Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC6 and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.
The nuclear envelope (NE)1 is a specialized subcompartment of the ER that forms the nuclear boundary in eukaryotes. It consists of inner and outer
membranes, nuclear pore complexes (NPCs), and the nuclear lamina (for review see Gerace and Burke, 1988 The nuclear lamina contains mainly a polymeric assembly of intermediate filament-type proteins called nuclear
lamins. Four major lamin isotypes have been described in
mammalian cells, lamins A, B1, B2, and C (for review see
Nigg, 1992 Two integral membrane proteins have been identified in
the nuclear pore membrane of higher eukaryotic cells,
gp210 (Gerace et al., 1982 The interphase ER comprises a continuous network of
cisternae and tubular membranes extending to the periphery of the cell from the NE (see Warren and Wickner,
1996 Two different models have been proposed to explain the
disassembly and reformation of nuclear membranes during mitosis. In one model, nuclear membrane proteins are
released into specific NE-derived vesicles distinct from
bulk ER membranes as a result of NE disassembly and are
reassembled in the NE by selective targeting of the NEspecific vesicles to the chromosome surfaces (Wilson and
Newport, 1988 To directly investigate the fate of nuclear membranes
during mitosis, we have used laser-scanning confocal microscopy and electron microscopy to compare the localizations of NE-specific integral membrane proteins to the distribution of bulk ER membranes during mitosis. Our
results demonstrate that integral proteins from both the
inner nuclear membrane and nuclear pore membrane become dispersed throughout ER membranes during mitosis
and are not restricted to a subpopulation of ER vesicles.
This indicates that nuclear membranes lose their identity
as a discrete subcompartment of the ER during mitosis
and strongly supports a model in which the sorting of specific membrane proteins to the NE at the end of mitosis is
driven by binding interactions at chromosome surfaces.
Cell Culture
All cell lines (COS-7, CV-1, normal rat kidney [NRK], NIH3T3, and
HeLa; American Type Culture Collection, Rockville, MD) were grown in
DME (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FBS
(Hyclone Laboratories, Logan, UT) and 100 U/ml penicillin-streptomycin (GIBCO BRL). Cultures were maintained at 37°C in the presence of 5%
CO2. To obtain mitotically enriched cell populations for confocal fluorescence microscopy, NRK cells growing on coverslips were incubated in medium containing 2 mM thymidine (Sigma Chemical Co., St. Louis, MO)
for 11 h to accumulate cells in S phase. The thymidine-containing medium
was replaced by normal growth medium for an additional 6 h, and cells
were then processed for microscopy. To obtain metaphase cells for immunogold electron microscopy, NRK cells growing on 150-cm2 plates were
incubated in medium containing 2 mM thymidine for 11 h. The thymidine
medium was removed, and 4 h later cells were incubated in medium containing 0.6 µg/ml nocodazole for a further 6 h. Mitotic cells were then collected by mechanical shake-off.
Antibodies
LAP1, LAP2, and gp210 were detected with the RL13 (Senior and Gerace, 1988 Lamin A was detected by an antipeptide polyclonal antibody raised
against a synthetic peptide comprising residues 572-585 of human lamin
A. This was obtained by coupling the peptide to Keyhole lymphet
hemocyanin and immunizing a rabbit as described previously (Melchior et
al., 1995 Immunofluorescence Microscopy
The localization of all proteins was examined by indirect immunofluorescence microscopy. NRK cells growing on glass coverslips were enriched in
mitotic populations by a thymidine presynchronization (see above). Cells
were fixed in 4% formaldehyde in PBS for 6 min at room temperature,
permeabilized with 0.2% Triton X-100 in PBS for another 6 min, and
treated with PBS containing 0.2% gelatin. Cells were then incubated with
primary antibodies diluted in PBS/gelatin for 1 h at room temperature,
washed in PBS, incubated for another 40 min at room temperature with
secondary antibodies diluted in PBS/gelatin, and washed in PBS. Monoclonal antibodies RL13 (mouse), RL20 (mouse), and RL29 (hamster)
were used at 25 µg/ml and were detected with rhodamine-conjugated goat
anti-mouse IgG or rhodamine-conjugated goat anti-hamster IgG (Molecular Probes, Inc., Eugene, OR). Polyclonal antisera against the ER (Louvard et al., 1982 Immunogold Electron Microscopy
To carry out double immunogold labeling of LAP1 and ER membranes in
mitotic cells, populations of metaphase-enriched NRK cells (see above)
were permeabilized in PBS containing 60 µg/ml digitonin for 5 min on ice
and then fixed in 4% formaldehyde in PBS with 1 mM MgCl2 for 20 min at
4°C. The cells were washed in PBS and incubated in PBS for 3 h at 4°C
with a mixture of the primary antibodies diluted in PBS and 0.5% BSA
(RL13 IgG [anti-LAP1] at 25 µg/ml and anti-ER antiserum [Louvard et
al., 1982 Localization of Integral Proteins of the NE in
Relation to Bulk ER Membranes during Mitosis by
Confocal Microscopy
We have investigated the fate of integral membrane proteins of the NE during mitosis by immunofluorescence
staining and laser-scanning confocal microscopy of mitotic
NRK cells to analyze whether nuclear membrane proteins
are restricted to a subcompartment of the ER during mitosis as they are during interphase. In this study, we compared the localization of proteins of both the inner nuclear membrane and nuclear pore membrane to the distribution
of bulk ER membranes. The ER was detected with two
fluorescent dyes that selectively label the ER, DiOC6 (Lee
and Chen, 1988 DiOC6 and a polyspecific anti-ER antibody raised
against salt and EDTA-extracted rough microsomes (Louvard et al., 1982
We next compared the localizations of LAP1, an integral protein of the inner nuclear membrane, and ER membranes during interphase and mitosis (Fig. 2). In interphase cells, LAP1 was localized in a nuclear rim pattern,
whereas the ER membranes were seen as an extensive reticular/vesicular network extending throughout the cell
(Fig. 2 A). As expected, overlap between interphase NE and ER labeling occurred at the nuclear rim (Fig. 2 A,
Merge). By prometaphase, when the NE has disassembled,
LAP1 appeared to be localized throughout the entire ER;
nearly every membrane structure labeled with the ER
probe also was labeled with the LAP1 antibody (Fig. 2 B,
compare LAP1 and ER). Moreover, there was a roughly uniform distribution of LAP1 throughout ER membranes
(i.e., the relative intensity of labeling of the various membrane structures is similar with both the LAP1 and ER
probes). Dispersion of LAP1 throughout ER membranes
appears to occur very close to the time of nuclear lamin
depolymerization, since all late prophase cells that we examined by double immunofluorescence staining contained LAPs and lamin in the portions of the NE that remained
assembled (Yang, L., and L. Gerace, unpublished observations).
The dispersion of LAP1 throughout ER membranes
persisted through metaphase (Fig. 2 C) and mid-anaphase
(Fig. 2 D, left cell). In late anaphase, when nuclear membranes begin to assemble around chromosomes, LAP1 became segregated from bulk ER membranes at the periphery of the chromosome masses (Fig. 2 D, right cell). Furthermore, in some late anaphase cells where LAP1 association with chromosomes was apparent, the LAP1 remaining in the peripheral ER was not uniformly localized
throughout, but appeared to be locally concentrated in
certain ER elements (Fig. 2 D, right cell). A similar phenomenon was seen for LAP2 (see below; data not shown). LAP1 was exclusively perinuclear by telophase when it
was separated from all peripheral ER membranes (Fig. 2
E). It should be noted that LAP1 has a higher concentration in the telophase NE relative to the ER label than in
the interphase NE (i.e., the NE appears green in the
merged images of Fig. 2 E, while it appears yellow in the
merged images of Fig. 2 A). This very likely is due to a substantial increase in the surface area of the NE from telophase to early G1 without concomitant synthesis of new
NE proteins, thereby decreasing the relative LAP1 concentration.
We obtained comparable localization results for LAP1
during mitosis using either a monoclonal antibody to
LAP1 (shown in Fig. 2) or a polyclonal antibody raised
against the nucleoplasmic domain of LAP1 (data not
shown; see Materials and Methods), lending confidence to
our findings. These results indicate that LAP1 becomes essentially randomized throughout the ER by prometaphase
when the NE is disassembled and again becomes concentrated in a discrete subdomain of the ER in late anaphase
when the NE reassembles.
We next extended this analysis to LAP2, another integral membrane protein of the inner nuclear membrane,
and gp210, an integral protein of the nuclear pore membrane. Similar to the results seen with LAP1, LAP2 and
gp210 appeared to be dispersed throughout all ER membranes in mitosis. In prometaphase (data not shown) and
metaphase (Fig. 3) cells, virtually all ER membranes were labeled in a roughly uniform fashion with antibodies to
LAP2 (Fig. 3 A) and gp210 (Fig. 3 B). The proteins were
resegregated to the nuclear periphery at the end of mitosis
(Gerace et al., 1982
As a control, we analyzed the localization in mitotic cells
of To directly visualize the mitotic dynamics of LAPs, we
expressed chimeras consisting of LAP1C and LAP2 fused
to green fluorescent protein (GFP) in cultured mammalian cells and examined the GFP fluorescence by confocal
light microscopy (Yang, L., and L. Gerace, unpublished
observations). The GFP chimeras were localized to the
NE in interphase cells and were dispersed throughout ER
membranes during mitosis, in agreement with the results
of immunofluorescence localization. Unfortunately, because the fluorescence intensity of membrane structures
labeled with the GFP-LAP fusion proteins was strongly
diminished after the proteins were distributed throughout the ER in mitosis, we were not able to carry out a real time
analysis of the mitotic dynamics of LAPs in the NE.
In summary, our results indicate that integral membrane
proteins of both the inner nuclear membrane (LAP1 and
LAP2) and nuclear pore membrane (g210) are dispersed
throughout the ER during mitosis in NRK cells, at the resolution of light microscopy. We have obtained similar results in several other cultured mammalian cell lines (CHO,
COS, and HeLa cells; data not shown), and we believe that the phenomenon we have described is a general property
of these NE proteins during mitosis.
Localization of NE and ER Membranes in Mitotic Cells
by Immunogold Electron Microscopy
To confirm and extend the results we obtained with confocal light microscopy, we carried out double immunogold
labeling of digitonin-permeabilized mitotic NRK cells to
localize LAP1 and ER membranes at the EM level (Fig.
4). The populations used for this analysis were selected
from nocodazole-arrested cultures and were highly enriched in metaphase-like cells. ER membranes were labeled with a rabbit polyclonal anti-ER antibody and 5-nm gold coupled to a secondary antibody, and LAP1 was detected with a mouse monoclonal antibody and 10-nm gold
coupled to a secondary antibody. The antibody concentrations were adjusted so that similar labeling densities were
obtained with the 5- and 10-nm gold particles. Both antibodies labeled two categories of intracellular membrane structures: discrete, relatively large (usually 50-500 nm in
diameter) vesicles with an obvious lumen (Fig. 4 A, large
arrowheads; Fig. 4 C, top row) and densely-staining aggregates that contained thin tubules and clusters of small vesicles (Fig. 4 A, small arrows; Fig. 4 C, bottom two rows).
The density of gold labeling was considerably higher for
the latter category of structures than for the former. At
least in part, this probably reflects the larger amount of membrane surface per unit area in aggregates of thin tubules and small vesicle clusters as compared to large, single vesicles.
Most membrane structures of both classes (i.e., discrete
vesicles and aggregates of thin tubules/small vesicles) that
were labeled with anti-ER antibodies also were labeled
with anti-LAP1 antibodies (Fig. 4 A, arrows and arrowheads, and gallery in Fig. 4 C). The gold labeling with antiER and -LAP1 antibodies was specific, as very little labeling of membranes was obtained in samples incubated with
gold-coupled secondary antibodies alone (Fig. 4 B). Furthermore, very little labeling of the peripheral ER was obtained with anti-LAP1/10-nm gold particles in interphase
cells (data not shown), where LAP1 is undetectable in the
peripheral ER by immunofluorescence microscopy (Fig.
2). Finally, the anti-ER and -LAP1 antibodies labeled only a fraction of all membrane structures in the permeabilized
cells (e.g., Fig. 4 A).
We confirmed the close colocalization of the ER and
LAP1 probes by quantitative analysis. In one analytical
method, a field containing circular windows with a diameter of 100 nm was randomly placed on prints of electron
micrographs. We found that 82% of the windows that contained at least two 5-nm gold particles (ER probe) also
contained at least one 10-nm particle (LAP1 probe) (n = 55). By contrast, only 3.8% of all random windows contained at least one 10-nm gold particle (n = 500). In a second method of analysis, we measured the distance from
each 5-nm gold particle (ER probe) to the nearest 10-nm
gold particle (LAP1 probe). We found that 68.2% of all
5-nm gold particles had a 10-nm gold particle localized
within a radius of 100 nm (n = 197). Considered together, these results indicate that LAP1 is located close to most of
the ER label. If LAP1 were restricted to a minor subset of
ER membranes, a much smaller fraction of the ER label
would be expected to have closely associated LAP1. In
conclusion, the findings from immunogold EM are in close
agreement with the results from confocal light microscopy
and confirm that LAP1 is dispersed throughout ER membranes during mitosis.
Order of Lamin and LAP Assembly at the
End of Mitosis
Our localization studies suggest that binding interactions
are likely to be important for localizing integral membrane
proteins to the reforming NE at the end of mitosis (see
Discussion). In principle, nuclear lamins could provide
binding sites to promote this process if lamins were to assemble around chromosomes at the same time as integral
membrane proteins. Although recent immunofluorescence localization studies with conventional light microscopy
showed that LAP1 and LAP2 (Foisner and Gerace, 1993 To reinvestigate this question, we carried out double immunofluorescence localization of lamin A and LAPs in
cultured NRK cells during late mitosis and examined specimens using confocal light microscopy to enhance the ability to visualize chromosome-associated lamins in the presence of disassembled cytosolic lamins. As shown in Fig. 5,
when LAP1 (A) and LAP2 (C) started to become concentrated at parts of the chromosome surfaces in late anaphase,
some lamin A also was concentrated in the same regions
of the chromosomes. By early telophase, we observed that
virtually all LAPs were concentrated at the chromosome
surfaces, while a significant fraction of lamins remained
unassembled (Foisner and Gerace, 1993
The Fate of Nuclear Membranes in Mitotic Cells
We have used light and electron microscope immunolocalization to investigate the fate of several integral membrane proteins of the NE during mitosis in cultured mammalian cells. We have examined two integral membrane
proteins of the inner nuclear membrane, LAP1 and LAP2,
and an integral protein of the nuclear pore membrane, gp210. By immunofluorescence staining and confocal microscopy, we found that the three NE markers are localized throughout all ER membranes after NE disassembly
in prometaphase and remain dispersed until the time of
NE reassembly in late anaphase. In agreement with the results of light microscopy, we found by immunogold EM that
LAP1 is detectable in most ER membranes in metaphase
cells. To a first approximation, the NE proteins appear to
be uniformly dispersed throughout the mitotic ER. However, our light and EM localization is not sensitive enough
to rule out the possibility that one or more of the markers
we have analyzed have a somewhat higher concentration
in certain elements of the ER than others (see discussion
of gp210 below). Nevertheless, it is clear that the NE
markers are not restricted to hypothetical NE-derived vesicles in mitotic cells since the NE comprises only ~5% of
the surface area of the interphase ER in BHK cells, a typical cultured mammalian cell line (Griffiths et al., 1989 We consider it likely that the three proteins we have
studied are representative of most if not all NE-specific integral membrane proteins since these three proteins have
the same problems of biogenesis and compartmentalization as do other proteins of the inner nuclear membrane
and nuclear pore membrane. Therefore, our data indicate
that NE membranes lose their identity as a distinct subcompartment of the ER during mitosis, and that disassembled nuclear membrane proteins are distributed throughout ER membranes in a relatively nonselective fashion.
In contrast to the findings of the present study, a number of authors have speculated previously that Xenopus
eggs (meiotic cells) and mitotic mammalian cells contain a
separate population of NE-derived vesicles that is distinct
from most ER membranes. However, these proposals
have been based on largely circumstantial evidence rather
than direct localization of specific proteins in intact cells,
and we now reconsider these previous findings in light of
our present results.
Experiments analyzing in vitro nuclear assembly in extracts from Xenopus eggs demonstrated that only ~20%
of ER membrane markers became assembled into nuclei
when the ability to form nuclear membranes was saturated
by the addition of a large amount of chromatin substrate
to the extracts (Wilson and Newport, 1988 Immunological studies of two lamin isotypes in Xenopus
eggs showed that a minor fraction of these lamins is membrane-associated in egg extracts and that the two lamin
isotypes present in the membrane-associated pool are
bound to different-sized vesicles (Lourim and Krohne,
1993 Studies with mammalian cultured cells showed that the
NPC protein gp210 accumulates in the reforming NE at
the end of mitosis subsequent to reassembly of the inner
membrane protein p58/LBR (Chaudhary and Courvalin,
1993 Mechanism of Nuclear Envelope Disassembly and
Reformation during Mitosis
Our findings directly demonstrate that integral membrane
proteins of the NE are dispersed throughout the ER during mitosis, as summarized in Fig. 6. Two processes (alone
or in combination) could lead to dispersion of NE proteins
throughout the ER during prometaphase. In one scenario,
the lamina and pore complexes could undergo disassembly
before NE vesiculation and loss of morphological continuity between the NE and the peripheral ER. Upon release of integral membrane proteins from their binding sites at
the NPC and lamina, the integral proteins could rapidly
diffuse throughout the membrane bilayer of the continuous NE/ER system. In a second scenario, the NE could
lose connections with the peripheral ER before disassembly of the NPC and lamina. In this case, integral proteins
of the NE could become dispersed throughout the ER by a
continuous fusion and fission among the disassembled
membranes of the NE and ER, similar to the continuous
fusion that occurs among tubular and cisternal elements of
the interphase ER (Lee and Chen, 1988
Our observations do not support models in which reassembly of the NE occurs by selective targeting of hypothetical NE-specific vesicles to the chromosome surfaces
(Wilson and Newport, 1988 We obtained evidence that in some late anaphase cells
where LAPs are partially assembled around chromosomes, a fraction of LAPs becomes concentrated in localized regions of the peripheral ER away from the chromosome surfaces. However, we do not know the quantitative
significance of this phenomenon, or whether LAPs are stably concentrated in these regions or are undergoing dynamic disassembly/assembly. Among other possibilities,
LAPs could become concentrated in localized regions of
the peripheral ER by a low level of lamin assembly at the
ER membrane surface. NE proteins from these regions,
either as aggregates or monomers, could become concentrated at the NE by the diffusion/binding model depicted in Fig. 6.
A diffusion/binding mechanism also could be responsible for assembly of integral membrane proteins at the inner nuclear membrane during interphase after their synthesis on the rough ER (see Wiese and Wilson, 1993 The accumulation of integral proteins of the inner nuclear membrane at the chromosome surfaces during NE
reformation could be directed by binding to lamins, lamina-associated proteins, or chromatin. All three of the
well-characterized inner nuclear membrane proteins of
mammalian cells (LAP1, LAP2, and p58/LBR) interact directly with lamins, and two of these (LAP2 and p58/LBR) also appear to bind to chromatin directly (see introduction). In this study, we used confocal microscopy to refine
the results of previous immunofluorescence microscopy
on nuclear lamin reassembly and found that a portion of
the lamin pool assembles around late anaphase chromosomes at the same time as integral membrane proteins of
the inner nuclear membrane. Thus, lamins, which themselves bind chromatin directly, are present at the reforming NE at an appropriate time to provide binding sites for
integral membrane proteins (and vice versa).
Considered together, these observations indicate that
NE reassembly in late anaphase is likely to be a highly cooperative process involving interactions of lamins and integral membrane proteins with the chromosome surfaces
and each other (Fig. 6). From this perspective it seems
likely that multiple proteins contribute to nuclear membrane assembly around chromosomes, and that individually many of these may be dispensable for this process.
Antibody inhibition studies have indicated that lamins facilitate the association of membrane with chromosome
surfaces (Burke and Gerace, 1986 In conclusion, our data indicate that the NE loses its
identity as a specialized subcompartment of the ER during
mitosis, when integral membrane proteins that are highly
concentrated in the NE during interphase become dispersed throughout ER membranes. Our findings strongly
support the possibility that NE reassembly involves diffusion of integral membrane proteins through a functionally continuous ER and subsequent accumulation at the chromosome surfaces by binding interactions. It is plausible
that a similar diffusion/binding mechanism is used by cells
to reconstitute other subcompartments of the ER (e.g.,
rough and smooth ER) that may become disassembled during mitosis.
;
Nigg, 1992
; Georgatos et al., 1994
; Rout and Wente, 1994
).
The outer nuclear membrane is continuous with peripheral rough and smooth ER and appears to be biochemically and functionally similar to bulk ER membranes. In
contrast, the inner nuclear membrane contains specific
proteins that are not detected in the peripheral ER (for review see Gerace and Foisner, 1994
) and is lined by the nuclear lamina. Inner and outer nuclear membranes are connected by a specialized "pore membrane" that is adjacent
to NPCs (see Wozniak et al., 1989
).
). The lamina is thought to serve as a structural
framework for the NE and an anchoring site at the nuclear
periphery for interphase chromosomes (for review see
Nigg, 1992
; Georgatos et al., 1994
). In vitro studies have
indicated that chromatin interacts directly with lamins
(Burke, 1990
; Glass and Gerace, 1990
; Hoger et al., 1991
;
Yuan et al., 1991
; Taniura et al., 1995
) and some minor
lamina-associated proteins (see below).
; Wozniak et al., 1989
) and
POM121 (Hallberg et al., 1993
). These proteins are speculated to have a role in anchoring the NPC to the pore
membrane and in nucleating NPC assembly (Gerace et al.,
1982
; Wozniak et al., 1989
). Several integral membrane proteins restricted to the inner nuclear membrane also
have been characterized in higher eukaryotes: lamina-
associated polypeptide (LAP)1 (Senior and Gerace, 1988
;
Martin et al., 1995
), LAP2 (Foisner and Gerace, 1993
; Furukawa et al., 1995
), p58/lamin binding receptor (LBR)
(Worman et al., 1988
, 1990), and otefin (Padan et al.,
1990
). LAP1 and LAP2 are tightly associated with the
lamina, as indicated by their resistance to extraction with a
combination of nonionic detergent and high salt (Senior
and Gerace, 1988
; Foisner and Gerace, 1993
). In vitro
binding studies have suggested that LAP1 and LAP2
(Foisner and Gerace, 1993
), as well as p58/LBR (Worman et al., 1988
), directly interact with lamins and that LAP2
(Foisner and Gerace, 1993
) and p58/LBR (Ye and Worman, 1996
) associate with chromatin. LAP1C and LAP2
each contain a single predicted transmembrane segment
and a large nucleoplasmic domain (Furukawa et al., 1995
;
Martin et al., 1995
). In contrast, p58/LBR, which is homologous to yeast sterol C14 reductase (see Georgatos et al.,
1994
), has eight predicted transmembrane segments. Although functions of specific integral membrane proteins of
the inner nuclear membrane have not been determined, it
is likely that certain lamin-binding integral membrane proteins have a role in the attachment of lamin filaments to
the inner nuclear membrane and in the structure or
higher-order arrangement of lamin filaments. They also
could play a role in reassembly of the NE at the end of mitosis.
). During mitosis, the ER undergoes disassembly to
vesicles and membrane tubules/cisternae to allow the partitioning of ER membranes to the daughter cells (Warren
and Wickner, 1996
). In higher eukaryotes, the NE breaks down during mitotic prophase into a form that cannot be
structurally distinguished from disassembled elements of
the peripheral ER (e.g., Porter and Machado, 1960
; Robbins and Gonatos, 1964; Roos, 1973
; Zeligs and Wohlman,
1979). The NE is reassembled during late anaphase by a
process that involves the association of membrane vesicles
and cisternae with the surfaces of the fused chromosome masses coupled with membrane fusion (Robbins and Gonatas, 1964
; Roos, 1973
; Zeligs and Wollman, 1979
). Pore
complexes are gradually inserted into the NE, beginning in
late anaphase and continuing until early G1 (Maul, 1977
).
Immunofluorescence microscopy has shown that certain
integral membrane proteins of the inner nuclear membrane, LAP1, LAP2 (Foisner and Gerace, 1993
), and p58/
LBR (Chaudhary and Courvalin, 1993
), accumulate at the
surfaces of chromosomes during the early stages of NE
reformation in late anaphase. However, the mechanism of
their reassembly is controversial, in part because the exact
fate of integral membrane proteins of the NE during mitosis is unknown (discussed in Gerace and Foisner, 1994
).
; Chaudhary and Courvalin, 1993
; Maison et
al., 1993
). In a second model, nuclear membrane proteins
become dispersed throughout all ER membranes during
mitosis and are sorted to the reforming NE by diffusion
through a functionally continuous ER coupled with binding to specific sites at the chromosome surfaces (discussed
in Gerace and Foisner, 1994
). Distinguishing between these models is important for understanding the mechanisms for assembly and maintenance of the NE and other
ER subdomains during the cell cycle.
Materials and Methods
), RL29 (Foisner and Gerace, 1993
; Furukawa et al., 1995
), and
RL20 (Greber et al., 1990
) monoclonal antibodies, respectively. The relevant hybridomas were grown in EXCELL 300 serum-free medium (JRH
Biosciences, Lenexa, KS), and monoclonal antibodies were purified from
the culture supernatants by ammonium sulfate precipitation followed by
chromatography on a protein G column (Pharmacia LKB Biotechnology
Inc., Piscataway, NJ). LAP1 and LAP2 were also detected with affinitypurified rabbit polyclonal antibodies raised against residues 1-320 of
LAP1C and residues 298-373 of LAP2. Immunofluorescence labeling of
interphase and mitotic cells with these antibodies gave results identical to
those obtained with the monoclonal antibodies. These fragments of
LAP1C and LAP2 were expressed in Escherichia coli as GST fusion proteins and purified from soluble bacterial lysates by chromatography on a
glutathione-Sepharose matrix (Pharmacia LKB Biotechnology, Inc.).
Rabbits were immunized with the purified fusion proteins as described
previously (Melchior et al., 1995
). To prepare affinity matrices for purifying anti-LAP1C (1-320) and anti-LAP2 (298-373) antibodies, the purified
GST fusion proteins were coupled to CNBr-activated Sepharose (Pharmacia LKB Biotechnology, Inc.) at 0.5-1.0 mg/ml according to the manufacturer's instructions. Antisera were first preadsorbed against GST-agarose to remove the anti-GST antibodies, and the resulting sera were then
used to obtain specific affinity-purified antibodies with the affinity matrices as described (Melchior et al., 1995
).
). The antibody specifically recognizes lamin A in Western blot
analysis of isolated rat liver NEs. The polyclonal anti-ER antiserum was
the gift of Dr. Daniel Louvard (Curie Institute, Paris, France). It was obtained by immunizing rabbits with EDTA and salt-stripped canine pancreas rough microsomes, and it recognizes four ER membrane proteins
with apparent molecular masses of 29, 58, 66, and 91 kD (Louvard et al.,
1982
). Dr. Michael Jackson (R.W. Johnson Pharmaceutical Institute, La
Jolla, CA) provided a rabbit polyclonal antiserum against calnexin (Ware et al., 1995
); Dr. Stephen Fuller (European Molecular Biology Laboratory, Heidelberg, Germany) provided an antibody against a 12-amino
acid peptide composing the COOH terminus of protein disulphide
isomerase (Vaux et al., 1990
); and Dr. Marilyn Farquhar provided a rabbit
polyclonal antiserum against
-mannosidase II (Velasco et al., 1993
).
),
-mannosidase II (Velasco et al., 1993
), protein disulphide isomerase (Vaux et al., 1990
), and calnexin (Ware et al., 1995
) were
used at a dilution of 1:200 and stained with rhodamine or fluorescein-conjugated goat anti-rabbit IgG (Molecular Probes, Inc.). For some of the experiments, after the secondary antibody incubation the ER was labeled by
DiOC6 or R6 (Molecular Probes, Inc.) at 0.5 µg/ml for 5 min at room temperature. The specimens were mounted in slowfade antifade solution
(Molecular Probes, Inc.) and examined with a laser-scanning confocal microscope (model MRC-600; BioRad Labs, Hercules, CA).
] at 1:100). The cells were then washed in PBS and incubated
overnight at 4°C with a mixture of goat anti-mouse IgG conjugated with
10-nm colloidal gold (for labeling LAP1) and goat anti-rabbit IgG conjugated with 5-nm colloidal gold (for labeling the ER) diluted in PBS and
0.5% BSA. After another wash in PBS, the cells were fixed in 2% glutaraldehyde for 30 min at 4°C, washed thoroughly, and then postfixed with 1% OsO4 for 1 h at room temperature. The samples were dehydrated and
embedded in Epon 812 resin as described (Guan et al., 1995
). Sections
were stained with 2% uranyl acetate for 1 min. Micrographs were recorded with an electron microscope at 80 kV (model 600; Hitachi America
Ltd., Brisbane, CA). As a nonspecificity control for the gold-coupled antibodies, some samples were incubated in PBS and 0.5% BSA instead of the
primary antibody solution before being incubated with the gold-coupled
secondary antibodies. Only cells showing clearly defined mitotic chromosomes were analyzed.
Results
; Terasaki and Reese, 1992
) and R6 (Terasaki
and Reese, 1992
), as well as with several polyclonal antibodies against ER proteins (see below).
) gave essentially coincident patterns of
fluorescence labeling in interphase and metaphase NRK
cells (Fig. 1, A and B, respectively), where arrays of tubular and vesicular membrane elements were seen in the optical sections. DiOC6 and a polyclonal antibody against protein disulfide isomerase, an abundant soluble protein
of the ER lumen (Vaux et al., 1990
), also gave nearly coincident labeling patterns in interphase and metaphase cells
(Fig. 1, C and D, respectively). Similar results were obtained by double labeling of cells with DiOC6 and an antibody against another major soluble protein of the ER lumen, calnexin (Ware et al., 1995
; data not shown). Finally, both DiOC6 and R6 gave coincident staining patterns in
interphase and metaphase cells (Fig. 1, E and F, respectively). These data indicate that under our staining conditions, DiOC6, R6, and the polyspecific anti-ER antibody
selectively label the ER in NRK cells. We have used these
three staining reagents interchangeably to detect the ER
throughout this study.
Fig. 1.
Localization of ER membranes with antibodies and fluorescent dyes. NRK cells were fixed and labeled for indirect immunofluorescence microscopy with a polyspecific anti-ER antibody (A and B) or anti-protein disulphide isomerase antibody
(anti-PDI, C and D) and were then counterstained with DiOC6.
Alternatively, the fixed cells were double stained with DiOC6 and
R6 (E and F). Shown are images of the labeling with DiOC6 (left
column), antibodies or R6 (center column), and the merge of the
two fluorescent channels (right column). Examples of interphase
(A, C, and E) and metaphase (B, D, and F) are presented. Bar, 10 µm.
[View Larger Version of this Image (28K GIF file)]
Fig. 2.
Comparison of the localizations of LAP1 and ER membranes throughout mitosis. NRK cultures enriched in mitotic cells were
fixed and examined by double immunofluorescence microscopy after labeling with the LAP1-specific monoclonal antibody RL13 and a
polyspecific anti-ER antibody. Shown is the labeling with RL13 (left column), the anti-ER antibody (center column), and the merge of
the two fluorescent images (right column). The figure presents representative examples of cells (two different cells in each row) in interphase (A), prophase (B, left cell), prometaphase (B, right cell), metaphase (C), mid-anaphase (D, left cell), late anaphase (D, right cell) and telophase (E). The NE in the prophase cell shown in B (left cell) is deformed due to invagination by the mitotic spindle. Bar, 10 µm.
[View Larger Version of this Image (57K GIF file)]
; Foisner and Gerace, 1993
). Comparable results on the localization of LAP2 in mitotic cells
were obtained using either monoclonal antibodies (shown
in Fig. 3) or polyclonal antibodies raised against a fragment of the nucleoplasmic domain of this protein (data
not shown; see Materials and Methods). As observed previously (Chaudhary and Courvalin, 1993
), we found that
assembly of LAP1 and LAP2 around chromosomes, which
occurred in late anaphase (Foisner and Gerace, 1993
), preceded the assembly of the majority of gp210 (data not
shown).
Fig. 3.
Comparison of the localization of ER membranes to the distributions of LAP2, gp210, and -mannosidase II in metaphase
cells. NRK cultures enriched in mitotic cells were fixed and examined by double labeling with DiOC6 and the LAP2-specific monoclonal antibody RL29 (A), DiOC6 and the gp210-specific monoclonal antibody RL20 (B), or R6 and a polyclonal antibody against the Golgi
enzyme
-mannosidase II (C). Shown is the labeling with the ER probes (left column), antibody probes (center column), and a merge of the two fluorescent images (right column). The green/red color representation for the antibody and R6 was computationally reversed in
the images presented in C for consistency. In A and B, two different metaphase cells are shown, and in C, a metaphase (left cell) and
anaphase (right cell) are shown. Bar, 10 µm.
[View Larger Version of this Image (56K GIF file)]
-mannosidase II, an integral membrane protein of the
Golgi complex (Velasco et al., 1993
). Golgi membranes
are known to remain separate from the ER during mitosis
(Warren and Wickner, 1996
) and therefore should present
a distribution distinct from ER membranes in confocal microscopy. As expected, in mitotic cells the antibody to
-mannosidase II labeled a set of membrane structures that largely did not overlap with ER membranes, even though
both membranes were extensively dispersed throughout
the cytoplasm (Fig. 3 C). This indicates that our microscope procedure would be able to clearly distinguish hypothetical NE-specific membranes, if they existed, as a population separate from bulk ER membranes.
Fig. 4.
Comparison of the localizations of LAP1 and ER membranes in metaphase cells by double immunogold labeling. Populations
of nocodazole-arrested metaphase NRK cells were processed for double immunogold labeling by incubation with the LAP1-specific
monoclonal antibody RL13 and a polyspecific anti-ER antibody, followed by 10-nm antibody-coupled gold (to detect the LAP1 probe)
and 5-nm antibody-coupled gold (to detect the ER probe). Shown are images of cells incubated with anti-LAP1 and anti-ER antibodies
followed by secondary gold-coupled antibodies (A and C) or cells incubated with only the secondary gold-coupled antibodies and without the primary antibodies (B). Examples of mitotic chromosomes (ch) used to identify mitotic cells are designated. A indicates examples of the two classes of antibody-labeled structures: large discrete vesicles (large arrowheads) and densely staining aggregates of small
vesicles and tubules (small arrows). C shows a gallery of the antibody-labeled membranes: large discrete vesicles (top row) and aggregates of small vesicles and tubules (bottom two rows). Bars: (A and B) 300 nm; (C) 100 nm.
[View Larger Version of this Image (149K GIF file)]
)
and p58/LBR (Chaudhary and Courvalin, 1993
) become
concentrated around chromosomes at the end of mitosis
before most lamins, these studies could not exclude the
possibility that a fraction of lamins associates with the
chromosome surfaces at the same time as the inner membrane proteins. Lamins exist in a large stoichiometric excess over integral membrane proteins of the inner nuclear
membrane, and the high concentration of disassembled
lamins in the cytosol would make it difficult to detect chromosome-associated lamins by conventional light microscopy (see Gerace and Foisner, 1994
).
; and data not
shown), while by mid-late telophase (Fig. 5, B and D),
most of the lamin pool had reassembled as well. These
data indicate that lamin and LAPs associate with the chromosome surfaces in late anaphase in a temporally and spatially coordinated fashion, even though much lamin remained unassembled in early telophase when the assembly of LAPs was essentially completed (Foisner and Gerace,
1993
). Thus, even though lamin and LAPs begin to associate with chromosome surfaces at the same time in late
anaphase, the half-time of assembly of the lamin pool appears to be longer than that of LAPs.
Fig. 5.
Comparison of the localization of lamins to the distribution of LAPs at the end of mitosis. NRK cultures enriched in
mitotic cells were labeled for double immunofluorescence microscopy with an antibody against lamin A and the LAP1-specific
monoclonal antibody RL13 (A and B) or the LAP2-specific monoclonal antibody RL29 (C and D). Shown are images of the lamin
labeling (left column), the LAP1 or LAP2 labeling (center column), or a merge of the two fluorescent images (right column).
Each row presents representative images of cells in late anaphase
(left cell) or mid-late telophase (right cell). Bar, 15 µm.
[View Larger Version of this Image (24K GIF file)]
Discussion
).
). The authors interpreted this to mean that the membrane proteins involved in NE assembly are present in a discrete population
of ER vesicles (Wilson and Newport, 1988
). However,
since ER membranes continuously undergo fusion in the
assembly extracts (Newport and Dunphy, 1992
) and the
NE is continuous with the ER, these results also are consistent with the possibility that NE proteins initially are
dispersed throughout ER vesicles and are subsequently
depleted from most ER elements during the assembly reaction by diffusion through the ER and binding to sites in
the reassembling NE (see below). Other studies with Xenopus nuclear assembly extracts showed that two separate
detergent-sensitive particulate fractions were involved in
NE assembly in this system, prompting the conclusion that
two separate vesicle populations are involved in nuclear
pore and membrane assembly (Vigers and Lohka, 1991
).
However, only one of the fractions could be prepared as
isolated membranes, raising the possibility that the active
component of the second fraction is a nonmembranous,
detergent-sensitive particulate structure.
). We have found that the membrane-associated B-type
lamins of CHO cells redistribute between a membrane
and a soluble state in cell homogenates (Gerace, L., unpublished). This raises the possibility that the membrane
associations observed for the different Xenopus egg lamins
could in part result from in vitro redistribution during
membrane isolation. Immunolocalization of the membrane binding sites for the lamins (which presumably are
nonexchangeable integral proteins) in whole cells would
be useful in this situation.
). The authors interpreted this to mean that gp210 and
p58/LBR are present in distinct vesicles in mitotic cells
(Chaudhary and Courvalin, 1993
). However, the data also
are consistent with the possibility that these proteins are
distributed throughout ER membranes in mitosis and accumulate in the NE asynchronously because of the asynchronous appearance of their appropriate binding sites in
the reforming NE. Most pore complex assembly occurs
during telophase and early G1 after assembly of nuclear
membranes (e.g., Maul, 1977
), and it is possible that the
binding sites for gp210 appear in the NE only at the time of pore complex assembly. Immunoblot analysis indicated
that the relative amounts of gp210 in a heavy vs light membrane fraction from metaphase cells were different from
the relative amounts of p58/LBR in the same two membrane fractions (Chaudhary and Courvalin, 1993
). Although these results could suggest that the two proteins differ somewhat in relative concentrations in heavy and
light membrane vesicles in mitotic cells, they do not demonstrate that they are present in physically distinct vesicles. Similarly, although lamin B and p58/LBR appear to
be more enriched in vimentin-associated vesicles in mitotic cells extracts compared to free vesicles (Maison et al.,
1993
), these results do not demonstrate the existence of an
NE-specific vesicle population. In conclusion, previous studies have not clearly addressed the fate of NE proteins
during mitosis. We believe that direct localization of NE
proteins in intact cells, as we have presented in this study,
is the only approach that can resolve this issue. It should
be noted that integral membrane proteins of the NE conceivably may not be distributed throughout the ER in Xenopus eggs as we have found in mitotic mammalian cells
because of the size differences between the two cells or
some other reason.
).
Fig. 6.
Model depicting the dynamics of integral membrane
proteins of the NE during mitosis. The interphase NE is morphologically continuous with the peripheral ER but is a specialized
ER subcompartment that contains integral membrane proteins
specific to the inner nuclear membrane (top) and nuclear pore
membrane (not shown). Integral proteins of the inner nuclear
membrane may be bound to lamins, lamina-associated proteins,
or chromatin. During mitosis when the NE is disassembled, integral membrane proteins of the NE are dispersed throughout all
ER membranes, and the NE loses its identity as an ER subcompartment (middle). We propose that integral proteins are resorted to the NE during late anaphase by diffusion through a
functionally continuous ER and subsequent association with
binding sites at the chromosomes (bottom). The binding sites for
inner membrane proteins may include lamins and chromatin
(bottom); those for nuclear pore membrane proteins may include
other pore complex components (not shown). These binding interactions would result in the net collection of nuclear membrane proteins at the reforming NE (bottom, arrows). Reformation of the NE may involve cooperative assembly of lamins and integral membrane proteins of the inner nuclear membrane (bottom).
[View Larger Version of this Image (24K GIF file)]
; Chaudhary and Courvalin,
1993
). Rather, they strongly support the possibility that
binding interactions at the chromosome surfaces account
for the accumulation of integral membrane proteins at the
reforming NE in late anaphase (Fig. 6). In a process analogous to that of NE disassembly (see above), integral membrane proteins could reach the reforming NE by two different mechanisms, working either separately or in concert.
In one case, the ER could exist as a continuous reticulum
in late anaphase, and integral proteins could accumulate in
the reforming NE by rapid diffusion through the continuous ER bilayer to binding sites at the chromosome surfaces. Morphological evidence supports the existence of a
highly interconnected ER reticulum in late mitotic cells
(Robbins and Gonatas, 1964
; Roos, 1973
; Zeligs and Wollman, 1979
; Stracke and Martin, 1991
). Alternatively, a
number of separate ER elements could exist in late
anaphase cells, and integral proteins of the NE could be rapidly exchanged between these elements by continuous
fusion/fission. After reaching the chromosome-associated
nuclear membranes, integral nuclear membrane proteins
could be removed from the diffusionally free pool by binding interactions.
).
Integral membrane proteins have the capacity to rapidly
move between outer and inner nuclear membranes (discussed by Gerace and Burke, 1988
; see also Powell and
Burke, 1990
). Morphological studies have indicated that
this diffusional exchange may occur around nuclear pore
membranes, since pore complexes appear to contain ~10nm-diam diffusional channels immediately adjacent to the
pore membrane (Hinshaw et al., 1992
). Diffusion of integral proteins to the inner membrane via the nuclear pore
membrane may occur during the later stages of NE assembly at the end of mitosis as well as during interphase.
; Ulitzur et al., 1992
), although the question of whether lamins are essential for
this process is not resolved (discussed in Gerace and Foisner, 1994
; Lourim and Krohne, 1994
). It is likely that the
abundance of individual inner membrane proteins and
their binding affinities for chromosomes and other NE
proteins will determine their relative importance in the
pathway of NE reassembly.
Received for publication 23 January 1997 and in revised form 31 March 1997.
Address all correspondence to Larry Gerace, Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037. Tel.: (619) 784-8514. Fax: (619) 784-9132.We are grateful to the following individuals for their gifts of antibodies: Drs. Marilyn Farquhar, Stephen Fuller, Michael Jackson, Daniel Louvard, and Kelly Moreman. We also wish to thank Christian Fritze, Susan Lyman, and Frauke Melchior for their helpful comments on this manuscript.
This work was supported by a grant from the National Institutes of Health to L. Gerace.
LAP, lamina-associated polypeptide; LBR, lamin binding receptor; NE, nuclear envelope; NPC, nuclear pore complex; NRK, normal rat kidney; GFP, green fluorescent protein.
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