Download Relation between nuclear envelope and nuclear lamina in nuclear

VOI. 40 NO. 6
SCIENCE IN CHINA (Series C)
December 1997
Relation between nuclear envelope and nuclear
lamina in nuclear assembly in vitro *
CAI Shutao (@#'@) ** and ZHAI Zhonghe
(aq$!J)
**"
(Life Sciences College of Peking University, Beijing 100871, China)
Received February 1, 1997
Abstract
Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to
study the process of nuclear lamina assembly in nuclear reconstitution i n vitro . The experimental results showed that
lamin was involved in the nuclear assembly i n v i t r o . The assembly of nuclear lamina was preceded by the assembly of
nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition
of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.
Keywords:
nuclear assembly in vitro, nuclear envelope, nuclear lamina, nuclear matrix.
Nuclear assembly is a universal phenomenon in life cycles, for example, the nuclear reconstitution after mitosis and meiosis. The process of nuclear assembly is a good model for studying the
function and organization of nucleus. However, the organism, even a single cell, is a complicated
system, in which so many factors interact with each other, and it is difficult to carry out manipulation and analyses. The cell-free nuclear assembly system derived from Xenopus laevis egg extracts can provide a good experimental model for the study of nuclear structure and f u n c t i ~ n " . ~ ] .
Nuclear lamina, considered as structural components of unclear matrix, is a 10-nm filament
network underlayered inner nuclear membrane. It is believed that nuclear lamina is structurally
and functionally related to nuclear envelope, chromatin lnd nuclear pore complex[31. The nuclear
lamina may provide a framework for nuclear envelope and chromatin in interphase nucleus[41. In
mitosis, the nuclear lamina disassembled and reassembled dynamically, while nuclear envelope
broke down in prophase, nuclear lamina disassembled and soluble lamin diffused into the cytoplasm; when nuclear envelope reappeared in telophase, the lamin reassembled around the chromatin to form the nuclear lamina of daughter nuclei[5961.It was suggested that the reversible disassembly and reassembly of nuclear lamina could regulate breaking down and reassembly of the
nuclear envelope[41. Cell-free nuclear assembly system makes it possible to study the relationships
between nuclear envelope and nuclear lamina i n vitro . Immunodepleting lamin A/C or lamin B
from CHO mitotic cell-free extracts results in inhibition of the assembly of nuclear envelope and
nuclear pore complex around the
In our previous reports, exogenous lambda DNA was added to Xenopus laevis egg extracts
* Project supported by the National Natural Science Foundation of China.
** Present address: Institute of Biophysics, the Chinese Academy of Sciences, Beijing 100101, China.
*** To whom correspondence should be addressed.
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NUCLEAR ENVELOPE & LAMINA ASSEMBLY
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cell-free system to induce nuclear assembly in v i t r ~ ' .~ The
'
nuclei assembled in vitro possess the
basic characteristics of normal nuclei[g1. In the study presented here, we apply in vitro nuclear
reconstitution system to studying the assembly of nuclear envelope and nuclear lamina. To investigate the role of nuclear lamina in nuclear assembly, we use anti-lamin antibodies to block the normal assembly of nuclear lamina. The experimental results demonstrate that the nuclear envelope
assembly is closely related with the nuclear lamina assembly.
1
Materials and methods
1 . 1 Nuclear reconstitution
' ~ ] some modificaNuclear reconstitution reaction was done by the method of ~ e w ~ o r twith
tionLgl.Xenopus laevis eggs were injected with 500-800 units of human chorionic gonadotropin
(HCG) the night before use. The eggs were dejelled with 2 % cysteine (pH7.8) and activated by
0 . 2 pg/mL ca2 ionophore A23187 in MMR ( 0 . 1 mol/L NaC1, 20 mmol/L KC1, 1 . 0 mmol/L
MgS04, 2 . 0 mmol/L CaC12, 5 . 0 mmol/L HEPES, 0 . 1 mmol/L EDTA, pH 7 . 8 ) plus 5 % Fi+
coll. Following activation the eggs were rinsed three times with 1/4 MMR plus 5 % Ficoll and
were allowed to incubate for another 20 min. The eggs were packed via 30 centrifugation at 100 g
in a solution containing 250 mmol/L sucrose, 2 . 5 mmol/L MgCl,, 50 mmol/L KC1, 100 pg cycloheximide, 5 pg/mL cytochalasin B, 1 mmol/L DTT. Then the eggs were lysed by centrifugation at 10 000 g at 4°C for 15 min, and the egg components were distributed within the tube with
yolk (bottom), cytosol (middle) and lipid ( t o p ) . The cytosol was removed with a syringe and
was centrifuged again as above. To prepare lambda DNA as a substrate for nuclear reconstitution,
the lambda DNA was heated for 5 rnin at 6 5 C , then cooled for 3 rnin at 22C . After this treatment, the DNA was noncovalently ligated (due to the sticky ends of lambda) into multimers. To
set up nuclear reconstitution reaction, the lambda DNA was incubated at 22°C in egg extracts
containing an ATP regenerating system consisting of 2 mmol/L ATP, 20 mmol/L phosphocreatine, and 50 pg/mL creatine kinase.
1.2
Nuclear matrix preparation
Nuclear matrix was prepared essentially according to the method of F ~ ~ [ ' O ' . The nuclei assembled in vitro were resuspended in cytoskeleton (CSK) buffer (100 mmol/L KC1, 300 mmol
/ L sucrose, 10 mmol/L PIPES, pH 6 . 8 , 3 mmol/L MgC12, 1 mmol/L EGTA, 1 . 2 mmol/L
PMSF and 0 . 5 % Triton X-100) and incubated at 4°C for 3-5 min. The nuclei were pelleted after centrifugation at 600 g for 5 min, and the supernatant, which contained soluble components
of egg extracts, was removed. The pellets were resuspended in RSB-Magik ( 4 2 . 5 mmol/L TrisHC1, pH 8 . 3 , 8 . 5 mmol/L NaC1, 2 . 6 mmol/L MgC12, 1 . 2 mmol/L PMSF, 1% Tween 40 and
0 . 5 % deoxycholic acid) for 5 rnin at 4°C . The eluted protein was removed in supernatant after
centrifugation at 600 g for 5 min. The chromatin fraction (DNA and histone) was removed from
nuclei after digestion for 20 rnin at 20°C in a buffer identical to the CSK buffer but containing 50
mmol/L NaCl instead of 50 mmol/L KC1 and 100 pg/mL DNaseI. Digestion in DNase I was terminated by addition of ammonium sulfate to the final concentration of 0 . 2 5 mol/L. The insoluble
nuclear matrix was pelleted at 1000 g for 5 min.
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SCIENCE IN CHINA (Series C)
Vol. 40
Indirect immunofluorescence staining
The in vitro assembled nuclei were fixed in - 20°C methanol for 10 min and treated with
- 20°C acetone for 5 min. After being rinsed with PBS, the nuclei samples were reacted with anti-lamin antibodies (C23, provided by Prof. Lin) for 1 h at 37'C . After being rinsed with PBS,
the nuclei were incubated with FITC-rabbit anti-mouse IgG for 30 min at 37°C. The samples
were observed with an Opton fluorescence microscope.
1.4
Inhibition of nuclear lamina assembly
m
Immunoblotting showed that anti-lamin antibodies ( C23 ) cross-reacted with lamin
in
Xenopus laevis egg extracts. An enough amount of anti-lamin antibodies was added to egg extracts (1 pL antibodiesz20 pL egg extracts) and pre-incubated for 30 min; then ATP regenerating system and exogenous lambda DNA were added to start the nuclear assembly reaction. At the
same time, normal nuclear assembly reactions were set up as a control.
1 . 5 Electrophoresis and immunoblotting
SDS-polyacrylamide gel was prepared according to the method of ~ a e m m l i ' " ~ .Immunoblot
were prepared after transfer of proteins to nitrocellulose paper according to the method of Towbin
et a1.[''I. Anti-280 ku nuclear matrix protein antibodies were generously provided by Prof. Penman.
1.6
Electron microscopy
The i n vitro assembled nuclei were fixed in 2. 5 % glutaraldehyde and post-fixed with 1%
OsO4, dehydrated through a series of graded ethanol, and embedded in Epon 812 resin[l3]. The
sections were examined with a JEM 100 electron microscope at 80 kV.
2 Results and discussion
Xenopus laevis eggs stored a large number of components essential for nuclear assembly,
such as enzyme, nucleotide, histone, membrane vesicles. During the induction of exogenous
DNA, nuclear assembly occurred in egg extracts cell-free system. For investigation of the assembly of nuclear lamina and nuclear matrix, it is necessary to use protein-free DNA, not to demembrane nuclei or chromatin, as a template. Our previous report demonstrated that the nuclei assembled i n v i t r o , induced by exogenous lambda DNA, possess typical nuclear envelope and chromatin-like
We showed that cell-free reconstituted nuclei could be labeled by anti-
lamin antibodies, which suggested that lamins were involved in nuclear assCmbly i n v i t r o C 8 ] .
At first, we investigated the process of nuclear lamina assembly in nuclear recqnstitution. Indirect immunofluore~cencestaining with anti-lamin antibodies showed that, before 60 min, chromatin was not labeled by anti-lamin antibodies (fig. 1( a ) , ( b ) ) , and after 4 h, the neo-nuclei
were positively stained by anti-lamin antibodies (figure 1(c) ,( d ) ) .
Immunoblotting data showed that Xenopus laevis egg extracts contained 68 ku lamin (fig.
2A). After selective extraction, the pellet of egg extracts showed negative reaction with antilamin antibodies (fig. 2B). The results suggested that lamin exists in soluble form in egg ex-
NUCLEAR ENVELOPE & LAMINA ASSEMBLY
No. 6
tracts. After adding exogenous lambda DNA
and ATP regenerating system to egg
extracts, the nuclear assembly reaction occurred in vitro . Immunoblotting showed
that lamin appeared in the nuclei assembled
in vitro (fig. 2 C ) . It suggested that lamin
is involved in nuclear assembly in vitro . Using non-ionic detergent and salt solution to
extract the nuclei assembled in vitro, lamin
remained in insoluble nuclear matrix fraction
as shown by immunoblotting (fig. 2 D ) .
The results described above demonstrated
that soluble lamin in egg extracts assembled
insoluble nuclear lamina in nuclear reconstitution in vitro.
-
Taking aliauots at different tirne of nuv
Fig. 1.
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Fluorescence micrograph showing chromatin assembled i n
vitro within 60 min. (a) Visualized by DNA fluorexent dye DAPI
staining; (b) not labeled by anti-lamin antibodies; (c) the nucleus
after 4 h in vitro assembly reaction; ( d ) positively stained by anti-
clear reconstitution reaction to prepare nuclelamin antibodies. x 1 000
ar matrix, immunoblotting showed that
lamin appeared in insoluble nuclear matrix fraction after 1 h (fig. 3 ) , while 280 ku nuclear matrix protein appeared in insoluble nuclear matrix fraction in 30 min (figure 4 ) .
In the previous electron microscopy studies, we noticed
that there exists time difference between nuclear matrix assembly and nuclear lamina assembly. The inner nuclear ma-
Fig. 2.
trix assembly preceded nuclear lamina assembly, which was
confirmed by the immunofluorescence microscopy may immunoblotting data presented here. The assembly of inner nuclear matrix may be a prerequisite for nuclear lamina assem. bly. It is consistent with the report that chromatin assembly
Immunoblotting showing: A, lamin
in Xenopus laevis egg extracts; B, after sequential extraction, no larnin remaining in the
pellet of egg extracts; C, lamin in the nuclei
assembled in egg extracts cell-freesystem; D,
preceded nuclear lamina assembly[21.
Adding anti-lamin antibodies to Xenopus laevis egg extracts system to inhibit normal assembly of nuclear lamina,
immunoblotting showed that the nuclei assembled in antilamin antibodies blocked egg extracts contained no lamin
(fig. 5D). The nuclear matrix fraction of the abnormally assembled nuclei showed negative reaction with anti-lamin antibodies (fig. 5E). These results suggested that anti-lamin antibodies
blocked nuclear lamina assembly in nuclear reconstitution in vitro .
Iamin in the nuclear matrix fraction of the nu-
cIei assembled in vitro .
We applied electron microscopy and immunofluorescence microscopy to investigating the effect of anti-lamin antibodies blocking nuclear assembly in vitro . Transmission electron microscopy
of Epon 812 resin ultrathin section showed that the nucleus, normally assembled in egg extracts,
possesses basic characteristics of the nucleus, such as nuclear envelope, nuclear pore complex and
chromatin-like structure (fig. 6 ) . Blocked by anti-lamin antibodies, the nuclei were assembled ab-
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Fig. 3 .
Vol. 40
SCIENCE IN CHINA (Series C)
Imrnunoblotting showing the pmess of soluble
Fig. 4 .
Immunoblotting showing the pmess of soluble
280 ku nuclear matrix protein assemble to insoluble nuclear
lamin assemble to insoluble nuclear lamina in cell-free nucle-
matrix in cell-free nuclear reconstitution reaction.
ar reconstitution reaction.
normally in egg extracts. Instead of double
membrane nuclear envelope, membrane vesicles
form multi-layer concentric double-membrane system around chromatin ( fig. 7 ) . Fluorescence microscopy data confirmed this result. The diameter
of chromatin mass, shown by DNA fluorescent
DAPI ( fig. 8 ( a ) ) , is much smaller than that of
the same nucleus under a light microscope (fig. 8
Fig. 5 .
Immunoblotting showing; A, larnin in Xenopus
laewis egg extracts; B; after pre-incubation with anti-lamin
antibodies for 30 min, lamin in Xenopus laevis egg extracts;
C, lamin in normal assembled nuclei in vitro; D, Iamin absent in the nuclei assembled in anti-lamin antibody blocked
( b ) ) , which indicates that there exists a multilayer membrane structure covering chromatin. Indirect immunofluorescence staining showed that
this nucleus was negatively stained by anti-lamin
antibodies.
Xenopus laevis egg extracts; E, larnin absent in the nuclear
matrix fraction of the nuclei assembled in anti-lamin antibody
blocked egg extracts.
Previous reports showed that anti-lamin antibodies were microiniected into mitotic PtK2 cell to
inhibit reassembly of nuclear lamina, resulting in chromosome deconsensation in t e l ~ ~ h a s e " ~ ] .
Futhermore, immunodepletion of lamin from cell-free system derived from mitotic CHO cell with
anti-lamin antibodies coupled with Staphylococcus aureus resulted in inhibition of nuclear enveIope
and nuclear pore complex around c h r o r n o ~ o m e ' ~However,
~.
this method will probably remove
lamin together with lamin bound nuclear membrane vesicles. The inhibition of nuclear envelope
assembly is caused not only by deficiency of lamin, but also by deficiency of membrane vesicles.
Adding anti-lamin antibodies to Xenopus laevis egg extracts cell-free system did not inhibit assembly of chromatin, but inhibited the assembly of nuclear envelope, and membrane vesicles in
egg extracts formed annulate lamellae[151. The experimental results described here demonstrated
that lamin was involved in nuclear assembly i n v i t r o . Adding anti-lamin antibodies to Xenopus
laevis egg extracts system before nuclear assembly reaction resulted in abnormal assembly of nuclear envelope. Membrane vesicles form multi-layer concentric double-membrane around the chromatin. The mechanism of the abnormal assembly of nuclear envelope described here is not clear.
No. 6
Fig. 6 .
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NUCLEAR ENVELOPE & LAMINA ASSEMBLY
TEM micrograph of Epon 812 resin section show-
ing the ultrastructure of the nucleus assembled in Xenopus
laevis egg extracts cell-free system.
Fig. 7 .
TEM micrograph of Epon 812 resin section show-
ing the ultrastructure of the nucleus abnormally assembled
in anti-lamin antibodies blocked Xenopus laewis egg extracts
cell-frei system.
Probably, the assembly of nuclear lamina is an important mid-step in nuclear assembly, and lack
of this step will result in abnormal assembly of membrane system, especially the double-membrane
of the nucleus. Nuclear lamina may play the role of framework for the nuclear envelope assembly
around chromatin. In normal nuclear assembly, lamin inducts membrane vesicles to the surface of
the chromatin to form double membrane envelope. While lacking lamin or nuclear lamina assembly, the recognition between nuclear envelope and chromosome is misleading, resulting in abnormal assembly of nuclear membrane.
Fig. 8.
Micrographs of the same nucleus imaged with different methods showing the structure of the nucleus abnormally
assembled in anti-lamin antibodies blocking Xenopus laevis egg extracts cdl-free system. The diameter of the chromatin
(a), visualized by DNA fluorescent DAPI staining, is much smaller than the nucleus imaged by a light microscopy ( b ) .
Indirect immunofluorescent microscopy showed the same nucleus negatively labeled by anti-lamin antibodies (c)
.
Immunodepletion or antibody blocking techniques can be used to study the function of the
protein in the cell. In the previous studies, most researchers microinjected antibodies into the cells
to observe the effect of the antibodies on the cell. Microinjection, however, is difficult to be manipulated, which limits its application. It is possible to apply immunodepletion or antibody blocking in cell-free system to studying the function of the protein related with nuclear assembly.
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Vol. 40
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