reactions were stopped, treated with proteinase K, and resolved by electrophoresis on a Tris borate-buffered, 0.8% agarose gel. The gel was dried and exposed for autoradiography and phosphoimaging with a FUJIX BAS 2000 (Fuji Photo Film Co., Tokyo, Japan)
to quantitate the amount of [32P]dCTP incorporation.
Preparation of Nuclear Matrices
Nuclear matrices were prepared from the nuclei assembled in vitro as described in (11). Nuclei were assembled in a 100-µl nuclear assembly reaction, and the reaction mixture was diluted with 650 µl of NWB containing
0.5% Triton X-100. DNase I (DPRF; Worthington Biochemical Corp., Freehold, NJ) was added to a final concentration of 8.3 µg/ml, and, after a 10min incubation at 20°C, an additional 750 µl of 4 M NaCl, 20 mM Hepes,
pH 7.4, 20 mM EDTA, and 1 mM DTT was added. After 10 min at 20°C, the
matrices were fixed at the same temperature by adding 160 µl of 10 mM
ethylene bis [succinimidyl succinate] (Pierce Chemical Co., Rockford, IL). The fixation was stopped after 7 min by the addition of 42 µl of 1 M Tris
HCl, pH 7.4.
Microscopic Analyses of Xenopus Nuclei Assembled
In Vitro
Nuclei assembled in vitro were fixed for 10 min at 20°C by diluting the assembly reaction mixture 10-fold in NWB and adding 0.1 vol of 100 mM
ethylene glycol bis [succinimidylsuccinate] in DMSO (35). Fixation was
stopped by adding 1 M Tris HCl, pH 7.4, to achieve a final concentration of
25 mM. Alternatively, nuclei were fixed with 4% paraformaldehyde in
NWB for staining with XMCM3 (33). Subsequently, nuclei were pelleted
onto coverslips as described elsewhere (37). After fixation, coverslips
were placed in 0.1% NP-40 or 0.1% Triton X-100 in PBS for 2 min, and then rinsed twice for 2 min in PBS at room temperature. 30-µl aliquots of
primary antibodies, diluted 1:20 in PBS, were overlaid onto coverslips. After a 30-min incubation at 37°C, coverslips were washed four times with
PBS and incubated for an additional 30 min at 37°C with a 1:50 dilution of
the appropriate fluorochrome-labeled secondary antibody. The coverslips
were then washed five times in PBS and mounted in 50 mM Tris Base, pH
9.0, 50% glycerol, and 2 mg/ml of p-phenylenediamine (Sigma Chemical Co.).
The rabbit polyclonal sera used for these studies were directed against
human lamins A and C (39), XORC2 (9; a gift from William Dunphy, California Institute of Technology, Pasadena), and XMCM3 (33; a gift from
Ronald Laskey, Cambridge University, UK). Monoclonal ascites and supernatants used included L6-5D5 directed against Xenopus lamin B3 (52),
CRL 1640 directed against DNA polymerase To determine the function of nuclear lamins, we sought a
method to perturb lamin organization. In previous studies,
we found that microinjection of bacterially expressed human LA into mammalian cells resulted in its incorporation
into the endogenous nuclear lamin structures (18). This
technique allowed us to follow the pathway of nuclear
lamin assembly in situ (18). We used this same technique
to introduce a mutant lamin protein into BHK-21 cells.
The mutant,
Human Lamin A Is Incorporated into the
Endogenous Lamin Structures of Xenopus Nuclei
Assembled In Vitro
While microinjection of As a control for the use of the Xenopus system, wildtype LA was added to the nuclear assembly reactions. Nuclei assembled under these conditions contain LA, which
colocalizes with the endogenous Xenopus lamin B3 (LB3)
(Fig. 3, a and b). Staining is present at the nuclear periphery and elsewhere through the nucleus when viewed by
conventional immunofluorescence. The LB3 staining pattern is similar to that seen in nuclei assembled without LA
(compare Fig. 3 b with Fig. 6 a). Furthermore, nuclei assembled in the presence of LA contain a normal DNA
staining pattern as indicated with Hoechst dye (Fig. 3 f).
They also possess a normal distribution of nuclear membrane, as shown by staining with the membrane intercalating dye DIOC6 (data not shown), and a nuclear envelope
with nuclear pore complexes, as suggested by the presence of a typical WGA staining pattern (Fig. 4, c and d). This
organization of human LA in a heterologous system is
consistent with results obtained in other laboratories. For
example, when human lamin RNA is expressed in Xenopus oocytes, the expressed protein is localized to the lamina of the germinal vesicle (28). Similarly, Xenopus lamins
expressed in mammalian cells integrate normally into the
lamina (15).
To determine if LA is stably incorporated into the endogenous nuclear lamina, nuclear matrices were prepared
from nuclei assembled in the presence of LA. Nuclei were
first treated with DNase I, and subsequently extracted
with 2 M NaCl and 0.1% Triton X-100 (see Materials and
Methods). The nuclear lamina is resistant to this digestion
and extraction procedure that has been shown to remove
~90% of the nuclear proteins and DNA (11). Indirect immunofluorescence shows that the LA within the lamina is
resistant to extraction and is therefore incorporated into
the lamina of these nuclei (Fig. 3 c).
Xenopus nuclei assembled in vitro replicate their DNA
once per cell cycle in a semiconservative manner (4, 30,
43). To determine whether the incorporation of LA affects
DNA replication, biotinylated dUTP was added to nuclear
assembly reactions (see Materials and Methods) containing LA. After fixation and staining with Texas red-
streptavidin, fluorescence microscopic observations show
that this nucleotide is incorporated in a fashion indistinguishable from control nuclei (compare Figs. 3, d and e,
and 6, a and b).
When the mutant human lamin
We also determined if nuclear protein import could take
place in disrupted nuclei. This involved the use of nuclei
that were assembled in the presence of Interestingly, the disrupted nuclei are smaller than those
formed in control assembly reactions or in reactions containing LA (e.g., compare Fig. 6 a and 6 b with 6 c and 6 d).
Nuclei assembled in the presence of In contrast with the results obtained with the buffer control (Fig. 6, a and b) or with LA (see Fig. 3 e), the addition
of
Disruption of Lamin Organization Alters the
Distribution of Factors Required for the Elongation
Phase of DNA Synthesis
To examine the effects of altered lamin organization on
the major steps of DNA replication, the distributions of
five proteins known to be involved in either the initiation
or the elongation phases of DNA replication were examined. The organization of factors involved in the initiation
phase of DNA replication was studied with antibodies directed against DNA polymerase The DNA polymerase
In contrast, the staining patterns produced by the antibodies directed against the elongation factors, PCNA and
the large subunit of RFC, were dramatically altered in nuclei assembled in the presence of
Disruption of Nuclear Lamin Assembly
Is Reversible
To determine if the
Disruption of Nuclear Lamin Organization after
Nuclear Assembly In Vitro
The microinjection of In this study we describe the use of a dominant negative
mutant human lamin to disrupt the organization of the nuclear lamin assemblies both in vivo by microinjection into
mammalian cells and in in vitro assembled Xenopus nuclei. We further demonstrate that a normal distribution of
nuclear lamins is required for DNA synthesis. Specifically,
the addition of The disruption of lamin organization alters the distribution of both PCNA and the large subunit of RFC, two essential cofactors for DNA polymerase At the present time, we believe that our results support
an effect on the elongation phase of DNA replication for
several reasons. Evidence suggesting that the initiation of
DNA synthesis does not rely on an intact lamin organization
comes from our immunofluorescence studies of XORC2,
XMCM3, and DNA polymerase Evidence indicating that the initiation of DNA synthesis
occurs in disrupted nuclei comes from the observations
that Further clues for the role of the nuclear lamins are derived from the immunofluorescence pattern of XMCM3
in Most of our understanding of the biochemistry of DNA
replication has come from the coupling of genetic studies
in yeast with in vitro studies of SV-40 replication (55). The
mechanisms involved in the regulation of DNA replication
in more complex genomes remain largely unknown. However, it appears from the data presented in this study and
elsewhere (17, 34, 44, 55) that, in higher eukaryotic cells,
the nuclear lamins play a vital role in this process. We have
previously reported that lamin B colocalizes with PCNA
in mammalian cells during S phase (39). In this report we
find that the disruption of nuclear lamin organization also alters the normal organization of RFC and PCNA, such
that all three proteins are found in the same aggregates.
These results suggest that lamins interact with the components of the strand elongation complexes. This interaction
could be direct, with lamins binding to a component of the
replication machinery, or indirect through unknown nuclear proteins or structural entities. This proposed interaction of PCNA with nuclear lamins is also supported by the
finding that PCNA is readily extracted from nuclei formed in lamin B3-depleted assembly reactions, but is resistant
to extraction in nuclei assembled in control reactions (27).
Traditionally, lamins have been thought to be located
exclusively at the nuclear periphery, which makes it difficult to model lamin involvement in DNA synthesis, since
much of the replication process takes place deep in the nucleoplasm. However, the presence of lamins in the nucleoplasm is supported by a number of studies (7, 18, 39), and
we have found lamin B3 within the nucleoplasm of control
Xenopus nuclei (Fig. 3 a). This lamin staining may be part
of a dynamic nucleoplasmic lamin network. Such a network could form a scaffold (24, 25) upon which replication
factors are assembled into functional units that facilitate
the formation of active elongation complexes and/or stabilize such complexes once they are formed. Such an organization would explain why perturbations in nuclear lamin
organization can block DNA replication and cause the abnormal distribution of RFC and PCNA.
The reduced size of nuclei assembled in the presence of
The Since it is known from in vitro studies that the central
In summary, the addition of exogenous normal and mutated lamins to the Xenopus nuclear assembly system has
provided evidence that a normal nuclear lamin organization is required to proceed from the initiation to the elongation phase of DNA replication. The assays used are relatively simple and should continue to provide further
structural and biochemical information about the role of
nuclear lamins in DNA replication. Furthermore, the availability of a soluble pool of nuclear components should allow us to fractionate the interphase extract and to determine whether lamin proteins are interacting directly with
elongation factors at replication forks or indirectly through
other unidentified nuclear components. This use of Xenopus extracts has already proven to be very important in
identifying and characterizing the interactions of factors involved in the initiation of DNA replication within nuclei
(1, 9, 10, 29, 33, 61,). We believe such an approach will also
help to elucidate the role of nuclear lamins in other processes such as postmitotic nuclear assembly, nuclear growth,
and the maintenance of the overall shape and structural
integrity of the nucleus.
helix, composed of heptad repeats. The
rod domains are primarily responsible for the higher order
lamin-lamin interactions that govern lamin assembly (20). The two non-
-helical end domains are also involved in
assembly and may interact with other nuclear structures
(see 40, for detailed discussion).
. This polymerase is responsible
for the elongation phase of DNA replication (59).
Materials and Methods
NLA were cloned in a pET-derived
vector and expressed in the NovaBlue (DE3) strain of E. coli (Novagen,
Madison, WI).
NLA lacks the first 33 amino acids of human lamin A. Both of the expressed proteins were purified by ion-exchange chromatography as described previously (38). The protein in column buffer (6 M
urea, 25 mM Tris, 2 mM EDTA, and 1 mM DTT) was dialyzed against PB
(300 mM NaCl, 25 mM Tris Base, pH 9, and 1 mM DTT). After dialysis,
SDS-PAGE analysis of the resulting protein solution showed the presence
of one major band of protein. In the case of
NLA, the protein had a molecular mass of ~69 kD (Fig. 1 a). This 69-kD protein reacted with a rabbit polyclonal antibody directed against human lamins A/C as demonstrated by immunoblotting (Fig. 1 b). SDS-PAGE and blotting analyses were carried out as described in (39). The relatively minor bands seen in the immunoblot (Fig. 1 b) are due to a small amount of proteolysis that is present in
all preparations of nuclear lamins (38). The same gel profiles are seen for
the wild-type LA protein, but the apparent molecular mass is 72 kD (not
shown). The protein solutions were aliquoted and stored at
80°C at a final concentration of 2 mg/ml. Before use in microinjection experiments
and nuclear assembly assays, samples were centrifuged at 20,000 g for 10 min at room temperature to remove insoluble material.
Fig. 1.
(a) SDS-PAGE of purified
NLA and (b) corresponding Western
blot using a polyclonal lamin A/C antibody (39). The major band runs at 69 kD, the predicted molecular mass of
NLA. There are also minor bands,
representing the proteolytic fragments
seen in nuclear lamin preparations expressed in E. coli (38). Numbers on
the left represent molecular mass size
markers in kD.
[View Larger Version of this Image (38K GIF file)]
NLA at a concentration of 1 mg/ml in PB (18). Controls
consisted of the injection of cells with PB. Cells were fixed in methanol 2 h
after microinjection and processed for immunofluorescence as described
previously (18). A rat polyclonal antibody directed against human lamins A
and C (39) or a rabbit polyclonal antibody directed against human lamin B
(39) was diluted 1:100 in PBS for use as a primary antibody for immunofluorescence. Secondary antibodies were diluted 1:50 in PBS and included
FITC-labeled goat anti-rat IgG (Jackson ImmunoResearch Laboratories,
Inc., West Grove, PA), tetramethyl rhodamine isothiocyanate-labeled goat anti-rabbit IgG, and lissamine rhodamine-labeled donkey anti-rabbit IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD).
NLA in PB were added to the assembly reaction to a final concentration of 200 µg/ml. Controls consisted
of the addition of an equal volume of PB to parallel assembly reactions. The volume of protein solution or PB alone added to an assembly reaction
was maintained at
10% of the final volume of the reaction mixture. 15 min after the addition of protein or PB to an assembly reaction, demembranated sperm chromatin was added to initiate nuclear assembly. Sufficient sperm chromatin was added to achieve a final concentration of 1,000 nuclei per µl. Unless otherwise specified, nuclei were either fixed for immunofluorescence or prepared for electrophoretic analysis at 90-120 min
after initiating the assembly reaction as described below.
NLA on assembled nuclei were studied by adding
NLA (200 µg/ml final concentration) at a time interval of 90 min after the
addition of sperm chromatin to a nuclear assembly reaction. After an additional 45 min, nuclei were fixed and processed for immunofluorescence
as described below.
(57; American Type Culture Collection, Rockville, MD),
RFC 11 directed against the large subunit of replication factor C (8; a gift from Bruce Stillman, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY), and PC10 directed against
PCNA (Boehringer Mannheim Biochemicals, Indianapolis, IN). The myc
9E10 epitope antibody was also used (13; American Type Culture Collection). The antibodies directed against PCNA, DNA polymerase
, XMCM3, and XORC2 have all been shown to react only with their targeted antigens in Xenopus extracts ( 9, 26, 27, 33). The antibody directed against human lamin A does not cross-react with Xenopus lamin B3 in immunofluorescence assays (data not shown). The secondary antibodies used were
FITC-labeled donkey anti-mouse IgG, tetramethyl rhodamine isothiocyanate-labeled donkey anti-rabbit IgG, and lissamine rhodamine-labeled
donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.).
Detection of bio-11-dUTP incorporation involved the incubation of fixed
nuclei for 30 min at 37°C in Texas red- or FITC-labeled streptadivin (Amersham Corp.) at a 1:500 dilution in the presence of 12.5 µg/ml of RNase
A (4; Sigma Chemical Co.). Nuclear membranes were stained with the lipophilic dye dihexyloxacarbocyanine (DIOC6) (Molecular Probes, Eugene, OR) at 2.5 µg/ml during the secondary antibody incubation and at
0.25 µg/ml in the mounting medium (44). Nuclear pore proteins were
stained by adding FITC-labeled WGA (Sigma Chemical Co.) at 50 µg/ml
during secondary antibody incubation (14). DNA was visualized by adding Hoechst dye (Molecular Probes) at 1 µg/ml to the mounting medium.
Microscopic observations were carried out on an Axiophot (Carl Zeiss,
Inc., Thornwood, NY) equipped with a 35-mm camera or an LSM 410 confocal microscope (Carl Zeiss, Inc.) equipped with an argon/krypton laser. Confocal micrographs were stored on optical disks, and micrographs
were printed on an UP-D8800 video printer (Sony Corp., Park Ridge, NJ).
Results
NLA Disrupts Nuclear Lamin Organization In
Mammalian Nuclei
NLA, lacks the NH2-terminal nonhelical
domain (33 amino acids) of LA, but it contains the entire
-helical rod and COOH terminus. Bacterially expressed
NLA cannot form typical LA paracrystals in vitro, showing that the NH2 terminus is required for higher order
lamin assemblies (21, 38). To determine the effects of
NLA in vivo, a solution of this mutant protein (1 mg/ml;
see Materials and Methods) was injected into the cytoplasm of BHK cells. Injected cells were fixed after 2 h and
then processed for immunofluorescence. As can be seen in
Fig. 2, there is a dramatic alteration in the distribution and
organization of the nuclear lamins. Instead of producing
the typical rim pattern associated with the nuclear envelope as well as nucleoplasmic foci (Fig. 2, a and b), lamins
A/C and B colocalize almost exclusively in large nucleoplasmic aggregates in injected cells (Fig. 2, c and d). These
results show that
NLA acts as a dominant negative mutant that can induce the rapid disruption of the endogenous lamin structures that are composed of both A- and
B-type lamins.
Fig. 2.
Double label immunofluorescence showing nuclear
lamin patterns in a normal interphase BHK cell and in a cell injected with NLA. Nuclei were stained for lamins A/C (LA) (a
and c) or lamin B (LB) (b and d). In the uninjected cell, there is a
distinctive lamin rim as well as less intense nucleoplasmic foci (a
and b), as previously described (39). In cells fixed 2 h after injection, the lamin rim is no longer obvious, and the lamin staining
for both lamins A/C and B appears mainly in the same foci (c and
d). Confocal optics showing sections through the mid-region of
the nucleus. Bar, 5 µm.
[View Larger Version of this Image (126K GIF file)]
NLA can disrupt the lamina of
individual BHK-21 cells, such studies are not amenable to
detailed biochemical and structural analyses, as only a limited number of cells can be injected, and it is difficult to
control the amount of protein injected (for a discussion of
this latter point, see 19). Similarly, others have shown that
transfection of lamin cDNA mutants into mammalian cells
can also result in similar nucleoplasmic structures and apparent disruption of the endogenous lamin organization, but the amount of protein expressed per cell and the percentage of cells expressing the protein are quite variable
(23). In light of these limitations, we have developed a hybrid system in which controlled concentrations of mutated
and wild-type human lamins are added to nuclear assembly extracts prepared from Xenopus laevis eggs (4, 30, 43).
One of the major advantages of this in vitro nuclear assembly system includes the ability to precisely control the
amount of protein added to the extract. In addition, large
numbers of nuclei can be prepared for morphological and
biochemical studies. We have used the human lamins in
this system because species-specific antibodies allow us to
distinguish between the endogenous Xenopus lamin B3
and the mutant human lamin.
Fig. 3.
Characterization of
nuclei assembled in reactions
containing human LA. (a
and b) Nucleus stained for
(a) human lamin A and (b)
Xenopus LB3. The human
LA does not disrupt the
lamin network and appears
to colocalize with the endogenous LB3. (c) Confocal microscopic image of a nucleus
after nuclear matrix extraction and staining for human
LA. LA staining is retained
in the peripheral lamina after
the matrix extraction protocol. (d-f) Fluorescence images from a triple label preparation of the same nuclei.
(d) Nucleus stained for human lamin A; (e) biotinylated dUTP incorporation pattern as shown by Texas
red-conjugated streptavidin
(REP); (f) Hoechst dye
showing the location of
DNA. The incorporation of lamin A does not appear to
alter DNA replication in the
extract. Bar, 5 µm.
[View Larger Version of this Image (78K GIF file)]
Fig. 6.
The addition of NLA to nuclear assembly reactions
inhibits DNA replication as shown by the reduced incorporation
of biotinylated dUTP. Fluorescence images of the LB3 pattern
and biotinylated dUTP incorporation in (a and b) control nuclei
and (c-f) nuclei formed in the presence of
NLA. (a, c, and e)
Immunofluorescence using the antibody against Xenopus LB3;
(b, d, and f) Texas red-conjugated streptavidin shows the patterns of incorporation of biotinylated dUTP. Disruption of lamin
organization greatly inhibited the incorporation of biotinylated
nucleotide when compared with control nuclei. However, all of
the lamin-disrupted nuclei do contain a faint punctate nucleoplasmic pattern of labeled nucleotide incorporation that is readily resolved by confocal microscopy. (a-d) Conventional optics (see f).
(e and f) Confocal optics. Bar, 5 µm.
[View Larger Version of this Image (94K GIF file)]
Fig. 4.
Double label fluorescence observations of nuclei stained
for different aspects of nuclear envelope structure and function. (a-f) Nuclei were assembled in interphase extracts containing: (a
and b) buffer control, (c and d) lamin A, and (e and f) NLA. Nuclei were stained for (a) lamin B3 or (c and e) human lamin A,
and (b, d, and f) the nuclear pore WGA binding proteins using fluorescently tagged WGA. Nuclei assembled under all three
conditions appear to have essentially normal distributions of
WGA binding proteins at the nuclear periphery. (g and h) Nucleus assembled in the presence of
NLA and stained for (g)
NLA and (h) the membrane dye DIOC6 (MEM). The nucleus
contains a disrupted lamin organization but retains normal membrane staining. Bar, 5 µm. (i and j) Import of wild-type lamin A
into (i) buffer control and (j)
NLA-disrupted nuclei. The wildtype lamin A was detected using the myc 9E10 epitope antibody
(13). Nuclei were assembled with or without
NLA, and 90 min
after the initiation of assembly, myc-tagged human lamin A was
added to the reaction. The nuclei were fixed 20 min later and
stained with the myc antibody. Both (i) control and (j)
NLAdisrupted nuclei show prominent myc staining, demonstrating
that the disrupted nuclei retain the ability to import protein. The
majority of the imported protein localizes to the characteristic
foci of
NLA-disrupted nuclei. Confocal optics showing sections
through the mid-region of nuclei. Bar, 5 µm.
[View Larger Version of this Image (48K GIF file)]
NLA Disrupts Nuclear Lamin Organization in Nuclei
Assembled In Vitro and Inhibits DNA Replication
NLA is added to the nuclear assembly reaction at the same concentration as that
used for the wild-type LA (200 µg/ml final concentration;
see Materials and Methods), normal lamin assembly is altered in >90% of the nuclei. Instead of producing a typical
nuclear lamin staining pattern, antibodies directed against
Xenopus LB3 and LA stain large nucleoplasmic spheroidal bodies (Fig. 5, a and b). Confocal microscopic analysis of nuclei assembled with
NLA shows that both Xenopus
LB3 and
NLA colocalize within these nucleoplasmic aggregates and that there is no obvious staining of the nuclear periphery (Fig. 5, d and e). However, the DNA of
these lamin-disrupted nuclei appears to be distributed normally as indicated by Hoechst dye (Fig. 5 c). Furthermore,
nuclear pore complexes, as indicated by WGA staining (Fig. 4, e and f), and the nuclear membranes, as indicated
by DIOC6 staining, (Fig. 4, g and h) appear to be normal.
Fig. 5.
Nuclei assembled in an interphase extract containing NLA. (a-c)
Conventional fluorescence images of
lamin and DNA patterns of a nucleus
stained for (a) human LA, (b) Xenopus
LB3, and (c) DNA. (d and e) Confocal images of a disrupted nucleus stained for (d)
human LA and (e) Xenopus LB3. The endogenous lamin structure has been disrupted and LB3 appears in foci colocalizing with
NLA. Bar, 5 µm.
[View Larger Version of this Image (86K GIF file)]
NLA for 90 min
(see Materials and Methods). Under these conditions,
100% (n = 84), of the in vitro assembled nuclei were disrupted. Subsequently myc-tagged wild-type LA was added
to the same extract at a concentration equivalent to 25%
of the mutant protein. 30 min later, the reactions were stopped and immunofluorescence assays demonstrated
that the wild-type lamin A was imported into 100% (n = 80) of the disrupted nuclei (Fig. 4, i and j). These observations demonstrate that the disrupted nuclei are able to import nuclear proteins. Furthermore, under the experimental conditions used, the nuclei maintained their disrupted
phenotype throughout the transport process. In contrast with the control nuclei, the myc-tagged lamin A colocalized
with
NLA in the nucleoplasmic aggregates in disrupted
nuclei (Fig. 4, i and j).
NLA also seem
more fragile than nuclei formed in either control or LAcontaining assembly reactions, as indicated by their increased tendency to break open when centrifuged at low
forces (see Materials and Methods; data not shown).
NLA to nuclear assembly reactions greatly inhibits DNA
replication. When examined with conventional fluorescence optics, the disrupted nuclei show greatly reduced or
no detectable incorporation of biotinylated dUTP after 90- and 180-min incubations (Fig. 6 d). However, confocal microscopy demonstrates that the majority of these nuclei do
exhibit a faint punctate nucleoplasmic pattern of biotinylated
dUTP incorporation (Fig. 6 f). To obtain a more quantitative
measure of the inhibition of DNA synthesis, 32P-labeled
dCTP was added to nuclear assembly reactions, in the presence or absence of
NLA. After a 90-min incubation,
the DNA was isolated and resolved by gel electrophoresis
(see Materials and Methods). Autoradiographic (Fig. 7 A)
and phosphoimage analysis (Fig. 7 B) demonstrates that
NLA reduces the level of 32P-incorporation by 94% in
disrupted nuclei.
Fig. 7.
The addition of NLA to nuclear assembly reactions
reduces [32P]dCTP incorporation by ~95%. (A) Autoradiogram
of an agarose gel showing the incorporation of 32P-labeled dCTP
into the DNA of nuclei formed in the presence of buffer control
or
NLA. After nuclear assembly, the samples were treated as
described in Materials and Methods and resolved on an 0.8%
agarose gel. The upper band is at the origin of the gel. (B) Quantitation of replication assays shown in A. The radioactive signal of
the dried gel was quantitated with a FUJIX BAS 2000 phosphoimager. The sum of the signal intensity/area value for both bands
in each lane was used to measure the total incorporation of radioactivity into DNA. The average value for four replicate assays
was plotted in a bar graph, where the vertical axis represents the
signal/area values (in thousands) determined by the imager. The
average value for samples containing
NLA was 2,292, with values ranging from 2,035-2,513. The average value for the control samples was 39,030, with samples ranging from 34,514-47,443.
The addition of
NLA to the nuclear assembly reaction reduced
the incorporation of 32P-labeled dCTP to ~5% of that found in
control reactions.
[View Larger Version of this Image (27K GIF file)]
, XORC2, and XMCM3.
DNA polymerase
is believed to catalyze the formation of primers at origins of replication (55). XORC2 has been
shown to be essential for DNA replication in the Xenopus
nuclear assembly extracts, and, as a result of its sequence
homology to the yeast ORC2, it is most likely involved in
the initiation of DNA replication (9). XMCM3 has been
characterized as a component of the licensing factor for
DNA replication in Xenopus nuclear assembly extracts, and it is thought to be required for the initiation of DNA
replication (10, 29, 33).
and XORC2 staining patterns
were unaffected in
NLA-disrupted nuclei (Fig. 8, a-d),
compared with nuclei formed in the presence of buffer
(data not shown). In both disrupted and control nuclei,
staining with these antibodies indicated that they colocalized with chromatin (not shown). This is in agreement with
previous studies (9, 26). These results suggest that the disruption of the lamin network does not affect the early stages of replication. The XMCM3 staining pattern of
NLA-disrupted nuclei was also coincident with chromatin (Fig. 8, e and f). This was true of 45-, 90-, and 180-min
nuclear assembly reactions. In addition, we noticed that, in
nuclei assembled in the presence of buffer, the XMCM3
staining colocalizes with chromatin at 45 min, but the fluorescence intensity decreases over time so that at 90 min it
is not detectable (data not shown). This loss of XMCM3
signal is identical to the results obtained by other groups and is believed to represent a displacement of the protein
from chromatin as replication proceeds (10, 29, 33). The
disruption of lamin organization apparently prevents this
XMCM3 displacement.
Fig. 8.
Nuclei formed in the presence of NLA, and then subsequently stained for
NLA or lamin B3 and one of several early
DNA replication markers. (a and b) Nucleus stained for (a)
NLA and (b) DNA polymerase
. (c and d) Nucleus stained for
(c) LB3 and (d) XORC2. (e and f) Nucleus stained for (e) LB3
and (f) XMCM3. The distribution of DNA polymerase
, XORC2,
and XMCM3 is not altered by the disruption of the lamin structure. Confocal microscopic images showing sections through the
middle of the nuclei. Bar, 5 µm.
[View Larger Version of this Image (82K GIF file)]
NLA. Both of these
proteins are required cofactors for DNA polymerase
, the
polymerase known to be responsible for chain elongation
during DNA synthesis (55, 59). As assayed by indirect immunofluorescence, these two factors are associated with
chromatin in nuclei assembled in control reactions (Fig. 9,
a and d) or in assembly reactions containing LA (data not
shown). However, when nuclei are assembled in the presence of
NLA, PCNA and RFC colocalize with the lamin
aggregates (Fig. 9, b, c, e, and f). These observations suggest that the inhibition of DNA replication resulting from
the disruption of nuclear lamin organization may be caused
by alterations in the localization and/or targeting of the
components of the DNA replication machinery responsible for chain elongation.
Fig. 9.
Staining patterns of lamin and
DNA replication factors involved in elongation in nuclei assembled in the presence
of (a and d) buffer or (b, c, e, and f)
NLA. (a) Control nucleus stained for
PCNA. (b and c) Nucleus assembled in
the presence of
NLA stained for (b)
PCNA and (c)
NLA. (d) Control nucleus
stained for RFC. (e and f) Nucleus assembled in the presence of
NLA stained for
(e) RFC and (f)
NLA. PCNA and RFC
distributions are altered from the control
as a consequence of lamin disruption such
that PCNA and RFC colocalize with lamin
aggregates in these nuclei. Confocal microscope showing sections through the
middle of the nuclei. Bar, 5 µm.
[View Larger Version of this Image (96K GIF file)]
NLA-induced alterations of nuclear
structure and function are reversible, nuclei were assembled in the presence of
NLA for 90 min. The disrupted
nuclei were then removed from nuclear assembly reactions
containing
NLA by centrifugation. These nuclei were resuspended in fresh extract to which no
NLA was added
and were assayed for DNA replication. The nuclei were fixed 90 min later for immunofluorescence or processed
for autoradiography (see Materials and Methods). As seen
by both biotinylated dUTP incorporation (Fig. 10, a and b)
and [32P]dCTP incorporation (data not shown), nuclear
DNA synthesis was "rescued" when the disrupted nuclei
were transferred to normal nuclear assembly reactions. Interestingly, although a few of the lamin aggregates remained in these nuclei, apparently normal nuclear lamina
staining was reestablished (Fig. 10 a).
Fig. 10.
(a and b) Lamin
and biotinylated dUTP incorporation in a nucleus
formed in the presence of
NLA, and subsequently
transferred to an interphase
extract containing biotinylated dUTP but lacking
NLA (see text). Confocal
micrographs showing sections through the middle of
the nucleus. (a) Nucleus
stained for Xenopus LB3. (b)
Pattern of biotinylated dUTP
incorporation as shown by binding of Texas red-conjugated streptavidin. The disrupted nuclei were transferred to a nuclear assembly
reaction lacking
NLA, where they form a lamin rim and replicate DNA. However, some lamin foci remain. (c) Postassembly disruption of the lamin structure of an in vitro assembled nucleus.
NLA was added 90 min after the onset of nuclear formation, a point at
which the nuclei have normal lamin organization and have largely completed DNA replication. The addition of
NLA disrupts the assembled LB3 staining pattern. Bar, 5 µm.
[View Larger Version of this Image (39K GIF file)]
NLA into cultured mammalian cells
demonstrates the capacity of this truncated protein to disrupt endogenous lamin organization in fully formed interphase nuclei. At 90 min, nuclei formed in the Xenopus nuclear assembly reactions under normal conditions contain
a normal lamin organization as indicated by lamin antibody staining (see, e.g., Fig. 4 a). DNA replication is complete or nearly complete, as indicated by the low level of
incorporation of a 5-min pulse of biotinylated dUTP at
this time interval (data not shown). To determine if
NLA
can disrupt the lamin organization once it is established,
NLA was added to the nuclear assembly reaction 90 min
after the initiation of nuclear assembly. The reaction was
allowed to continue for an additional 45 min (see Materials
and Methods). We found that the addition of
NLA under
these conditions induced a dramatic disruption of the endogenous lamina in >90% of the nuclei observed. This resulted in the formation of nucleoplasmic aggregates containing LB3 (Fig. 10 c) and
NLA (data not shown). The
addition of
NLA at both earlier and later time points (45, 120, and 150 min) after the initiation of nuclear assembly
also resulted in the formation of nucleoplasmic aggregates
indistinguishable from those seen in Fig. 10 c. In all cases,
these aggregates appear to be structurally identical to
those formed when nuclei are assembled in the presence of
NLA (see Figs. 3-6). However, it should be noted that
nuclei disrupted 90 min after the initiation of assembly are
larger than nuclei formed in the presence of
NLA.
Discussion
NLA, a mutant human lamin, to the Xenopus laevis nuclear assembly system blocks the formation
of a normal lamina at the nuclear periphery. Instead, the
endogenous LB3 and
NLA are found as constituents of
the same large aggregates dispersed throughout the nucleoplasm. Under these conditions, DNA synthesis is dramatically reduced to ~5% of its normal level. These results are
consistent with those obtained from immunodepletion
studies demonstrating that LB3 is required for DNA synthesis in in vitro assembled nuclei (17, 34, 45). The nuclei
formed in LB3-immunodepleted extracts or
NLA-containing extracts described in this study have other features
in common, including the fact that nuclear membranes and
pores appear to assemble in a relatively normal fashion
(34, 45). However, the dominant negative approach introduced in this study avoids one of the major problems inherent in the immunodepletion experiments: that the block
in DNA replication could be caused by the removal of other
proteins associated with lamin B3 in the assembly extract. In the experiments presented in this study, no components
are removed from the nuclear assembly system.
during the elongation phase of replication (55). Normally both the large
subunit of RFC (see Fig. 9) and PCNA (26) are distributed
along chromatin. In
NLA-disrupted nuclei, these cofactors are reorganized, along with the nuclear lamins, to
form nucleoplasmic aggregates. These aggregates are not
obviously associated with chromatin, and this in turn may have an inhibitory effect(s) on the assembly and function
of the elongation machinery. Alternatively, disruption of
lamin organization may also alter an aspect of the initiation process itself that we have been unable to detect.
. These three proteins are thought to be involved in the initiation and primer formation steps of DNA replication (9, 10, 29, 33, 55). The disruption of the nuclear lamin structure does not detectably
alter the distribution of DNA polymerase
and XORC2
or the initial distribution of XMCM3, and all three of these
factors remain associated with chromatin. Interestingly, during nuclear assembly, it appears that XORC2 and XMCM3
along with two other initiation factors, RFA and FFA, bind to chromatin before the assembly of either higher order lamin structures or the nuclear membrane (1, 9, 10, 29,
33, 60, 61). These findings suggest that both the sites of initiation of DNA replication, as well as the organization of
initiation cofactors at these sites, take place early in the
process of nuclear assembly. In addition, the results reported here indicate that the location, organization, and
function of these origins of replication may be independent of the presence of normal lamin organization.
NLA dramatically reduces but does not eliminate incorporation of biotinylated dUTP or [32P]dCTP. The very
faint punctate pattern of biotinylated nucleotide incorporation in disrupted nuclei (Fig. 6 f) is reminiscent of the
centers of DNA synthesis observed in normal nuclei at the onset of replication (24, 25, 37). Therefore, it is possible that the pattern and low levels of nucleotide incorporation
seen in the disrupted nuclei described in this study may result from sites of primer and initial strand synthesis. This is
also consistent with the unaltered distribution of DNA
polymerase
in the lamin-disrupted nuclei. However, at
this point, we cannot eliminate the possibility that the low
level of incorporation of biotinylated nucleotide detected
in disrupted nuclei may be due to other processes such as
DNA repair.
NLA-disrupted nuclei. Normally, in control nuclei,
XMCM3 is displaced from chromatin as replication progresses and a dramatic decrease in fluorescence intensity is
observed (10, 29, 33). However, in the
NLA-disrupted nuclei reported in this study, the fluorescence intensity of
XMCM3 staining appeared unchanged throughout the entire assembly reaction. This suggests that the disruption of
the lamin structure prevents the dissociation of XMCM3
from chromatin, presumably by arresting replication before the normal displacement of this factor. Interestingly, it has been proposed that XMCM3 may be involved in the
switch between the initiation and elongation phases of replication (60). Taken together, these results suggest that
disruption of lamin organization blocks replication after
the initiation of DNA synthesis and prevents the switch
from the initiation to the elongation phase of DNA replication. However, the precise elucidation of the point at
which lamin disruption blocks DNA synthesis requires a
more complete understanding of the steps involved in DNA
replication.
NLA provides evidence that the nuclear lamins are involved in the growth of the nucleus after its initial assembly. In addition, the increased fragility of the nuclei assembled in the presence of
NLA supports the idea that the
nuclear lamin structure also provides a mechanical support system for the nucleus. These findings are consistent
with previous reports of the small size and increased fragility of nuclei assembled in the absence of LB3 (34, 45).
NLA-induced disruption of lamin structure is
most likely related to the dynamic characteristics of lamins
in vivo. These properties of nuclear lamins are very similar
to those found for other types of intermediate filament
systems (36, 46, 50, 56, 58). Specifically, it has been shown
that during interphase the nuclear lamins do not form a
static polymer in vivo, but rather they are in a state of dynamic equilibrium between subunits and polymer. For example, microinjected nuclear lamins are rapidly incorporated into endogenous lamin polymers (18). Similarly, the
results of fluorescence recovery after photobleaching experiments demonstrate that lamin assemblies undergo continuous subunit exchange in living cells (49).
-helical rod domain is required for normal lamin-lamin
interactions, it is probable that
NLA and LB3 interact
through their highly conserved rod domains to form heterocomplexes. However, the in vitro assembly of higher
order lamin structures such as tetramers and larger oligomeric complexes requires the NH2-terminal domain that is
missing in
NLA (20, 38). Therefore, the
NLA/LB3 heterocomplexes most likely cannot be incorporated into
higher order lamin complexes. In the presence of excess
NLA, the normal process of subunit exchange could produce a large pool of heterocomplexes. In turn, this could
drive the equilibrium in the direction of disassembly, ultimately resulting in the disruption of the endogenous lamin
structure described in this study.
Please address all correspondence to Robert D. Goldman, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. Tel.: (312) 503-4215. Fax: (312) 503-0954. e-mail: r-goldman{at}nwu.edu
Received for publication 24 October 1996 and in revised form 29 January 1997.
This work is supported by grant CA31760 from the National Cancer Institute.We thank Ms. Satya Khuon for help in preparing some of the micrographs and Ms. Laura Davis for help in manuscript preparation.
IF, intermediate filament; LA, lamin A; LB3, lamin B3; DIOC6, dihexyloxacarbocyanine.