1293
Journal of Cell Science 111, 1293-1303 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS7217
Nuclear envelope disassembly in mitotic extract requires functional nuclear
pores and a nuclear lamina
Philippe Collas
Department of Biochemistry, Norwegian College of Veterinary Medicine, PO Box 8146 Dep. 0033 Oslo, Norway
(e-mail: [email protected])
Accepted 17 February; published on WWW 20 April 1998
SUMMARY
Using sea urchin embryonic and in-vitro-assembled nuclei
incubated in sea urchin mitotic extract, I provide evidence
for a requirement for functional nuclear pores and a
nuclear lamina for nuclear envelope disassembly in vitro.
In interphase gastrula nuclei, lamin B interacts with p56,
an integral protein of inner nuclear membrane crossreacting with antibodies to human lamin B receptor.
Incubation of gastrula nuclei in mitotic cytosol containing
an
ATP-generating
system
rapidly
induces
hyperphosphorylation of p56 and lamin B. Subsequently,
p56-lamin B interactions are weakened and the two
proteins segregate into distinct nuclear envelope-derived
vesicles upon disassembly of nuclear membranes and of the
lamina. Nuclear disassembly is accompanied by chromatin
condensation. Blocking nuclear pore function with wheat
germ agglutinin or antibodies to nucleoporins prevents p56
and lamin B hyperphosphorylation, nuclear membrane
breakdown and lamina solubilization. These events are not
rescued by permeabilization of nuclear membranes to
molecules of 150,000 Mr with lysolecithin. In-vitroassembled nuclei containing nuclear membranes with
functional pores but no lamina do not disassemble in
mitotic cytosol in spite of p56 hyperphosphorylation.
Nuclear import of soluble lamin B and reformation of a
lamina in interphase extract restores nuclear disassembly
in mitotic cytosol. The data indicate a role for functional
nuclear pores in nuclear disassembly in vitro. They show
that p56 hyperphosphorylation is not sufficient for nuclear
membrane disassembly in mitotic cytosol and argue that
the nuclear lamina plays a critical role in nuclear
disassembly at mitosis.
INTRODUCTION
1995)
promotes
lamina
depolymerization
through
solubilization of lamins A/C into the cytosol and release of
lamin B mostly in a membrane-associated form (Gerace and
Blobel, 1980).
Despite the development of cell-free systems promoting NE
disassembly (Lohka and Maller, 1985; Miake-Lye and
Kirschner, 1985; Suprynowicz and Gerace, 1986; Newport and
Spann, 1987; Dessev et al., 1989, 1991; Nakagawa et al., 1989;
Maus et al., 1995; Pfaller and Newport, 1995), the contribution
of specific structural components of the NE to this process has
remained unexplored. A role of nuclear pores has been
suggested, as entry of the p34cdc2 regulator cyclin B into the
nucleus (and thereby nuclear disassembly) in mitotic prophase
is inhibited by blocking pore function with wheat germ
agglutinin (Kumagai and Dunphy, 199; Pines and Hunter,
1991). However, evidence for nuclear pore requirement in NE
disassembly is lacking. Similarly, the role of the nuclear lamina
in mitotic NE breakdown has not been investigated, perhaps
due to the lack of an experimental system. Circumstantial
evidence suggests that nuclear membrane and lamina
disassembly are biochemically separable processes. In
Xenopus egg extracts, lamina solubilization precedes (MiakeLye and Kirschner, 1985) and may even occur without
(Newport and Spann, 1987) nuclear membrane breakdown. In
The nuclear envelope (NE) consists of a double membrane
interrupted by nuclear pores and a lamina. The inner nuclear
membrane interacts with the lamina and chromatin and
contains specific integral proteins including lamin B receptor
(LBR; Worman et al., 1988, 1990) and lamina-associated
polypeptides (LAPs; Foisner and Gerace, 1993). The Nterminal region of LBR contains two globular domains
proposed to interact with B-type lamins in a phosphorylationdependent manner (Appelbaum et al., 1990; Ye and Worman,
1994; Nikolakaki et al., 1996) and HP1-type proteins (Ye et al.,
1997). The lamina of most somatic nuclei consists of A/C- and
B-type lamins, whereas many undifferentiated nuclei (Guilly
et al., 1987), including embryonic nuclei (Benavente et al.,
1985; Stewart and Burke, 1987; Holy et al., 1995) and in-vitroassembled pronuclei (Collas et al., 1996) contain only B-type
lamins.
A landmark of mitosis is the disassembly and reassembly of
nuclear membranes and the lamina (Marshall and Wilson,
1997). NE disassembly correlates with phosphorylation of
proteins of the inner nuclear membrane (Courvalin et al., 1992)
and nuclear pores (Favreau et al., 1996). In addition,
phosphorylation of nuclear lamins (Fields and Thompson,
Key words: Nuclear envelope breakdown, Lamin B, Lamin B
receptor, Phosphorylation
1294 P. Collas
mitotic Drosophila embryos, the nuclear membrane is partially
disrupted at prometaphase, but remains throughout mitosis
(Stafstrom and Staehelin, 1984), while the lamina disassembles
at metaphase (Paddy et al., 1996).
In this study, sea urchin embryonic nuclei and in-vitroreconstituted nuclei with complete or altered NEs were
exposed to a mitotic sea urchin extract to investigate nuclear
pore and nuclear lamina requirements for NE disassembly.
Previous studies have shown that the inner nuclear membrane
of interphase sea urchin gastrula nuclei harbors a 56 kDa
integral membrane protein (p56) cross-reacting with antihuman LBR antibodies, which is believed to tether the NE with
the lamina and chromatin (Collas et al., 1996). Furthermore,
the lamina of sea urchin gastrulae and in-vitro-assembled male
pronuclei consists of a single detectable B-type lamin (Holy et
al., 1995; Collas et al., 1996). These NE markers were used in
these experiments to demonstrate that functional nuclear pores
and a nuclear lamina are necessary for NE disassembly in vitro.
MATERIALS AND METHODS
Buffers, reagents and antibodies
Buffer N consisted of 50 mM Hepes (pH 7.2), 250 mM sucrose, 50
mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM PMSF. Lysis
buffer and membrane wash buffer were as described (Collas and
Poccia, 1997). Anti-bovine serum albumin (BSA) antibodies were
from Sigma (St Louis, MO). Nuclear localization signal (NLS)
peptides were from Multiple Peptide Systems (San Diego, CA; Collas
and Aleström, 1996). The chicken polyclonal antibody against sea
urchin lamin B (W3-1; a gift from Dr J. Holy, University of
Minnesota; Holy et al., 1995) recognizes a 70 kDa lamin B in
Strongylocentrotus purpuratus gastrula nuclei (Holy et al., 1995) and
a 65 kDa lamin B in Lytechinus pictus male pronuclei (Collas et al.,
1996). Affinity-purified rabbit anti-human LBR polyclonal antibodies
(a gift from Dr J.-C. Courvalin, Institut Jacques Monod, Paris, France)
were as described and recognized a 56 kDa integral membrane protein
(p56) in sea urchin NEs (Collas et al., 1996). The anti-nucleoporin
antibody mAb414 (a gift from Dr M. Rout, The Rockefeller
University) was as described (Davies and Blobel, 1986).
Mitotic egg extracts
L. pictus eggs were fertilized and cultured at 15°C for 100 minutes,
at which time >95% of the eggs were in metaphase, as judged by DNA
labeling. Eggs were washed in lysis buffer, the buffer removed and
eggs homogenized through a 22-gauge needle. The lysate was
centrifuged at 10,000 g for 10 minutes and the supernatant cleared at
150,000 g for 3 hours (Collas et al., 1996). The supernatant (‘mitotic
cytosol’) was stored at −80°C. Interphase cytosolic extracts were
prepared as above at 15 minutes postfertilization. In order to detect
solubilized lamin B in the cytosol, mitotic cytosols were
immunodepleted of soluble lamin B prior to NE disassembly reactions
(Collas et al., 1996).
Embryonic and sperm nuclei and in-vitro-reconstituted
nuclei
Gastrula L. pictus embryos grown at 15°C for 44 hours were washed
in buffer N and homogenized with 35 strokes of a tight-fitting pestle
in a Dounce glass homogenizer. Nuclei were pelleted at 1,000 g
through 1 M sucrose, washed, respun through sucrose and
resuspended in buffer N at 107 nuclei/ml. Nuclei were stored at −80°C
in 70% glycerol/buffer N. To assess their integrity, nuclei were
examined by phase-contrast microscopy or incubated with 0.1 mg/ml
FITC-Con A for 30 minutes and examined by fluorescence
microscopy (Newmeyer et al., 1986).
Sperm nuclei from L. pictus were demembranated with 0.1% Triton
X-100 (Collas and Poccia, 1997). To produce nuclei surrounded by an
envelope devoid of nuclear pores and of a lamina (‘poreless and
laminaless nuclei’), demembranated L. pictus sperm nuclei were
incubated in an L. pictus fertilized egg cytoplasmic extract (10,000 g
supernatant) containing an ATP-generating system (2 mM ATP, 20 mM
creatine phosphate, 50 µg/ml creatine kinase) and 100 µM GTP (Collas
and Poccia, 1997). Although these nuclei are surrounded by
membranes, they lack detectable pores and a lamina and remain small
(Collas and Poccia, 1997). Swollen male pronuclei with a complete
NE containing nuclear pores and a lamina were produced in
cytoplasmic extract by fusion of additional vesicles to the membranes
of poreless and laminaless nuclei, and ATP-induced import of soluble
lamin B into these nuclei (Collas et al., 1996; Collas and Poccia, 1997).
Nuclei with membranes containing functional pores but no detectable
lamina (‘laminaless nuclei’) were produced by fusion of vesicles
selectively depleted of lamin B-containing vesicles (Collas et al., 1996)
with the membranes of poreless and laminaless nuclei, in interphase
cytosol immunodepleted of soluble lamin B (Collas et al., 1996).
Nuclear envelope disassembly
Nuclei were incubated in mitotic cytosol (~1000 nuclei/µl) at room
temperature. NE disassembly was initiated by addition of an ATPgenerating system and allowed to proceed for 1 hour. In some
experiments, inhibitors of nuclear pore function were added to cytosol
30 minutes before adding the ATP-generating system. Aliquots were
taken to examine chromatin after staining DNA with 0.1 µg/ml
Hoechst 33342 and membranes with 10 µg/ml of the lipophilic dye
dihexyloxacarbocyanine iodide (DiOC6). Chromatin was considered
to condense when nuclei acquired a compact and irregular
morphology. For immunoblotting analyses, nuclei were centrifuged at
500 g through 1 M sucrose.
Lysolecithin permeabilization of nuclear membranes
To permeabilize nuclear membranes, lysolecithin was added to 250 µl
of nuclear suspension to a concentration of 0.75 µg/ml and incubated
for 15 minutes at room temperature. Excess lysolecithin was quenched
with 1 ml of 3% BSA, then nuclei sedimented and washed in buffer
N. To assess NE permeabilization, nuclei were labeled with 2 mg/ml
of 150 kDa FITC-conjugated dextran. Nuclear membranes following
lysolecithin treatment were examined by labeling nuclei with 10
µg/ml of the lipophilic dye DiOC18.
BSA-peptide conjugates and nuclear import assay
Synthetic NLS peptides were conjugated to BSA at a coupling ratio
of ~50 peptides per BSA molecule as described (Collas and Aleström,
1996). Nuclear import reactions were performed for 2 hours in
interphase extract containing ~1000 nuclei/µl and 2 mg/ml BSA-NLS
conjugate, without ATP to monitor BSA-NLS binding to the NE, or
with an ATP-generating system to assess translocation into nuclei.
NE-bound and intranuclear BSA-NLS was detected by indirect
immunofluorescence. To quantify nuclear import of BSA-NLS,
photographs were taken in a TIFF format under subsaturating
fluorescence and intra nuclear fluorescence intensity measured in a
2×2 µm2 random area using the OptiLab/Pro software (Graftek,
Mirmande, France). The same area was moved to a random location
near the nucleus to measure background fluorescence. Nuclear import
was expressed as mean difference (±s.d.; n=50) between intranuclear
and background fluorescence per µm2.
Immunofluorescence and immunoblotting procedures
Immunofluorescence microscopy was performed as described (Collas
et al., 1996), with all antibodies used at a 1:100 dilution. p56, lamin
B and BSA were detected using secondary antibodies conjugated to
TRITC (p56) or FITC (lamin B, BSA). Microscopic observations,
photography and immunoblotting analyses were done as described
(Collas et al., 1996).
Nuclear disassembly in vitro 1295
32P-labeling
and immunoprecipitation of p56 and lamin B
To immunoprecipitate p56 and lamin B from NE-derived vesicles, a
nuclear disassembly reaction was performed in 200 µl mitotic cytosol,
nuclei centrifuged through sucrose, and the supernatant (containing
cytosol and NE-derived vesicles) diluted to 1 ml with membrane wash
buffer and centrifuged at 150,000 g for 30 minutes to sediment
vesicles. Vesicles were solubilized for 30 minutes in
immunoprecipitation buffer (0.1% Triton X-100, 10 mM Tris-HCl
(pH 7.5), 0.2 M NaCl, 100 µM sodium vanadate and protease
inhibitors). p56 and lamin B were immunoprecipitated from the
solubilized material and immune complexes solubilized in SDS
sample buffer (Collas et al., 1996). Immunoisolation of p56 and lamin
B from NE-derived vesicles was performed without detergent.
Interphase and mitotic 32P-labeling of p56 and lamin B was
performed by incubating nuclei at room temperature for 10 minutes
in 200 µl of interphase or mitotic cytosol, respectively, containing 0.75
µCi/µl [32P]ATP and an ATP-generating system. A 10-minute
incubation was sufficient to achieve significant levels of 32P
incorporation into p56 and lamin B before complete NE breakdown.
To immunoprecipitate 32P-labeled p56 and lamin B from nuclei,
nuclei were sedimented, washed and extracted for 30 minutes at 4°C
in nuclear extraction buffer (1% Triton X-100, 10 mM Tris-HCl (pH
7.5), 0.4 M NaCl, 100 µM sodium vanadate and protease inhibitors).
Nuclei were pelleted and p56 and lamin B immunoprecipitated from
the extracted material as above. Immune complexes were subjected to
SDS-PAGE in 12% polyacrylamide gels and 32P-labeled proteins
detected by autoradiography. Identity of p56 and lamin B on
autoradiograms was confirmed by their detection on parallel blots (not
shown).
RESULTS
Disassembly of the NE in mitotic cytosolic extract
Nuclei were prepared from L. pictus gastrulae. Embryonic
nuclei were chosen because they disassemble more readily than
differentiated somatic nuclei in vitro (Newport and Spann,
1987). Integrity of isolated nuclei was assessed by phase
contrast microscopy and staining with FITC-Con A, which
labels only damaged NEs (Newmeyer et al., 1986). Over 90%
of nuclei examined were intact, as judged by the lack of FITCCon A staining (Fig. 1).
A mitotic cytosolic extract, which promotes NE
disassembly, was prepared from naturally synchronized mitotic
L. pictus embryos. Gastrula nuclei incubated in mitotic extract
containing an ATP-generating system were examined at regular
intervals after DNA staining with Hoechst 33342 and
membrane labeling with the lipophilic dye DiOC6. As shown
in Fig. 2A, membrane labeling was greatly attenuated at 10
minutes and disappeared at 20 minutes, suggesting removal of
the nuclear membranes. Some chromatin condensation was
noticed at 20 minutes and evident at 60 minutes, as shown by
compacted chromatin morphology. Replacing ATP by 2 mM
of the ATP analogues ATPS or AMP-PNP did not promote
nuclear membrane disassembly, indicating a requirement for
ATP hydrolysis.
NE disassembly in mitotic extract was further documented
by double immunofluorescence analysis of the transmembrane
protein p56 and of the peripheral protein lamin B. All p56
labeling disappeared from nuclei within 20 minutes of
incubation, confirming early nuclear membrane solubilization
(Fig. 2B). By contrast, significant lamin B labeling was
observed at 20 minutes, which disappeared at 60 minutes.
Fig. 1. Morphology of isolated L. pictus gastrula nuclei.
(A) Frozen/thawed isolated gastrula nuclei were examined by phase
contrast. (B) Integrity of the nuclear membranes was assessed by
incubating nuclei with 0.1 mg/ml FITC-Con A (inset, DNA). FITCCon A labeling reflects a damaged envelope (arrow). Bars, 5 µm (A),
10 µm (B).
Solubilization of p56 and lamin B was also examined by
immunoblotting nuclear and supernatant fractions obtained at
progressive stages of disassembly (Fig. 2C, Nuclei). As a result
of their solubilization from nuclei, p56 and lamin B were
detected in supernatant fractions containing cytosol and NEderived vesicles (Fig. 2C, Cytosol+MVs). The apparent Mr of
lamin B detected in the cytosol/MV fraction in Fig. 2C was
shifted from 65 kDa to 68 kDa, suggesting modification of the
protein.
Phosphorylation of p56 and lamin B in mitotic extract was
demonstrated by 32P incorporation into these proteins during
NE breakdown. Gastrula nuclei were incubated in interphase
or mitotic extract containing [32P]ATP for 10 minutes, at which
time the NE was still present. Nuclei were recovered through
sucrose, extracted with 1% Triton X-100/0.4 M NaCl, and p56
and lamin B immunoprecipitated from the extracted material
using anti-LBR and anti-lamin B antibodies. When nuclei were
incubated in interphase cytosol before extraction,
autoradiography of the immunoprecipitated material showed
that p56 and lamin B were phosphorylated and coprecipitated
with either antibody (Fig. 3, I). However, when nuclei were
incubated in mitotic cytosol, p56 and lamin B appeared
hyperphosphorylated and did not coprecipitate (Fig. 3, M),
suggesting that the two proteins were either weakly associated
or dissociated before extraction. Lamin B and p56 were not
phosphorylated when nuclei were incubated in lysis buffer
containing [32P]ATP (Fig. 3, Buffer), indicating the absence of
endogenous nuclear-associated p56 or lamin B kinase activity.
These results indicate that the mitotic extract induces rapid
hyperphosphorylation of p56 and lamin B in nuclei and a
weakening of their interactions preceding their solubilization.
Lamin B and p56 segregate into distinct NE-derived
vesicles in vitro
Since both p56 and lamin B were detected in the mitotic
cytosol/MV fraction after NE disassembly, the distribution of
these proteins between soluble (cytosolic) and NE-derived
membrane vesicle (MV) fractions was investigated. Gastrula
nuclei were incubated in mitotic cytosol for 1 hour, sedimented,
the supernatant fractionated by ultracentrifugation, and both
1296 P. Collas
Fig. 2. Nuclear membrane and lamina disassembly in mitotic extract. (A) DNA and membrane labeling of gastrula nuclei incubated in mitotic
cytosol containing an ATP-generating system. DNA was labeled with Hoechst and membranes with DiOC6. (B) Double immunofluorescence
localization of p56 and lamin B in gastrula nuclei incubated in mitotic cytosol as in A, using anti-LBR and anti-lamin B antibodies,
respectively. (C) Immunoblotting analysis of nuclei incubated in mitotic cytosol as in A, using anti-LBR and anti-lamin B antibodies. Cytosol
and NE-derived vesicle fractions (Cytosol+MVs) were also immunoblotted using the same antibodies. Bar, 10 µm.
soluble and insoluble fractions examined by immunoblotting
using anti-LBR and anti-lamin B antibodies. As expected from
an integral membrane protein, p56 was detected exclusively in
the vesicle fraction (Fig. 4A, left panel). Lamin B was detected
in both soluble and vesicle fractions (Fig. 4A, right panel),
suggesting that part of lamin B remained associated with NEderived vesicles.
A putative association of lamin B and p56 in sedimented
Fig. 3. Hyperphosphorylation and dissociation of p56 and lamin B
upon NE disassembly in mitotic extract. Gastrula nuclei were
incubated for 10 minutes in mitotic extract (M), interphase extract
(I), or lysis buffer (Buffer), each containing [32P]ATP and an ATPgenerating system. p56 and lamin B were extracted from nuclei with
1% Triton X-100/0.4 M NaCl, immunoprecipitated from the
extracted material with anti-LBR and anti-lamin B antibodies (IP),
and phosphorylation assessed by autoradiography of the
immunoprecipitates. Migration of molecular mass standards is
indicated in kDa on the left.
Nuclear disassembly in vitro 1297
vesicles was investigated by solubilization of the insoluble
material under mild conditions (0.1% Triton X-100/0.2 M
NaCl) followed by immunoprecipitation of p56 and lamin B.
Immunoprecipitates (P) and supernatants (S) were
immunoblotted with each antibody. Fig. 4B (left panels) shows
that anti-lamin B antibodies precipitated lamin B but not p56,
while anti-LBR antibodies precipitated p56 but not lamin B.
Neither protein was detected in material precipitated with preimmune antibodies (Fig. 4B, right panels).
To determine whether p56 and lamin B disassembled from
nuclei colocalized in the same vesicles or segregated into
different vesicles, p56 and lamin B were immunoisolated from
NE-derived vesicles in the absence of detergent (Collas et al.,
1996). Immunodepletions were complete, as judged on blots
of immunoprecipitates and supernatants, and selective since
lamin B- and p56-containing vesicles were not coprecipitated
by anti-LBR and anti-lamin B antibodies, respectively (Fig.
4C). Neither p56-containing vesicles nor lamin B-containing
vesicles were precipitated by preimmune antibodies (not
shown). Moreover, immunofluorescence analysis of NEderived vesicles using anti-LBR and anti-lamin B antibodies
showed that p56 and lamin B did not colocalize within these
vesicles (Fig. 4D). These results indicate that, in mitotic
extract, p56 and lamin B are hyperphosphorylated, dissociate
prior to disassembling from nuclei and segregate into distinct
NE-derived vesicles.
Role of nuclear import in NE disassembly in mitotic
extract
It was previously shown that L. pictus sperm chromatin
incubated in interphase L. pictus egg extract decondenses and
becomes enclosed by a nuclear membrane with no apparent
pores and no lamina (Collas and Poccia, 1997). These nuclei
are referred to as ‘poreless and laminaless nuclei’.
Supplementing the extract with ATP and additional membranes
promotes nuclear swelling to produce male pronuclei with a
complete NE, containing pore complexes and a lamina,
provided that soluble lamins are present in the extract (Collas
et al., 1996). The putative disassembly of the poreless and
laminaless nuclei in mitotic cytosol, i.e., the possibility that the
nuclear membrane can be disassembled ‘from the outside’, was
investigated. Nuclei were incubated for 1 hour in mitotic
cytosol then examined by DNA and membrane labeling, and
immunofluorescence using anti-LBR antibodies. Poreless and
Fig. 4. Localization of p56 and lamin B in different NE-derived vesicles in vitro. (A) Following disassembly of gastrula nuclei, soluble
(cytosol) and NE-derived vesicle (MV) fractions were isolated and immunoblotted using anti-LBR and anti-lamin B antibodies. Migration of
molecular mass standards is indicated in kDa on the left. (B) Isolated NE-derived vesicles were solubilized with 0.1% Triton X-100/0.2 M
NaCl, and p56 and lamin B immunoprecipitated from the solubilized material. Control precipitations were done using preimmune antibodies
(Ctl lam, Ctl LBR). Immunoprecipitates (P) and supernatants (S) were immunoblotted using anti-LBR and anti-lamin B antibodies. (C) p56 and
lamin B were immunoisolated from NE-derived vesicles in the absence of detergent. Immunoprecipitates (P) and supernatants (S) were
immunoblotted using anti-LBR and anti-lamin B antibodies. HC, IgG heavy chain. (D) Immunofluorescence analysis of p56 and lamin B in
NE-derived vesicles using anti-LBR and anti-lamin B antibodies. The marked area in the overlap view (Merge) is enlarged in the right panel.
Bar, 5 µm.
1298 P. Collas
Fig. 5. Nuclei with no pores and no
lamina do not disassemble in mitotic
extract. Poreless and laminaless
nuclei and male pronuclei with a
complete NE were incubated in
mitotic cytosol for 1 hour and
examined by membrane labeling or
immunofluorescence using anti-LBR
antibodies (p56). Insets, DNA. Bar,
10 µm.
laminaless nuclei remained intact, in contrast to pronuclei,
which disassembled in a manner similar to gastrula nuclei (Fig.
5). Thus, requirements for nuclear pores and a lamina for NE
disassembly in mitotic extract were investigated.
A likely mode of access of p56 and lamin B kinase(s) or
their cofactors from the cytosol to their substrate is import into
the nucleus through nuclear pores. Since nuclear import is
inhibited by the lectin wheat germ agglutinin (WGA; Finlay et
al., 1987), disassembly of gastrula nuclei was investigated in
the presence of 0.5 mg/ml WGA for 1 hour in mitotic extract.
As shown in Fig. 6A, the chromatin remained fully
decondensed, and nuclear membranes and the lamina were
Fig. 6. Blocking nuclear import with WGA
inhibits NE disassembly in mitotic extract.
(A) Gastrula nuclei were incubated for 1 hour in
mitotic cytosol containing WGA, or WGA plus
the WGA ligand TCT, and analyzed after DNA
and membrane labeling or by double
immunofluorescence using anti-LBR (p56) and
anti-lamin B antibodies. Input nuclei (not shown)
were similar to those shown in Fig. 2.
(B) Permeabilization of gastrula nuclei treated
with 0.75 µg/ml lysolecithin to a 150 kDa FITCconjugated dextran. Nuclei were labeled with 10
µg/ml DiIC18 (Membrane) and 2 mg/ml FITCdextran (FITC-dex). (C) Immunoblotting analysis
of lysolecithin-treated (Lyso +) and intact (Lyso
−) gastrula nuclei incubated for 1 hour in mitotic
cytosol with or without WGA, using anti-LBR
and anti-lamin B antibodies. Bars, 5 µm.
intact as judged by membrane labeling and double
immunofluorescence analysis of p56 and lamin B. No p56 nor
lamin B was released from nuclei, as seen on immunoblots of
cytosol/MV fractions (not shown). In contrast, in cytosol
containing WGA and 1 mM of the WGA ligand N,N′,N″triacetylchitotriose (TCT), the chromatin condensed and the
NE disassembled (Fig. 6A). Inhibition of NE disassembly was
also obtained after blocking nuclear import with the antinucleoporin antibody mAb414 (not shown). These results
suggest therefore that nuclear pore function is essential for NE
disassembly in mitotic extract.
To verify that the lack of NE disassembly observed with
Nuclear disassembly in vitro 1299
WGA was only due to blocking nuclear pore function, nuclei
were incubated in mitotic cytosol containing WGA for 1.5
hours, then 1 mM of the WGA ligand TCT added and nuclei
examined after another hour of incubation. Over 80% of nuclei
were disassembled, as judged by DNA staining and
immunofluorescence analysis of p56 and lamin B (not shown).
Thus the lack of NE disassembly observed with WGA did not
result from degradation or cycling of the extract out of Mphase, but only from blocking nuclear import. This confirms
that functional pores are necessary for NE disassembly in vitro.
Additional evidence to support this conclusion was provided
by experiments in which pores were blocked with WGA, but
access of the cytosolic p56 and lamin B kinase or cofactors to
the nucleus was allowed by permeabilizing the NE of gastrula
nuclei with 0.75 µg/ml lysolecithin prior to incubation in
mitotic cytosol. Lysolecithin treatment was mild, as judged by
a persisting membrane labeling, yet nuclei were permeable to
a 150 kDa FITC-conjugated dextran (Fig. 6B). Regardless of
NE permeabilization with lysolecithin, nuclei incubated in
WGA-containing cytosol retained p56 and lamin B, in contrast
to nuclei incubated in control WGA-free cytosol (Fig. 6C).
Disassembly of lysolecithin-treated nuclei in WGA-free
cytosol (Fig. 6C) indicated that lysolecithin itself was not
inhibitory. These results indicate that blocking nuclear pore
function with WGA prevents NE disassembly even though the
NE is permeabilized.
Laminaless nuclei do not disassemble in mitotic
extract
To address the question of a putative role of the nuclear lamina
in NE disassembly in vitro, laminaless nuclei (containing
membranes with functional pores but no lamina) were formed
by incubating poreless and laminaless nuclei for 40 minutes in
interphase cytosol selectively depleted of soluble and
membrane-bound lamin B, in the presence of an ATPgenerating system and 100 µM GTP. Under these conditions,
vesicles fused with the existing nuclear membranes but no
nuclear swelling occurred and no lamina was formed (not
shown; Collas et al., 1996).
The competence of these laminaless nuclei for import was
Fig. 7. Nuclei with functional pores but without a lamina do not disassemble in mitotic extract. (A) Nuclear import of BSA-NLS conjugates
into poreless and laminaless nuclei, laminaless nuclei, male pronuclei (all assembled in vitro), and gastrula nuclei. Nuclei were incubated for ~2
hours in interphase extract containing BSA-NLS, without ATP (−ATP) to assess BSA-NLS binding to the NE, or with an ATP-generating
system (+ATP) to promote BSA-NLS import inside nuclei. BSA was detected by immunofluorescence using anti-BSA antibodies and FITCconjugated secondary antibodies. (B) Nuclear import of BSA-NLS was quantified by measuring intranuclear FITC fluorescence intensity on
photographs of nuclei. Amount of BSA imported is expressed as mean ± s.d. nuclear fluorescence (in arbitrary units/µm2) after background
subtraction (n=50 per group). (C) Laminaless nuclei are not disassembled in mitotic extract. Laminaless nuclei and male pronuclei were
incubated in mitotic cytosol and disassembly assessed by DNA staining (insets), membrane labeling and immunofluorescence using anti-LBR
antibodies. Bars, (A) 10 µm, (C) 5 µm.
1300 P. Collas
assessed by monitoring import of BSA conjugated to NLS
peptides. Nuclei were incubated for ~2 hours in interphase
cytosol containing BSA-NLS conjugates without ATP to assess
substrate binding to the NE, or with an ATP-generating system
to monitor translocation across the NE (Newmeyer et al.,
1986). Immunofluorescence analysis of nuclei using anti-BSA
antibodies showed that laminaless nuclei imported BSA-NLS
in an ATP-dependent manner, as did pronuclei and gastrula
nuclei (Fig. 7A). However, no NE binding nor translocation of
BSA-NLS occurred with poreless and laminaless nuclei (Fig.
7A). Quantification of nuclear import of BSA-NLS conjugates
showed that laminaless nuclei, pronuclei and gastrula nuclei
imported the substrate to similar levels (Fig. 7B). Nuclear
import of BSA-NLS was inhibited by WGA, but not
WGA/TCT (not shown). Thus, laminaless nuclei are capable
of karyophilic substrate import to the same extent as gastrula
nuclei and therefore contain functional pores.
Disassembly of laminaless nuclei in mitotic cytosol was
investigated by DNA and membrane labeling and p56
immunofluorescence. Fig. 7C shows that, in contrast to male
pronuclei, laminaless nuclei failed to disassemble after >1 hour
in mitotic cytosol. A putative correlation between the failure to
disassemble and a lack of phosphorylation of p56 was
investigated
by
autoradiography
of
anti-p56
immunoprecipitates from detergent/salt extracts of laminaless
nuclei. The results show that p56 of laminaless nuclei was
hyperphosphorylated similarly to p56 of pronuclei (not shown;
see Fig. 8C, first lane). These observations indicate that nuclei
with functional pores but no lamina do not disassemble in
mitotic extract despite hyperphosphorylation of p56.
The critical role of the lamina in NE disassembly was
demonstrated by restoration of NE breakdown after
reformation of a lamina in laminaless nuclei. To reconstitute
a lamina, laminaless nuclei were incubated for 1 hour in
interphase cytosol containing endogenous lamins and an
ATP-generating system. A lamina assembled, as shown by
immunofluorescence and immunoblotting analysis of nuclei
using anti-lamin B antibodies (Fig. 8A,B, Input), likely as a
result of import of soluble lamin B through nuclear pores
(Collas et al., 1996). Incubation of nuclei with a reconstituted
lamina in mitotic cytosol promoted nuclear membranes and
lamina disassembly, as judged by immunoblotting of nuclei
and cytosol/MV fractions using anti-LBR and anti-lamin B
antibodies (Fig. 8B, M-phase). Autoradiography of p56 and
lamin B immunoprecipitated from a detergent/salt extract
from nuclei with a reconstituted lamina showed that both
proteins were phosphorylated and dissociated (Fig. 8C).
These results indicate that reconstitution of a lamina was
essential to restore the ability of nuclei to disassemble in
mitotic extract.
DISCUSSION
The results of this study demonstrate a requirement for
functional nuclear pores and a lamina in NE disassembly in
vitro. Several lines of evidence support this conclusion. (1)
Blocking nuclear pore function prevents p56 and lamin B
hyperphosphorylation, dissociation of both proteins and
nuclear membrane and lamina disassembly. (2) Nuclei with
functional pores but no lamina do not disassemble in mitotic
Fig. 8. Reformation of a nuclear lamina in laminaless nuclei restores
NE disassembly in mitotic cytosol. (A) Laminaless nuclei were
incubated for 2 hours in interphase cytosol containing an ATPgenerating system and endogenous lamin B (+Lamins), or in lamin
B-depleted interphase cytosol (−Lamins). Reformation of a nuclear
lamina was examined by immunofluorescence using anti-lamin B
antibodies. Bar, 5 µm. (B) Nuclei with a reconstituted lamina
(+Lamina) and laminaless nuclei (−Lamina) were incubated in
mitotic cytosol (M-phase), and nuclei and cytosol/MV fractions
immunoblotted using anti-LBR and anti-lamin B antibodies.
(C) Laminaless nuclei and nuclei with a reconstituted lamina were
incubated for 10 minutes in [32P]γATP-containing mitotic cytosol,
and p56 and lamin B phosphorylation determined as in Fig. 3.
cytosol. (3) Reconstitution of a lamina into laminaless nuclei
restores NE disassembly.
Segregation of p56 and lamin B into distinct NEderived vesicles in vitro
In interphase nuclei, interactions between integral proteins of
the inner nuclear membrane and nuclear lamins are believed to
tether the NE with the lamina (Worman et al., 1988; Foisner
and Gerace, 1993). Likewise, as in male pronuclei assembled
in vitro (Collas et al., 1996), the inner nuclear membrane
integral protein p56 associates with lamin B in embryonic
nuclei, as shown by coprecipitation of both proteins following
their extraction from gastrula nuclei with detergent and salt.
Remarkably, LBR and lamin B do not coprecipitate from nuclei
of cultured vertebrate somatic cells in spite of their association
(Courvalin et al., 1992). This difference may be related to
different lamin contents in differentiated cells and
undifferentiated or embryonic cells that do not express A/Ctype lamins (Guilly et al., 1987; Stewart and Burke, 1987).
The association of p56 with lamin B in nuclei exposed to
mitotic cytosol appears to be weakened by phosphorylation of
Nuclear disassembly in vitro 1301
either or both proteins, and the two proteins dissociate from
one another prior to their release from nuclei. The nature of the
embryonic sea urchin p56 and lamin B kinase remains
undetermined; however, the kinase is most likely of cytosolic
rather than nuclear origin, since cytosol (interphase or mitotic)
is required for p56 and lamin B phosphorylation (Fig. 3).
Alteration of the affinity of LBR (or p56) for the lamina and/or
chromatin at mitosis is supported by the conversion of LBR
from a detergent-resistant into a detergent-extractable form
after phosphorylation (Bailer et al., 1991). After release from
nuclei, p56 and lamin B segregate into distinct NE-derived
vesicles, while part of lamin B becomes soluble, in agreement
with previous in vivo and in vitro observations (Chaudhary and
Courvalin, 1993; Collas et al., 1997). Furthermore, in contrast
to previous studies that examined total mitotic vesicles (Meier
and Georgatos, 1994), I have analyzed vesicles exclusively
derived from the NE and provide evidence for the segregation
of nuclear membrane and lamina components, rather than their
association during mitosis. Segregation of nuclear membrane,
pore and lamina components at mitosis is corroborated by
independent disassembly of nuclear membranes and the lamina
in Drosophila embryos (Stafstrom and Staehelin, 1984; Paddy
et al., 1996) and Xenopus egg extracts (Newport and Spann,
1987), and sequential binding of nuclear membranes and
lamins to chromatin during nuclear assembly after metaphase
(Chaudhary and Courvalin, 1993; Buendia and Courvalin,
1997) and in vitro (Newport et al., 1990; Jenkins et al., 1993;
Collas et al., 1996).
Requirement for nuclear pores in NE disassembly
Inhibition of nuclear pore function with WGA or antibodies to
nucleoporins prevents p56 and lamin B hyperphosphorylation,
NE disassembly and lamina solubilization in mitotic extract,
suggesting a role for nuclear pores in NE disassembly in vitro.
This contention apparently disagrees with findings of Gant and
Wilson (1997) that illustrate breakdown in mitotic extract of
Xenopus nuclei devoid of nuclear pores, which are assembled
in vitro in the absence of proteins of <50 kDa, including ADP
ribosylation factor (ARF). This difference may be explained by
the fact that most nuclei assembled in ARF-depleted extract
have discontinuous membranes (Gant and Wilson, 1997) and
that a subset of size-fractionated cytosol may create alterations
in nuclear membranes, facilitating their disassembly in mitotic
extract.
Nuclear pores may play a functional and a structural role in
NE disassembly. The present results corroborate implications
of Kumagai and Dunphy (1991), which suggest that nuclear
pores in G2 and early prophase provide access for cyclin B to
the nucleus (Pines and Hunter, 1991), thereby regulating
activation of p34cdc2 needed for phosphorylation of lamins
(Peter et al., 1990) and other proteins required for NE
disassembly. Nuclear pores may also be structurally important
for nuclear disassembly in that phosphorylation of pore
proteins in late prophase (Macaulay et al., 1995; Favreau et al.,
1996) may alter poreless pore, pore-nuclear membrane and
pore-lamina interactions. Moreover, absence of nuclear pores
or inhibition of pore function prevents nuclear membrane and
lamina disassembly, even after permeabilization of nuclear
membranes to components of 150,000 Mr with lysolecithin.
This suggests nuclear pores may not merely provide access of
soluble components implicated in nuclear disassembly, but
perhaps concentrate these factors to a threshold required for
activity, at the level of the NE or inside the nucleus. The role
of ran/TC4, a GTPase implicated in nuclear import (Melchior
et al., 1993), in mitotic p34cdc2 activation supports this view
(Kornbluth et al., 1994).
A putative role for the nuclear lamina in NE
disassembly
A consensus idea is that phosphorylation of lamin B on Ser
residues in the N- and C-terminal domains of lamin B is
required for mitotic lamina disassembly. A model for lamina
disassembly elicited by multisite phosphorylation (Fields and
Thompson, 1995) postulates that phosphorylation of lamin B
in the C-terminal region disrupts interactions between adjacent
lamin B polymers. This phosphorylation event may facilitate
subsequent phosphorylation of the N-terminal domain,
promoting disassembly of lamin B polymers (Peter et al.,
1990).
The respective role of lamin B and LBR phosphorylation in
NE disassembly has not been addressed previously. The
present data show that p56 is phosphorylated in interphase and
hyperphosphorylated in mitotic extract. In contrast, in cultured
vertebrate somatic cells, LBR is not hyperphosphorylated at
mitosis, although it is phosphorylated at different sites in
interphase and mitosis (Courvalin et al., 1992). This suggests
that LBR hyperphosphorylation may facilitate NE breakdown
in rapidly dividing embryonic cells. In nuclei containing
functional pores but no lamina, p56 is also
hyperphosphorylated in mitotic extract. Yet the NE does not
disassemble, arguing p56 hyperphosphorylation in itself is not
sufficient to promote nuclear membrane breakdown.
A provocative result of this study is the absence of
disassembly of nuclei reconstituted with an envelope
containing functional pores but no lamina. Two lines of
evidence suggest that the lack of lamina was responsible for
the failure to disassemble. First, there was no defective
nucleocytoplasmic transport since import of a karyophilic
substrate was quantitatively similar in laminaless nuclei and
pronuclei assembled in vitro or in gastrula nuclei (see also
Jenkins et al., 1993). Second, reconstitution of a lamina in
laminaless nuclei by incubation in interphase cytosol was able
to restore NE disassembly in mitotic extract. Nonetheless,
because in vitro assembly of a nuclear lamina is associated
with nuclear swelling (Collas et al., 1996), it is difficult to
assess whether restoration of the ability of the NE to
disassemble is due to reconstitution of the lamina per se, or
parallel import of other factors promoting nuclear disassembly.
In any event, these observations should be placed in parallel
with the fact that the yeast NE, which does not disassemble at
mitosis, does not contain any lamin-like proteins (Mewes et al.,
1997).
What may be the role of the lamina in nuclear disassembly?
In mitotic mammalian and Xenopus cell extracts, lamina
solubilization precedes NE breakdown (Miake-Lye and
Kirschner, 1985; Newport and Spann, 1987), suggesting that
initiation of lamina disassembly may be necessary for NE
breakdown. A possibility is that the lamina indirectly plays a
structural role in this process. There is strong evidence to
suggest that lamina assembly is required for proper formation
of the nuclear matrix (Zhang et al., 1996). Thus a simplistic
hypothesis is that absence of a lamina prevents nuclear
1302 P. Collas
disassembly through the lack of a nuclear matrix. It remains to
be shown however, whether the nuclear matrix acts in mitotic
nuclear disassembly. The lamina may also play a structural role
in vesicularizing the nuclear membranes, by facilitating the
segregation of lamin B and LBR into separate vesicles.
Another possibility is that the lamina creates a specialized
domain between the chromatin and the inner nuclear
membrane, reminiscent of the LBR/lamin/RS kinase complex
identified in avian erythrocyte NEs (Nikolakaki et al., 1996).
This domain may constitute a site where mitotic kinases and
other components involved in NE disassembly may dock. The
formation of domains within the nucleus, essential for nuclear
function and cell cycle regulation supports this idea (Ellis et
al., 1997; Jenuwein et al., 1997). In this respect, the nuclear
lamina may promote specific cell cycle events, perhaps by
acting as a novel check point controlling disassembly of the
NE.
The author thanks Drs J.-C. Courvalin for anti-human LBR
antibodies, J. Holy for W3-1antibodies, M. Rout for mAb414, D.
Poccia (Amherst College) for providing sea urchin gametes, and M.
Lohka (University of Calgary) and J.-C. Courvalin for valuable
discussions.
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