2253
Journal of Cell Science 112, 2253-2264 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0406
Sequential recruitment of NPC proteins to the nuclear periphery at the end of
mitosis
Khaldon Bodoor1, Sarah Shaikh1, Davide Salina1, Wahyu Hendrati Raharjo1, Ricardo Bastos1,*,
Manfred Lohka2 and Brian Burke1,‡
1The
Cancer Biology Research Group and 2Departments of Cell Biology and Anatomy and Biological Sciences, The University of
Calgary, Faculty of Medicine, 3330 Hospital Drive NW, Calgary AB, Canada T2N 4N1
*Present address: Institute Jacques Monod, Dept Biologie Cellulaire, Univ. Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France
‡Author for correspondence (e-mail: [email protected])
Accepted 13 April; published on WWW 10 June 1999
SUMMARY
Nuclear pore complexes (NPCs) are extremely elaborate
structures that mediate the bidirectional movement of
macromolecules between the nucleus and cytoplasm. With
a mass of about 125 MDa, NPCs are thought to be composed
of 50 or more distinct protein subunits, each present in
multiple copies. During mitosis in higher cells the nuclear
envelope is disassembled and its components, including
NPC subunits, are dispersed throughout the mitotic
cytoplasm. At the end of mitosis, all of these components are
reutilized. Using both conventional and digital confocal
immunofluorescence microscopy we have been able to
define a time course of post-mitotic assembly for a group of
NPC components (CAN/Nup214, Nup153, POM121, p62
and Tpr) relative to the integral nuclear membrane protein
LAP2 and the NPC membrane glycoprotein gp210. Nup153,
a component of the nuclear basket, associates with
chromatin towards the end of anaphase, in parallel with
the inner nuclear membrane protein, LAP2. However,
immunogold labeling suggests that the initial Nup153
chromatin association is membrane-independent. Assembly
of the remaining proteins follows that of the nuclear
membranes and occurs in the sequence POM121, p62,
CAN/Nup214 and gp210/Tpr. Since p62 remains as a
complex with three other NPC proteins (p58, 54, 45) during
mitosis and CAN/Nup214 maintains a similar interaction
with its partner, Nup84, the relative timing of assembly of
these additional four proteins may also be inferred. These
observations suggest that there is a sequential association of
NPC proteins with chromosomes during nuclear envelope
reformation and the recruitment of at least eight of these
precedes that of gp210. These findings support a model in
which it is POM121 rather than gp210 that defines initial
membrane-associated NPC assembly intermediates.
INTRODUCTION
import/export signals (Dworetzky et al., 1988; Feldherr et al.,
1984).
In addition to the central framework or ring-spoke complex,
NPCs also possess extensive peripheral structures extending
into both the cytoplasm and the nuclear interior (Ris, 1991).
Projecting from the cytoplasmic ring are eight short (about 100
nm) filaments, which contain docking sites for proteins en
route to the nucleus (Panté and Aebi, 1996; Richardson et al.,
1988). Similarly, the nucleoplasmic face of the central
framework of the NPC is capped by a basket-like structure that
is formed by eight 50-100 nm long filaments joined at their
distal ends by a 30-50 nm diameter ring. Like the cytoplasmic
filaments, the nuclear basket interacts with molecules transiting
the NPC (Kiseleva et al., 1996). From both morphological and
functional perspectives, the effect of these peripheral structures
is to endow the NPC with an overall asymmetry about an axis
parallel to the plane of the nuclear membranes.
Biophysical analyses of amphibian oocyte NPCs have
indicated a mass of about 125 MDa (Reichelt et al., 1990),
leading to the widely held view that these structures may be
Nuclear pore complexes (NPCs) are large and extremely
elaborate structures that mediate the bidirectional traffic of
macromolecules across the nuclear envelope (Davis, 1995;
Doye and Hurt, 1997; Görlich and Mattaj, 1996). Extensive
high-resolution EM observations of amphibian oocyte NPCs
and, more recently, of purified yeast NPCs (Yang et al., 1998),
have now laid the foundations of a consensus model of NPC
architecture (Goldberg and Allen, 1995; Panté and Aebi, 1995),
the central feature of which is a massive symmetrical
framework (with dimensions of approximately 120×80 nm)
embedded in the double membranes of the nuclear envelope.
This framework appears as eight radial multi-domain spokes
connected at their distal ends, and on both their nuclear and
cytoplasmic faces, by multisubunit rings (Akey and
Radermacher, 1993; Hinshaw et al., 1992). The whole
assembly embraces a large central gated channel, of as yet illdefined structure, that may accommodate particles of up to 26
nm diameter, provided that they bear specific nuclear
Key words: Nuclear pore complex (NPC), NPC protein, Mitosis,
Nuclear envelope
2254 K. Bodoor and others
composed of multiple copies of as many as 50-100 distinct
protein subunits. During the past few years roughly 15
vertebrate NPC proteins, or nucleoporins, have been identified
and characterized at the molecular level. However, these
probably represent somewhat less than 50% of the total mass
of the NPC (Bastos et al., 1995; Doye and Hurt, 1997; Rout
and Wente, 1994). For the majority of these proteins little is
known of their precise location within the 3-D structure of the
pore complex and information concerning interactions and
specific functions is still sketchy.
Some insight into the interactions between different
nucleoporins may be gained by examining the molecular
mechanisms and pathways of NPC formation. During
interphase, de novo assembly of NPCs takes place in order to
accommodate nuclear and nuclear envelope growth and is a
process that is common to all nucleated cells. However, in
higher cells with an open mitosis (Gant and Wilson, 1997) the
nuclear envelope (including NPCs) also undergoes a cycle of
disassembly followed by reassembly in order to allow the
condensed chromosomes access to the mitotic apparatus. This
cycle involves the phosphorylation-dependent depolymerization
of the nuclear lamina and, at least in mammalian somatic cells,
the dispersal of integral membrane constituents of both the
lamina and NPCs throughout the peripheral endoplasmic
reticulum. At the same time the soluble components of NPCs
are at least partially disassembled into subcomplexes and
dispersed throughout the mitotic cytoplasm (Finlay et al., 1991;
Grandi et al., 1997). Not surprisingly, a number of
nucleoporins have been shown to be targets of mitotically
activated protein kinases (Favreau et al., 1996; Macaulay et al.,
1995). Towards the end of mitosis, all of these disassembled
components are reutilised in the formation of a new nuclear
envelope within each daughter cell around the mass of newly
segregated chromatids.
The steps of NPC assembly following mitosis are still poorly
understood. Ultrastructural observations of post-mitotic
nuclear envelope assembly indicate that the association of
nuclear membranes with the chromosome surface precedes
formation of NPCs (Goldberg et al., 1997; Macaulay and
Forbes, 1996). Moreover, Courvalin and colleagues
(Chaudhary and Courvalin, 1993) have found that the nuclear
pore complex glycoprotein gp210 (Gerace et al., 1982;
Wozniak et al., 1989) is recruited to reforming nuclear
envelopes relatively late in mitosis when compared with the
inner nuclear membrane protein, lamin B receptor (LBR;
Worman et al., 1990, 1988). While LBR recruitment
commences at the end of anaphase, gp210 does not begin to
concentrate at the nuclear periphery until late telophase. In
other studies, high resolution scanning EM analyses on nuclear
envelopes assembled in vitro have revealed a plausible series
of morphologically distinct NPC assembly intermediates that
are constructed in regions of intact nuclear membranes
(Goldberg et al., 1997). Based on all of these observations, it
would be reasonable to anticipate that at the end of mitosis
soluble nucleoporins would associate with the chromosome
surface only after the nuclear membranes have associated with
chromosomes and perhaps even after gp210. As will be
described below, this does not appear to be the case.
In this study we have addressed the issue of when individual
nucleoporins are recruited to the nuclear envelope at the end
of mitosis. By following the fate of a variety of soluble
nucleoporins, including Nup153, Tpr, p62 and CAN/Nup214
as well as the integral NPC membrane protein POM121
(reviewed by Bastos et al., 1995), we have found that during
the post-mitotic reassembly of the nuclear envelope these
proteins relocate to the chromosome periphery in a stepwise
manner beginning in late anaphase and continuing through
telophase into early G1 phase. With the exception of Tpr, all of
these proteins were observed to accumulate at the chromatin
periphery well in advance of gp210. In contrast, all but Nup153
lagged behind the inner nuclear membrane protein LAP2. This
latter observation indicates that the recruitment of Nup153 may
not be directly dependent upon other membrane-associated
nucleoporins or nuclear envelope components. Finally,
POM121 is recruited to the nuclear periphery very early,
coincident with LAP2. Taken together, these results are most
consistent with a model in which it is POM121, rather than
gp210, that organizes the early stages of post-mitotic NPC
assembly within the reforming nuclear membranes.
MATERIALS AND METHODS
Cell culture
BHK, NRK and HeLa cells were maintained in a 7.5% CO2
atmosphere at 37°C. All of the cells were cultured in DMEM (Gibco
BRL, Gaithersburg, MD) containing 10% fetal bovine serum
(HyClone, UT), 100 µg/ml of penicillin/streptomycin (Gibco BRL)
and 2 mM glutamine (Gibco BRL). CHO cells were maintained in
MEM-alpha containing the same supplements.
Antibodies
The QE5 monoclonal antibody and a guinea pig polyclonal antibody
that recognizes CAN/Nup214, as well as an affinity-purified rabbit
anti-peptide antibody against Nup153, have been described in an
earlier publication (Panté et al., 1994). The rabbit anti-peptide
antibody specific for human lamin A has also been previously
described (Burke, 1990). The polyclonal rabbit anti-rough
endoplasmic reticulum antibody has a specificity identical to that
reported by Louvard et al. (1982) and has been described in earlier
publications (Burke et al., 1983). Human autoantibodies against the
nuclear pore complex protein gp210 were gifts from Dr Jean-Claude
Courvalin (Institute Jacques Monod, Paris) and Dr Marvin Fritzler
(University of Calgary, Alberta, Canada). The monoclonal antibody
against lamin B2 was provided by Dr Erich Nigg (University of
Geneva). Dr Larry Gerace (Scripps Research Foundation) provided
both the monoclonal and polyclonal antibodies against LAP2. Dr
Michael Hendzel (University of Calgary) provided the rabbit antiacetylated histone H4. The rabbit polyclonal antibody against p62 was
raised against recombinant protein fused to glutathione-S-transferase.
The monoclonal antibody SA1 that is specific for Nup153 was
prepared using conventional procedures against the C-terminal
domain of human Nup153, also expressed as a GST fusion protein.
Antibodies against green fluorescent protein (GFP) were obtained
from Clontech, Inc. (Palo Alto, CA). The rhodamine- and FITCconjugated secondary antibodies were purchased from Tago, Inc.
(Burlingame, CA). For use in double-label experiments, the secondary
antibodies were cross-adsorbed, as appropriate, against either mouse
or rabbit IgG covalently coupled to cyanogen bromide-activated
Sepharose CL 4B.
Immunofluorescence microscopy
Cells grown on glass coverslips were fixed with formaldehyde and
labeled with antibodies according to previously described procedures
(Ash et al., 1977). In short, cells were fixed for 20 minutes at room
temperature in 3% formaldehyde (prepared from paraformaldehyde
dissolved at 80°C in phosphate-buffered saline). Following PBS
NPC protein assembly in mitosis 2255
washes, the fixed cells were permeabilized for 5 minutes at room
temperature with 0.2% Triton X-100 in PBS and labeled with
appropriate primary and secondary antibodies. In addition most
samples were stained with the DNA-specific Hoechst dye #33258 to
reveal the cell nuclei. For differential permeabilization experiments
(in which antibodies do not have access to the nuclear interior) cells
were instead treated for 15 minutes at 4°C with 0.004% digitonin in
PBS (Panté et al., 1994). The digitonin was prepared from a 10% stock
solution in dimethyl sulphoxide. Specimens were observed and
photographed under appropriate illumination with a Leica DMRB
microscope equipped with ×63 PL APO NA1.4 and ×100 FLUOTAR
NA 1.32 objectives. Images were collected using a Princeton
Instruments (Princeton, NJ) MicroMax cooled CCD camera linked to
an Apple Macintosh 8100 PPC computer running IP Lab Spectrum
software (Signal Analytics Inc.). Figures were later composed using
Adobe Photoshop 3.0 and Deneba Canvas 3.5. For digital confocal
microscopy, specimens were observed at an Optovar setting of 2.0
using a Zeiss Photomicroscope lll equipped with ×63 PlanApo NA1.4
and ×100 Neofluor NA 1.32 objectives lenses and a Kodak MegaPlus
model 1.6 camera linked to a Touch TX4664 computer. Series of
images acquired at 0.5 µm focal intervals were digitally processed
using Micro-Tome version 3.1 (VayTek, Inc.) to yield stacks of
confocal slices.
Immunoprecipitations and immunoblotting
BHK cells grown in 35 mm tissue culture dishes were labeled
overnight, as appropriate, with 50 µCi 35S-Trans Label (ICN) in 1 ml
growth medium containing methionine and cysteine at 10% of their
normal concentrations. Alternatively, for 32PO4 labeling, cells were
grown for 1 hour in phosphate-free medium containing 1 mCi/ml
32PO (carrier free, ICN). For immunoprecipitation analysis, the cells
4
were washed once in PBS and then lysed in a buffer containing 50
mM triethanolamine (TEA), 500 mM NaCl, 0.5% Triton X-100, 1
mM DTT, 1 mM PMSF and 1:1000 CLAP (10 mg/ml in DMSO of
each of the following: chymostatin, leupeptin, antipain and pepstatin).
The lysate was centrifuged for 10 minutes in an Eppendorf centrifuge
at 4°C. To the supernatant were added 5 µl of appropriate
antiserum/ascites fluid and 20 µl protein A-Sepharose (50%
suspension). The mixture was then rotated overnight at 4°C. The
following morning the beads were washed five times in either the
same buffer or in a higher stringency buffer containing 50 mM TEA,
100 mM NaCl, 0.5% Triton X-100, 0.1% SDS, 1 mM DTT, 1 mM
PMSF and 1:1000 CLAP (Panté et al., 1994). After two final washes
in 50 mM Tris, pH 7.4, the beads were suspended in SDSpolyacrylamide gel sample buffer and fractionated by electrophoresis
(Laemmli, 1970). On completion of electrophoresis gels, where
necessary, were blotted onto nitrocellulose filters, usually BA85 from
Schleicher and Schuell (Burnette, 1981), employing a semi-dry
blotting apparatus manufactured by Hoeffer Scientific Instruments
Inc. (San Francisco, CA). Filters were blocked, labeled with primary
antibodies and then developed with peroxidase-conjugated secondary
antibodies exactly as previously described (Burke et al., 1982).
Alternatively, radioactive gels were examined by fluorography
following impregnation with Amplify™ (Amersham) and exposure to
Kodak X-Omat AR X-ray film. For some experiments cleared cell
lysates were first fractionated on a Superose 6 column that was
equilibrated in the high-salt lysis buffer, 50 mM TEA, 500 mM NaCl,
0.5% Triton X-100, 1 mM DTT, 1 mM PMSF and 1:1000 CLAP.
Individual fractions were then processed for immunoprecipitation as
described above.
Immuno-electron microscopy
CHO cells were synchronized using a single thymidine block and then
accumulated in prometaphase employing nocodazole as previously
described (Burke and Gerace, 1986). The arrested cells, collected by
‘shake-off’ (Tobey et al., 1967), were washed free of the drug and
allowed to continue through mitosis for another 30-40 minutes until
the majority were in late anaphase/early telophase. The progress of
the cells was monitored every few minutes by fluorescence
microscopy following staining with Hoechst dye (#33258). At the
appropriate time, the cells were fixed for 1 hour in 8% formaldehyde,
dehydrated in a graded ethanol series and embedded in LR White
resin. Polymerization was accomplished at 60°C. Silver/grey sections
were picked up on formvar-coated copper grids and labeled first with
purified SA1 IgG followed by goat anti-mouse IgG conjugated with
10 nm colloidal gold (Polysciences). After staining with uranyl acetate
and lead citrate, sections were examined using a Zeiss 902 electron
microscope equipped with a Gatan cooled CCD camera. For
quantitation, images collected at a nominal magnification of ×50,000
were displayed on an Apple Macintosh 6100 PPC using NIH Image.
Gold particles over defined regions of the section (chromatin interior,
chromatin periphery with or without membranes, cytoplasm; see Fig.
4) were counted and expressed as number per unit surface area
(pixels×104). Pixel size at this magnification was 2.42 nm.
Construction of cell lines expressing POM121-GFP
POM121-GFP in the mammalian expression vector pcDNA was
introduced into HeLa cells using the Lipofectamine reagent exactly
as described by the manufacturer (Gibco BRL). 24 hours posttransfection the cells were put under selection in medium containing
800 µg/ml G-418. After about 1 week the cells were trypsinized and
then sorted by FACS into high, medium and low expression pools.
Cells from the low expression pool were plated at low density and
individual clones isolated using glass cloning cylinders. One of these
clones, designated HeLap121G, was expanded and used for further
double- and triple-label experiments.
RESULTS
Studies by Chaudhary and Courvalin (1993) have previously
shown that the NPC glycoprotein gp210 is recruited to
reforming HeLa nuclear envelopes towards the end of
telophase, significantly later than the inner nuclear membrane
proteins LBR and LAP2. These are first detected at the
nuclear periphery at the end of anaphase. Preliminary
immunofluorescence observations employing a polyspecific
anti-nucleoporin monoclonal antibody (QE5) suggested to us
that, contrary to expectation, one or more members of the Olinked glycoprotein family (including CAN/Nup214, Nup153
and p62) reassociates with newly segregated chromatids well
in advance of gp210 (K. Bodoor and B. Burke, unpublished
data).
To further clarify the issue of nucleoporin assembly, we first
examined the recruitment of Nup153, a protein that is a
component of the basket structure which forms the
nucleoplasmic face of the NPC. This was carried out using a
monoclonal antibody, SA1, raised against recombinant human
Nup153 (McMorrow et al., 1994). The specificity of this
monoclonal antibody is demonstrated in Fig. 1A. Double
immunofluorescence analysis of mitotic NRK cells using the
SA1 antibody (Figs 1, 2) indicates that Nup153 associates with
newly segregated chromatids well in advance of gp210 (Fig.
1). This recruitment of Nup153 occurs near the end of anaphase
and therefore represents an early event in nuclear reformation.
Results obtained in independent experiments employing an
affinity purified polyclonal anti-peptide antibody (Fig. 3)
confirm the timing of Nup153 recruitment.
Further double-label analyses of mitotic NRK cells revealed
that recruitment of the three soluble proteins, Nup153,
2256 K. Bodoor and others
Fig. 1. (A,B) Specificity of the SA1 monoclonal antibody raised
against recombinant Nup153. (A) The blot represents a whole-cell
extract of NRK cells probed with SA1 in which only Nup153 is
detected. Dashes represent the positions of molecular mass markers
(200, 117, 96, 67, 45 and 31 kDa). (B) Indirect immunofluorescence
labeling of interphase NRK cells with SA1 reveals a pattern typical
of an anti-nucleoporin antibody. (C) Double indirect
immunofluorescence microscopy of a telophase HeLa cell using SA1
and a human auto-antiserum against gp210 indicates that recruitment
of gp210 to the nuclear periphery follows that of Nup153.
CAN/Nup214 and p62, could actually be temporally resolved.
These experiments demonstrate that Nup153 begins to
concentrate at the nuclear periphery well in advance of p62
(Fig. 2). Similarly Nup214 recruitment lags behind that of p62
(Fig. 2). From these results it is possible to establish a sequence
(in the order Nup153, p62, Nup214) in which the three Olinked glycoproteins are recruited to the nuclear periphery
during the later stages of mitosis. In a second set of
experiments we examined the recruitment of these three
soluble nucleoporins relative to that of the inner nuclear
membrane protein LAP2. Gerace and co-workers (Yang et al.,
1997) have shown that association of LAP2-containing
membranes with chromatin and concentration of LAP2 at the
chromatin periphery commences at the end of anaphase. In this
respect LAP2 behaves identically to LBR (Buendia and
Courvalin, 1997; Chaudhary and Courvalin, 1993; Ellenberg et
al., 1997). From the series double-label immunofluorescence
experiments in Fig. 3, it is evident that the recruitment of p62
and, by extension, Nup214, to the chromosome periphery
follows that of LAP2 (Fig. 3), whereas the recruitment of
Nup153 is roughly coincident with that of LAP2. Additional
double label experiments confirm that all of these proteins
(both LAP2 and the three O-linked glycoproteins) begin to
associate with the newly segregated chromatids in advance of
gp210 (Fig. 1 and data not shown).
The association of both Nup153 and LAP2 with the nuclear
periphery, although roughly coincident, can be readily
distinguished. While LAP2 initially associates with the polar
and lateral margins of the chromatids, a behaviour also noted
for LBR (Buendia and Courvalin, 1997; Chaudhary and
Courvalin, 1993; Ellenberg et al., 1997) and for nuclear
membranes (Robbins and Gonatas, 1964; Roos, 1973;
Zatsepina et al., 1977; Zeligs and Wollman, 1979), Nup153
exhibits a less restricted initial distribution (Figs 2, 3) and
instead appears to coat most available chromatid surfaces, with
the possible exception of telomeric regions. Post-embedding
immunogold labeling of lightly fixed early telophase cells
reinforces this view (Fig. 4). While the overall level of labeling
is low, morphometric analysis reveals that both membranecoated and uncoated surfaces of chromatids in these cells
exhibit similar amounts of associated Nup153 (Fig. 4D).
In spite of the fact that the appearance of LAP2 at the
chromatin surfaces initially parallels that of Nup153, the halftime of accumulation of LAP2 is noticeably shorter. This is
manifested by the fact that LAP2 is essentially cleared from
the cytoplasm by the end of telophase whereas some
cytoplasmic Nup153 is still detectable in early G1 (Fig. 3).
These differences in the distribution of Nup153 and LAP2 on
chromosomes, as well as their contrasting kinetics of
recruitment, argue that the two proteins behave independently
of each other.
While the three soluble nucleoporins (Nup153,
CAN/Nup214, p62) were found to concentrate at the
chromosome periphery ahead of the membrane-associated
gp210, this is certainly not a universal property of such
proteins. Tpr, a coiled-coil protein, which is attached to the
nuclear basket (Cordes et al., 1997), can first be detected at the
nuclear periphery relatively late, more or less concurrent with
gp210, and continues to be imported into the nucleus well into
G1 (Fig. 5). In addition, Tpr accumulates first within the
nucleoplasm in what appear to be fibrillar structures and
subsequently associates only relatively slowly with the nuclear
envelope.
Our results demonstrate that some soluble NPC components,
such as Nup153, p62 and CAN/Nup214, most likely associate
with the chromosome periphery prior to the completion of
active NPCs. Others, such as Tpr (as well as components of
the nuclear lamina), associate with the chromosome periphery
rather late and may therefore require a functional NPC in order
to cross the nuclear membranes. Unfortunately, it is not yet
feasible to determine the precise time when individual NPCs
become functional or what minimal assembly of NPC proteins
is required. However, it is possible to determine when the
nuclear membranes become continuous around the
chromosomes in each daughter cell and therefore the point at
which nuclear proteins can be accumulated or when proteins
larger than about 40-60 kDa no longer have free access to the
chromosome surface by simple diffusion through the
cytoplasm. To accomplish this, we employed a differential
permeabilization protocol, which takes advantage of the fact
that low concentrations of digitonin will permeabilize the
plasma membrane but leave the nuclear membranes intact. In
this way large molecules such as antibodies should not have
access to the nucleoplasm of digitonin-permeabilized
interphase cells. As shown in Fig. 6, an antibody against
acetylated histone H4 labels chromosomes in both Triton X100- and digitonin-treated mitotic cells. Interphase nuclei, in
NPC protein assembly in mitosis 2257
Fig. 2. Fluorescence microscopy
of mitotic NRK cells triplelabeled with (A) SA1 (antiNup153), rabbit anti-p62 and
Hoechst dye (DNA), or with (B)
guinea pig anti-CAN/Nup214,
rabbit anti-p62 and Hoechst dye
(DNA). In each series, a
progression from prometaphase
(top) to telophase or
telophase/early G1 (A) (bottom)
is shown. The data indicate that
Nup153 is recruited to the
chromatin periphery ahead of
p62 in late anaphase (A).
Similarly p62 recruitment
precedes that of
CAN/Nup214 (B).
contrast are only labeled following Triton treatment. When
employed in a double-label experiment with various anti NPC
antibodies, it is clear that histone H4 labeling is lost at about
the time that gp210 (or Tpr) is just beginning to concentrate at
the nuclear periphery and shortly after the onset of
CAN/Nup214 recruitment. This indicates the point at the
telophase/G1 boundary when the nuclear envelope becomes
sealed (Fig. 6). Proteins that associate with the chromosome
periphery before this time may do so by diffusion, whereas
those that associate after that time must either diffuse through
an NPC (for proteins less than 40-60 kDa) or be transported
through the NPC by an NLS-mediated import process.
At least some of the soluble nucleoporins that follow the
recruitment of LAP2 must associate, either directly or
indirectly, with integral membrane components. Our finding
that gp210 is a relatively late arrival at the nuclear periphery
when compared with the three NPC O-linked glycoproteins,
suggests that it does not play a part in the early recruitment of
these proteins. Consequently we decided to analyze the
behaviour of POM121, the only other known vertebrate NPC
2258 K. Bodoor and others
Fig. 3. Fluorescence microscopy of mitotic NRK cells triple-labeled with a monoclonal antibody against LAP2 (RL29), Hoechst dye (DNA)
and either rabbit anti-Nup153 (A,B) or rabbit anti-p62 (C). The panels in A are conventional fluorescence micrographs of early telophase (top)
and late telophase (bottom) cells. Those in B are digital confocal micrographs of prometaphase (p) and anaphase (arrow) cells. It is evident that
Nup153 exhibits a far less restricted distribution than LAP2 at early stages during nuclear reformation, coating most of the available chromatin
with the possible exception of telomeric regions. The open arrow in A indicates regions of chromatin that are clearly coated with Nup153 but
which lack detectable LAP2. The conventional fluorescence micrographs in C, showing a progression from metaphase (top) to telophase
(bottom), indicate that p62 follows closely behind LAP2. Furthermore, the distribution of p62 on the polar and lateral margins of the
chromatids in early telophase cells more closely resembles that of LAP2 rather than Nup153 (A).
membrane protein and which itself is a member of the Olinked glycoprotein family (Hallberg et al., 1993). To date we
have been unable to obtain suitable antibodies against
POM121 and consequently we have had to adopt an
alternative strategy. Hallberg and colleagues have previously
used POM121-green fluorescent protein (GFP) expression
constructs to study POM121 targeting (Soderqvist et al.,
1997). The GFP moiety, fused to the POM121 cytoplasmic
domain, does not appear to interfere with appropriate
localization to NPCs. Based on these studies we have
constructed several cell lines that constitutively express a fulllength POM121-GFP fusion protein. A serious concern with
this approach is that overexpression of the fusion protein could
potentially yield misleading results. Certainly high expression
of nucleoporins such as Nup153 (Bastos et al., 1996) and
CAN/Nup214 (Bastos et al., 1997) as well as POM121 itself
(Soderqvist et al., 1996) leads to a host of ultrastructural
abnormalities. In an attempt to obviate this we used a FACS
to sort stably transfected cells into high, medium and low
expression pools. Cells from the low expression pool were
then plated at very low density and individual clones isolated.
Those clones that we chose for further study exhibited nuclear
envelope-associated GFP fluorescence that was barely
detectable (by eye) in the microscope. However, the POM121GFP fusion protein could be readily observed by indirect
immunofluorescence microscopy using anti-GFP antibodies, a
testament to the relative sensitivities of these procedures. As
shown in Fig. 7, POM121-GFP was found to concentrate at
the nuclear periphery well in advance of gp210. Additional
double-label experiments (not shown) employing all of the
antibodies described here indicate that POM121 recruitment
is roughly coincident with LAP2 and is certainly no later than
p62. This timing suggests that POM121 may play a key role
in the initial stages of NPC reassembly.
The data described so far indicate that there is a stepwise
recruitment of the three soluble O-linked glycoproteins at the
end of mitosis that occurs in the order Nup153, p62 and
CAN/Nup214 and commences towards the end of anaphase.
Several studies have shown that p62 is a component of a
larger NPC-derived subcomplex containing three additional
NPC protein assembly in mitosis 2259
Fig. 4. Immuno-electron microscopy of interphase (A) and early telophase (B,C) CHO cells employing the SA1 (anti-Nup153) monoclonal
antibody. The relatively mild fixation conditions required to achieve adequate and reproducible labeling gave poor structural preservation of
NPCs. However, nuclear membranes were still easily discernible. Both membrane-coated (B) and uncoated (C) regions of chromatin could be
observed in the mitotic cells and exhibited associated gold particles. (D) A quantitative analysis of gold distribution. In the interphase sample in
which 150 gold particles were counted the nuclear envelope-associated gold was defined within an 80-100 nm strip abutting the inner nuclear
membrane (the nuclear basket structure, of which Nup153 is a component, may extend this distance into the nucleoplasm). Gold labeling
(arrows) within this region was statistically significant when compared to gold over the cytoplasm (Cy) or nuclear (N) interior (two-sample ttests; all P values<0.001). Analyses of mitotic cells in which 635 gold particles were counted and where the chromatin surface, either with (+)
or without (−) membrane, was defined in terms of a 100 nm strip centered on the chromatin/cytoplasm interface, yielded similar results. Both
coated and uncoated chromatin surfaces exhibited statistically significant levels of labeling when compared with the cytoplasm or chromatin
(Ch) interior (two-sample t-tests; all P values<0.001). Labeling is expressed as number of gold particles per 104 pixels. The pixel size at the
displayed magnification is 2.42 nm. The Total Gold in D represents all of the gold particles counted (i.e. 150 or 635) divided by the total
calculated cellular surface on all of the sections that were analyzed. Taken together, these data suggest that Nup153 may become chromatin
associated during the early phases of nuclear reassembly.
nucleoporins, p58, p54 and p45 (Finlay et al., 1991; Guan et
al., 1995; Kita et al., 1993). Since the integrity of this
subcomplex is preserved in mitotic cells, recruitment of
p58/54/45 must naturally coincide with that of p62. Similarly,
CAN/Nup214 is tightly associated with another nucleoporin,
Nup84, in interphase cells (Bastos et al., 1997; Fornerod et
al., 1997). If this interaction is maintained during mitosis it
is obvious that recruitment of these two proteins to the
reforming nuclear envelopes will also be coupled. To test this
we carried out immunoprecipitations from interphase and
mitotic populations of CHO cells that had been labeled with
35S-Trans Label. When low stringency wash conditions are
used, Nup84 coprecipitates with CAN/Nup214 from
interphase cell lysates (Bastos et al., 1997; Panté et al., 1994).
Fig. 8A demonstrates that the same is also true when mitotic
cells are analyzed in this way. Indeed the Nup84CAN/Nup214 stoichiometry is identical in both mitotic and
interphase precipitates, indicating that the interaction
between these two proteins is preserved during mitosis.
Fig. 5. Fluorescence microscopy of mitotic HeLa cells triple-labeled
with Hoechst dye (DNA) and a monoclonal antibody against Tpr, in
combination with human anti-gp210 or rabbit anti-p62. The cells are
in either early G1 phase (top two rows) or late telophase/G1 (bottom
row). Recruitment of Tpr is clearly a late event in nuclear envelope
reformation.
Similar analyses of cells labeled with 32PO4 confirmed
previous reports that Nup153 and CAN/Nup214 both exhibit
increased phosphorylation during mitosis (Fig. 8B) while p62
remains unphosphorylated (Favreau et al., 1996; Macaulay et
al., 1995). These results also reveal that Nup84, like its
partner CAN/Nup214, is hyperphosphorylated in mitotic cells
and consistent with the 35S data, both phosphoproteins
2260 K. Bodoor and others
Fig. 6. Indirect immunofluorescence micrographs of fixed NRK cells
permeabilized, as indicated, with either 0.004% digitonin on ice or
0.2% Triton X-100 at room temperature. Cells were first labeled with
a rabbit antibody against acetylated histone H4, after which they
were fixed and permeabilized with Triton X-100. They were then
labeled with either human anti-gp210 (A) or guinea pig antiCAN/Nup214 (B) plus the appropriate secondary antibodies as well
as Hoechst dye (DNA). The digitonin-permeabilized cells are only
labeled with the anti-histone antibody during mitosis when the
nuclear envelope has broken down and the antibody has access to the
chromosomes. Following Triton X-100 permeabilization, however,
all cells, both interphase and mitotic are labeled. This data shows that
histone labeling is lost at about the time that gp210 begins to
concentrate at the nuclear periphery and indicates the latest time by
which functional NPCs must be present.
remain associated (Fig. 8B). In addition to these
coimmunoprecipitation studies, we also subjected mitotic and
interphase cell extracts to gel filtration analysis. We had
previously reported that Nup153, as extracted from interphase
cells, behaves as a large (approximately 0.5-1 MDa) particle
migrating ahead of the p62 complex (Panté et al., 1994). As
Fig. 7. Immunofluorescence microscopy of HeLap121G cells which
stably express very low levels of a POM121-GFP fusion protein. The
cells were labeled with a human anti-gp210 as well as with Hoechst
dye (DNA). POM121-GFP was imaged by direct GFP fluorescence.
The three panels show an interphase cell (left) as well as a cell in late
telophase. The micrographs indicate that POM121-GFP is recruited
to the reforming nuclear envelope ahead of gp210.
shown in Fig. 8C, Nup153 in mitotic cell extracts exhibits the
identical behavior, indicating that it is released from the NPC
during prometaphase apparently as a large homo-oligomer.
The logical conclusion that can be drawn from all of these
results is that in common with p62 and its associated proteins
CAN/Nup214, and Nup84 are recruited to the reforming
nuclear envelope as a complex. In view of this it would seem
that at least eight components of the NPC (Nup153,
CAN/Nup214, Nup84, p62, p58, p54, p45 and POM121)
begin to associate with the chromosome periphery prior to
gp210 and before the nuclear membranes form an intact seal
around the chromosomes.
NPC protein assembly in mitosis 2261
Fig. 8. Analysis of nucleoporin complexes in mitotic cells.
(A) Low stringency immunoprecipitation analysis of 35Slabeled NRK cells employing QE5 (which recognizes
CAN/Nup214, Nup153 and p62). I and NI refer to immune
and non-immune samples, respectively. The appearance of
Nup84 in both the mitotic and interphase immunoprecipitates
is by virtue of its association with CAN/Nup214. (B) Low
stringency QE5 immunoprecipitates of 32P-labeled NRK
cells. The lower panel shows a blot of the same gel probed
with an antibody against Nup84, indicating equivalent
loading of each lane. It is evident from this experiment that
Nup84, in addition to CAN/Nup214 and Nup153, is
hyperphosphorylated in mitotic cells. (C) Fractionation of
35S-labeled interphase and mitotic cell extracts by gel
filtration chromatography on a Superose 6 column. 1 ml
fractions (numbers 9-19) were collected and analyzed by low
stringency immunoprecipitation using QE5. Nup153 from
both interphase and mitotic cells migrates as a high
molecular mass complex ahead of p62.
Fig. 9. Diagram summarizing the order in which
various nuclear envelope and NPC proteins
concentrate at the nuclear periphery during the later
phases of mitosis. The solid boxes indicate protein
complexes while the hatched box signifies that LAP2
and POM121 likely reside in the same membrane
compartment. The asterisk is a reminder that the
timing of POM121 recruitment was established using a
POM121-GFP fusion protein. The gradients represent
the slow accumulations of A- and B-type lamins,
which continue into early G1.
DISCUSSION
In the experiments described here, we have shown that there is
a stepwise association of nuclear pore complex proteins with
chromosomes at the end of mitosis. This process begins in late
anaphase with the recruitment of Nup153 and continues
through to late telophase when the recruitment of both gp210
and Tpr commences. Consistent with the results presented
here, proteins recognized by the widely used anti-nucleoporin
monoclonal antibody 414 (which binds several O-linked
glycoproteins including p62; Davis and Blobel, 1986) also
accumulate at the chromatin periphery in advance of gp210
(Jean-Claude Courvalin, personal communication). The
precise order in which the various NPC proteins reappear at
the reforming nuclear envelope is Nup153, POM121, p62 and
CAN/Nup214 followed finally by gp210 and Tpr (Fig. 9).
Since p62 (Finlay et al., 1991) and CAN/Nup214 (this paper)
remain in multimeric complexes with other proteins when
NPCs are disassembled, we can reasonably infer when p58,
p54 and p45 (components of the p62 complex) as well as
Nup84 (component of the Nup214 complex) return to the
nuclear periphery (Fig. 9). In this way at least seven peripheral
NPC proteins, in addition to the membrane protein POM121,
associate with the chromatin in advance of gp210 during postmitotic NPC assembly. Although differences in the affinities of
our various antibodies may have prevented us from detecting
trace amounts of some of the NPC proteins at the chromatin
periphery, our results indicate that the major portion of the
NPC proteins that we have examined are recruited in a
sequential fashion. This finding is consistent with the notion
that the presence of a particular NPC protein at the chromatin
periphery is required for the subsequent association of another
NPC protein (or protein complex). Although we do not, at
present, have any data with which to address this issue, the
sequential recruitment of NPC proteins may well reflect the
appearance of discrete intermediates in the assembly in vitro
of mature NPCs that have been documented by Wilson, Allen
and collaborators (Goldberg et al., 1997). If this is the case,
then nucleoporins such as Nup153 or POM121 could
potentially define sites of NPC assembly, which subsequently
recruit additional hierarchies of NPC proteins.
In contrast to the behavior of the majority of soluble NPC
proteins described here, Tpr associates with the chromosomes
either in parallel with or shortly after gp210. In addition, our
2262 K. Bodoor and others
differential permeabilization studies suggest that the major
portion of Tpr becomes nuclear associated only after the
chromosomes are enclosed with a continuous envelope. Taken
together, these results suggest that Tpr must require signal
mediated transport through newly functional NPCs prior to its
appropriate localization and assembly. The same is also true of
the nuclear lamins, the bulk of which remain cytoplasmic into
early G1 (data not shown). This is despite the fact that A-type
lamins initially associate with newly segregated chromatids in
parallel with Nup153 (Yang et al., 1997; data not shown).
We also compared the recruitment of NPC proteins to that
of inner nuclear membrane components. In earlier
experiments, Chaudhary and Courvalin (1993) found that LBR,
an integral protein of the inner nuclear membrane, accumulates
at the nuclear periphery well in advance of gp210. As recently
documented by Yang et al. (1997) and confirmed by our own
data, another inner membrane integral protein, LAP2, behaves
in a manner that is indistinguishable from LBR. Both of these
proteins begin to concentrate on the lateral and polar margins
of the segregated chromatids at the end of anaphase. Several
ultrastructural studies on mitotic cells during the past 30 years
have consistently shown that the first nuclear membranes
associate with chromatids at this time and exhibit an initial
distribution that is identical to that seen for LBR and LAP2
(Robbins and Gonatas, 1964; Roos, 1973; Zatsepina et al.,
1977; Zeligs and Wollman, 1979). It is most likely therefore,
that the recruitment of these two integral proteins reflect the
commencement of nuclear membrane assembly.
Results from several groups indicate that at the onset of
mitosis in mammalian somatic cells, integral nuclear membrane
proteins such as LAP2 and LBR redistribute throughout the
peripheral ER, which is in direct continuity with the nuclear
membranes. These proteins remain mobile within the ER
membrane until they reestablish interactions with either
chromatin or the nuclear lamina during nuclear envelope
reformation (Ellenberg et al., 1997). A recent report (Yang et
al., 1997), as well as our own unpublished data, suggests that
gp210 behaves in a similar fashion and colocalizes with
authentic ER markers in mitotic cells. However, Buendia and
Courvalin (1997) have shown in subcellular fractionation
experiments that a significant proportion of gp210 may be
recovered from mitotic cells in a vesicle class that is distinct
from those containing LBR and ER markers. Even though on
face value these findings present two mutually exclusive views
of nuclear membrane protein distribution in mitotic cells, they
could nevertheless be reconciled, were these various nuclear
membrane proteins to either enter or form discreet membrane
microdomains within the mitotic ER. If this were to be the case,
the formation of a heterogeneous microsomal vesicle
population during homogenization and fractionation might be
anticipated. This type of heterogeneity might also result from
differential rates of release of nuclear membrane proteins into
the peripheral ER during prophase as nuclear envelope
breakdown commences. Proteins that become mobile early,
might access the entire peripheral ER. Those released late might
not be able to do this since the ER itself is thought to undergo
some degree of fragmentation. In this way, it is possible that
certain nuclear membrane proteins could be enriched in
separate, but otherwise largely identical ER membranes.
Ultimately, however, this is an issue that should be addressed
by quantitative immuno-EM analysis of mitotic cells.
This model in which nuclear membrane components such as
LAP2, LBR and gp210 disperse throughout the peripheral ER
during mitosis has important consequences in terms of our
view of NPC protein recruitment and assembly. Since the
nuclear membranes account for roughly 5% of the ER surface
area (measured in BHK cells, which are ultrastructurally very
similar to the NRK and HeLa cells used in this investigation),
nuclear membrane components will be diluted by a factor of
about 20 during mitosis, assuming a uniform if not actually
homogeneous distribution throughout the peripheral ER
(Griffiths et al., 1989). Consequently, when we observe the
recruitment of LAP2-containing membranes at the end of
anaphase, these should also contain gp210, albeit at no more
than about 5% of its normal concentration as determined for
the nuclear envelope as a whole. In this way, gp210 should
mimic any other ER protein, which indeed it appears to do. At
present we cannot evaluate the role, if any, of the small amount
of gp210 that must be in close proximity to chromosomes as a
result of the binding of LAP2-containing membranes.
However, this fraction may still be free to diffuse within the
ER/nuclear membranes and, consequently, might not have a
function early in NPC assembly. Nevertheless, this is an issue
that certainly deserves closer scrutiny.
The accumulation of gp210 within the nuclear envelope that
we observe relatively late in mitosis is likely to involve a
process of lateral diffusion and capture, which should only
occur when specific binding sites become available. The most
likely candidates for the formation of gp210 binding sites are
the nucleoporins that have already been recruited to the nuclear
periphery. We propose that just as the mobility of LBR in the
ER/nuclear membranes becomes restricted when LBR binds to
chromatin, the mobility of gp210 would also become restricted
by binding, either directly or indirectly, to the nucleoporins that
have accumulated on chromosomes or on the nuclear
membranes. In the case of the latter this is likely to involve
POM121, given its early concentration in the reforming nuclear
envelope. Similarly we would argue that POM121 itself would
be captured by other soluble NPC proteins (such as Nup153)
that may associate with or are organized by the surfaces of the
chromatids very early in the nuclear assembly process. In this
way, these soluble nucleoporins in combination with POM121
would define initial membrane-associated assembly
intermediates. These could well represent some of the
structures observed by Goldberg et al. (1997). Presumably,
gp210 would be unable to bind to these early assembly
intermediates of the NPC, but could be accommodated in a preNPC at some time soon after the recruitment of CAN/Nup214.
The suggestion that the recruitment of NPC membrane proteins
might ultimately be a function of interactions with chromatinassociated soluble nucleoporins had previously been implied
by the findings of Sheehan et al. (1988). In a series of
ultrastructural investigations they noted what appeared to be
‘pre-pores’ attached to the surfaces of sperm chromatin
following extended incubation in Xenopus egg extracts. With
the availability of antibodies specific for a variety of NPC
proteins, this issue would perhaps now be worth revisiting.
The fusion of the inner and outer nuclear membranes to form
an aqueous channel across both membranes provides the only
morphological marker for NPC assembly in situ and has been
used in ultrastructural studies on mammalian mitotic cells over
the last 30 years or more to document the appearance of NPCs
NPC protein assembly in mitosis 2263
during telophase. The relationship between the recruitment of
NPC proteins and this fusion event, however, remains an
important unresolved issue. As a consequence, we do not
actually know at what point functional NPCs first appear at the
end of mitosis. In addition, the identity of the protein or proteins
that promote the fusion event remains unknown. Of the two
vertebrate integral proteins of the NPC, gp210 seems the more
likely candidate since it possesses a large lumenal domain that
displays characteristics of certain viral envelope fusion proteins.
However, given its relatively late recruitment one would have
to argue that gp210 must be able to function as an effective
fusogen even at the low concentration which we believe must
be present at the earliest stages of nuclear membrane formation.
Such a scenario is by no means out of the question. An
additional possibility that should also be considered, however,
is that NPC assembly including the membrane fusion event
could be facilitated by one or more accessory proteins, that do
not actually form part of the mature NPC. Proteins of this kind
would function essentially as pre-NPC chaperones.
Since we have established an order in which at least some
nucleoporins are recruited into NPCs it is now possible to
predict how assembly of one might affect another. For instance,
from our results we would anticipate that depletion of in vitro
assembly extracts of the p62 complex would have little effect
on the association of Nup153 with the chromatin periphery, but
might well interfere with the subsequent recruitment of
CAN/Nup214 (and Nup84). Conversely, depletion of
CAN/Nup214 may have no effect on p62 assembly but might
result in the formation of nonfunctional or defective NPCs,
which fail to accumulate Tpr. In the future it should be possible
to extend these studies to other vertebrate NPC proteins such
as Nup98 (Powers et al., 1997; Radu et al., 1995), Nup107
(Radu et al., 1994), Nup93 (Grandi et al., 1997) and Nup155
(Radu et al., 1993). Such analyses carried out both in vivo and
in vitro should help shed new light on the mutual interactions
of these various proteins. This in turn will be an important key
to our understanding of the mechanisms of nucleocytoplasmic
transport at the level of the NPC.
We would like to thank the following for their very generous gifts
of antibodies: Drs Jean-Claude Courvalin, Marvin Fritzler, Larry
Gerace, Michael Hendzel and Erich Nigg. We are also grateful to Dr
Einar Hallberg for the POM121-GFP cDNA. This work was supported
by grants from the MRC and AHFMR (to B. B.) and NSERC (to M.
L.).
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