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REMODELLING THE WALLS
OF THE NUCLEUS
Brian Burke* and Jan Ellenberg ‡
The nuclear envelope (NE) acts as a selective barrier around the genome and as a scaffold to
organize DNA in the nucleus. During cell division, the NE is broken down and chromosome
confinement is taken over by microtubules. After chromosome segregation, a new NE is
reassembled in each daughter cell. In this complex cycle of disassembly and reassembly, the fate
of the NE is intimately linked to the activity of the mitotic spindle. The finding that components of
the nuclear membrane become distributed throughout a continuous endoplasmic reticulum
during mitosis indicates new mechanisms by which nuclear membrane domains are established,
and highlights unique problems in the establishment of NE topology.
LAMINS
Lamins are rod-shaped proteins
of the intermediate filament
class. They consist of a head and
tail domain that flank a
conserved α-helical rod domain.
Lamins form parallel homoand probably heterodimers
which, in turn, can polymerize
in a head-to-tail fashion. These
linear polymers are thought to
associate laterally into 10-nm
lamin fibres, which form the
fibrous lamina meshwork in the
nuclear periphery.
*Department of Anatomy
and Cell Biology, The
University of Florida,
Gainesville, Florida 32610,
USA. ‡Gene Expression and
Cell Biology/Biophysics
Programmes, European
Molecular Biology
Laboratory, D-69117,
Heidelberg, Germany.
Correspondence to J.E.
e-mail: jan.ellenberg@
embl-heidelberg.de
doi:10.1038/nrm860
Intracellular compartmentalization has provided
eukaryotes with unique opportunities for the regulation
of many cellular processes. Nowhere is this more obvious than in the enclosure of the chromosomes in a
nuclear envelope (NE) This effectively separates —
both temporally and spatially — the nuclear processes
of gene transcription and replication from translation,
which is generally a cytoplasmic event. Clearly, control
of communication between the nucleus and cytoplasm
is a prerequisite for normal eukaryotic cell function.
This enhanced regulatory potential of eukaryotes, however, comes at the price of greatly complicating the
mechanics of cell division.
To complete mitosis successfully, microtubules of
the mitotic spindle must gain access to the chromosomes. To accomplish this, the spindle can either be
assembled in the nucleus, as occurs in yeast, or the NE
can be partially or completely broken down to allow the
chromosomes to engage with a cytoplasmic spindle.
This second option is referred to as ‘open mitosis’ and,
in vertebrate cells, involves the disassembly and dispersal of all the main elements of the NE. On completion
of mitosis, components of the NE are reused in the formation of nuclei within each daughter cell.
The breakdown and subsequent reassembly of the
NE is remarkably complex, and involves coordination
of activities as diverse as membrane fusion and microtubule-based movement. In recent years, a consensus
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
model of nuclear dynamics has been formulated that
accounts for virtually all changes in NE architecture that
occur during mitosis1–5. This article provides a synopsis
of this new view of NE dynamics in M phase and the
molecular mechanisms that underlie them, and focuses
mainly on results from vertebrate systems.
The nuclear envelope protein network
A complete catalogue of the protein composition of the
NE in vertebrates is not yet available, but proteomics
approaches are starting to fill the gaps6,7. It is now clear
that we should add chromosomes to the three classic
NE components (BOX 1), and we can then define four
main classes of proteins that organize the nuclear
periphery: LAMINS, inner nuclear membrane (INM) proteins, nuclear pore complex (NPC) proteins and
peripheral chromatin proteins (see TABLE 1 and FIG. 1).
Class I. Class I comprises the nuclear lamins, which are
type-V INTERMEDIATE-FILAMENT PROTEINS that can be subdivided into B-type (ubiquitous) and A/C-type lamins
(found only in differentiated cells) on the basis of their
primary sequence8,9. These proteins have a well-established function. They form a peripheral fibrous meshwork — the nuclear lamina — that supports the INM10.
Intranuclear lamins have proposed functions in DNA
replication11,12, RNA polymerase II transcription13 and
nuclear organization14.
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Box 1 | Architectural concepts of the nuclear envelope over the past 100 years
INTERMEDIATE FILAMENT
PROTEINS
Intermediate filaments are
protein fibres with a diameter of
~10 nm that represent the third
class of cytoskeletal polymers,
after microtubules and actin.
Intermediate filaments can be
subdivided into five classes.
Classes I–IV are found in the
cytoplasm and contain, for
example, keratins,
neurofilaments and vimentin.
Class V is nuclear and is
comprised exclusively of nuclear
lamins.
METAZOANS
The nuclear envelope (NE) has been defined to consist of three distinct structural components — the nuclear
membranes, the nuclear pores and the underlying nuclear lamina111 (FIG. 1). In the 1960s, fractionation and purification
of the NE from cells became possible112, and we have learned much about its molecular composition since then. This
understanding began with biochemical studies on lamins9, nuclear membrane proteins later 2,113 and, in the case of yeast,
has culminated in an almost complete inventory of the nuclear pore complex (NPC) proteins7,25.
The first insight about the fate of the NE in dividing cells came from transmitted-light-microscopy observations in the
early days of cell biology114, when fading and re-emergence of membranous material — ‘Kernbläschen’ (or nuclear
bubbles) — on the chromosome surface was observed in dividing cells of echinoderm embryos115. Six decades later,
pioneering electron-microscopic studies on mitotic plant and animal cells57,58,116 started to address the fate of NE
components during the open mitosis of METAZOANS at the ultrastructural level. Those studies indicated an intimate
relationship of nuclear membranes to membrane tubules and CISTERNAE of the endoplasmic reticulum (ER) in dividing
cells. In addition, the duplicated CENTROSOMES were noted to be closely associated with the NE and often buried in an
invagination or ‘Hof ’ in prophase58,116.
The next big step in mechanistic understanding came in the 1980s, with the finding that NE proteins are
phosphorylated specifically in mitosis40. Nuclear assembly and breakdown were reconstituted in amphibian oocyte
extracts68,117 — a system that led to a wealth of biochemical data. The fragmented membrane homogenates of this system
led to the conclusion that membrane vesicles function as the natural precursors for nuclear assembly. Curiously, the
studies in oocyte extracts largely ignored the earlier electron-microscope observations in somatic cells, except for a more
contemporary study118, which indicated that, in rat thyroid gland cells, the ER-membrane network is broken down into
small, round fragments. On this basis, such membrane vesiculation and fusion of vesicles to reform larger membrane
surfaces was proposed as the primary mechanism of nuclear remodelling33. Only recently have studies in intact somatic
cells revisited the fate of NE components in mitosis, and shown that the ER serves as the reservoir for nuclear membrane
proteins in M phase19,30,44. Given this turbulent history, we are quite likely to witness the emergence of new concepts or
rediscoveries of long-forgotten ones for other aspects of nuclear dynamics in the future.
Organisms that consist of more
than one cell.
CISTERNAE
Flat sheets of endoplasmic
reticulum that enclose a lumen
like a hollow pancake.
CENTROSOMES
The microtubule-organizing
centres in animal cells.
NUCLEAR BASKET
A fishtrap-like structure on the
nuclear side of the nuclear pore
that is made up of eight fibrils
joined by a distal ring.
FLUORESCENCE RECOVERY
AFTER PHOTOBLEACHING
(FRAP). A technique in which a
pool of fluorescent molecules is
destroyed locally by highintensity laser irradiation. After
this ‘photobleach’, the exchange
of the non-fluorescent
molecules with the surrounding
fluorescent molecules is
monitored and measured as
‘recovery’ of fluorescence in the
bleached area.
M-PHASE PROMOTING FACTOR
(MPF). The complex of a B-type
cyclin and cyclin-dependent
kinase 1, which is also referred to
as cdc2 or p34, depending on the
species. Only the complex of
both proteins in a specific state
of phosphorylation is active, and
this is the main enzyme that is
responsible for entry into
M phase in both meiosis and
mitosis.
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| JULY 2002 | VOLUME 3
Class II. Class II NE components consist of a growing
list of INM proteins and protein families (TABLE 1; FIG. 1).
These specialized integral membrane proteins are
found specifically in the INM and not (or only at low
levels) in the endoplasmic reticulum (ER) and the
secretory pathway. Most of these proteins act as adaptors that link the INM to the lamina and/or chromatin
by direct interactions through their nucleoplasmic
domains2,15. The nucleoplasmic domains contain several sequence motifs that mediate these interactions.
The nuclear magnetic resonance (NMR) structure of
one of these domains, the LEM domain (found in
LAP2, emerin and MAN1; TABLE 1), shows that variations of this domain can bind directly to either DNA
or a chromatin protein16,17.
The adaptor function of these proteins provides a
mechanism for their appropriate localization on the
basis of selective retention. In this scheme, proteins that
are mobile within the ER can access the INM through
the membrane continuities at the periphery of each
NPC — this is a signal-independent process. However,
only those proteins that can specifically interact with
nuclear components, lamins or chromatin, for example,
are retained and concentrated. This model predicts that
integral INM proteins should show a reduced mobility
on localization to the NE and, indeed, this has been confirmed in dynamic imaging studies of live cells that
express several green-fluorescent-protein (GFP)-tagged
INM proteins18–20. Further support for the model is provided by the finding that localization of emerin to the
INM depends, in part, on the expression of A-type
lamins21,22 and that it can be inhibited by cytoplasmic
domains that are too bulky to fit through the periphery
of the NPC23. The concept of selective retention is also
important in nuclear membrane dynamics in mitotic
cells (see below and BOX 2).
Class III. Class III comprises NPC proteins, also known
as nucleoporins. This group contains two transmembrane, and more than 20 soluble proteins that constitute
the NPC (FIG. 1). This very large protein complex forms
an aqueous transport channel through the NE, and
thereby connects the inner and outer nuclear membranes (ONM) with a domain that is also referred to as
the pore membrane (POM).
The NPC has an estimated maximum mass of 125
MDa (REF. 24). It resembles a flat, hollow cylinder that is
embedded in the NE (termed the ‘spoke-ring complex’)
and is ~120 nm wide and ~40 nm long. This cylinder
contains a central ~40-nm-wide channel, which contains proteinaceous material that is sometimes referred
to as the ‘central plug’. From the walls of the cylinder
emanate eight cytoplasmic and nuclear filaments, and
the latter are joined by a distal ring to form the NUCLEAR
25
BASKET . The function of the NPC is nucleo-cytoplasmic
transport, but it is also an important structural unit of
the NE, and is probably involved in nuclear organization
in general26,27. Unlike the INM proteins, the two transmembrane nucleoporins gp210 and POM121 interact
only with other nucleoporins, and not with nuclear
lamins or chromosomes. However, soluble nucleoporins
of the nuclear basket bind to both lamins and chromatin26,28, and the lamina directly connects to the nucleoplasmic face of the spoke-ring complex29.
Class IV. Class IV consists of chromatin proteins that
mediate interactions of peripheral chromatin with
members of the lamina and/or INM-protein classes.
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Table 1 | Proteins that organize the nuclear envelope
Protein class
Name
Interacts with
Lamins
Lamin A and B
Lamin A
Lamin B
LAP1 and 2 families, histones, DNA
Emerin, pRB
LBR, LAP2β
Reference
Integral INM
LBR
LAP1α, β and γ
LAP2 β
LAP2 γ, δ and ε
Emerin
MAN1
Nurim
LUMA
RFBP
Nesprins
Lamin B, DNA, HP1
Lamins
Lamin B, DNA, BAF
Lamins
Lamin A, Lamin B, BAF
BAF?
?
?
?
Actin?
NPC
Nup153
gp210
POM121
Lamin B
Nuclear membranes
Nuclear membranes
Chromatin
BAF
HP1 α, β and γ
LAP2α
H2A/H2B
Methyl H3
*(DNA), emerin, MAN1, LAP2β
(Methyl H3), LBR
(DNA), lamin A
(DNA), lamins
LBR?
15,119 –121
15,122,123
15
15
15
15
15
15,123
15
6
124
125,126
28,102
97
127
15,123
15,128
15
15
129, but see
130,131
*Parentheses denote interactions that group a protein into one of the four classes of nuclear
envelope protein. These interactions do not link two structural elements of the nuclear envelope.
this organization is that the NE is a complex crosslinked
structural protein network. Recently, the dynamic and
mechanical properties of this network have been
addressed by time-lapse imaging and FLUORESCENCE
RECOVERY AFTER PHOTOBLEACHING (FRAP) of GFP-tagged
proteins in living cells. This approach found the lamina and pores to be immobile and stably bound for
many hours in interphase NEs30,31, and could show
that the NE behaves as an elastic, mechanically quite
stable entity30,32.
The stable structure of the NE, which encloses and is
connected to the chromosomes, poses a fundamental
problem for cell division in metazoan cells, in which the
microtubules remain exclusively cytoplasmic throughout the cell cycle. For spindle microtubules to gain
access to chromosomes, these cells have to undergo an
open mitosis, and break down their NE at the onset of
M phase. After the spindle has accomplished chromosome segregation, new NEs have to be assembled
quickly in the daughter cells (FIG. 2). Although our
insight into NE assembly has grown considerably over
the past five years, we are only starting to work out the
mechanism of NE breakdown (NEBD).
Tearing down the walls: NE breakdown
This class is small, and comprises three isoforms of heterochromatin protein 1, two other peripheral chromatin
proteins, and core histones (TABLE 1; FIG. 1).
In summary, a network of direct and indirect protein–protein interactions connects the four structural
units of the nuclear periphery — the lamina, INM,
nuclear pores and chromosomes. The prediction from
In dividing cells, the NE is ruptured during NEBD,
which defines the transition from prophase to
prometaphase in the cell-division cycle33 (FIGS 2b,c).
According to the textbook model33, the NE vesiculates
after the lamina has depolymerized as a result of mitotic
phosphorylation. However, studies in several systems
point to a different mechanism that involves mechanical
interactions of the NE, with spindle microtubules and
disassembly of the NPC being key steps in NEBD 32,34–37.
Cytoplasm
a Nuclear pore proteins
b INM proteins
c Lamins
d Chromatin proteins
Nucleus
50 nm
Figure 1 | Topology of the nuclear envelope. The nuclear envelope (NE) consists of four
structural units. The inner and outer nuclear membranes (light yellow) are joined at the nuclear
pore complex (light purple). The inner nuclear membrane (INM) is anchored by transmembrane
proteins to the underlying layer of lamina (light green) and to the peripheral chromatin (dark red).
The right side shows examples from each of the four classes of NE protein (labelled a–d).
Nucleoporins are represented by the soluble protein Nup358 (RanBP2, dark purple), a main part
of the cytoplasmic filaments, and gp210 (dark purple), a transmembrane protein with a large
lumenal domain in the perinuclear space. LAP2 (yellow) represents INM proteins, and has a
transmembrane domain, a lamin-interacting region, and two LEM domains that interact with DNA
and with the chromatin protein, which is represented by BAF (light red). These (and further) mutual
interactions between all four structural units form the highly crosslinked structural protein network
of the NE. The figure is drawn approximately to the known molecular scale of the structures and
molecules that are shown.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
Mitotic phosphorylation of NE proteins. Many biochemical studies have shown that NE proteins are subject to mitotic phosphorylation by the M-PHASE PROMOTING
cdc2
FACTOR (MPF) kinase p34
. This is believed to abolish
the structural protein–protein interactions that are necessary for NE integrity, and lead to its disassembly.
Nuclear lamins are phosphorylated by protein kinase C
and, subsequently, by cdc2, which results in their
depolymerization38,39 and dispersal in mitotic cells30,40,41.
Nucleoporins42,43 and several INM proteins2 are also
substrates of cdc2. In the case of the nucleoporins, this
presumably causes their release from, and subsequently
the disassembly of, the NPC, which explains the dispersed localization of nucleoporins that is observed in
mitotic cells30,44,45. The effect of phosphorylation is less
clear for INM proteins, but it is also assumed to abolish
their ability to interact with lamins and/or chromatin,
which allows the INM to detach from chromosomes.
Vesiculation versus ER absorption. The fate of nuclear
membrane proteins has been controversial, based on
results in different experimental systems1,2. Studies that
used fractions of Xenopus laevis oocytes have shown
that vesicles that are enriched in NE proteins can be isolated and are competent to assemble the NE in vitro46.
These studies gave rise to the model that vesiculation is
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Box 2 | Selective retention
Selective retention is a mechanism for establishing the steady-state localization of a
protein in a subcellular compartment. A protein that has no interactions is free to
diffuse and will be distributed homogenously in its compartment, whether this is the
cytoplasm or a membrane system, such as the endoplasmic reticulum (ER)–nuclear
envelope (NE). However, a protein that can bind to a cellular structure that is accessible
from its compartment will be retained at the site of binding. Its concentration there will
be determined by the number of binding sites, the strength of the interaction and the
ability of the protein to reach the binding site, typically by diffusion. This happens, for
example, for cytoplasmic proteins that interact with the cell surface, or for ER
membrane proteins that can interact with chromatin. In the latter case, in interphase
cells, the transmembrane protein can freely diffuse between the ER and the inner
nuclear membrane (INM). In the INM, it has access to its binding site on chromatin,
and is therefore retained and concentrated.
This mechanism underlies ‘targeting’ of INM proteins, although the movement of
individual molecules is driven by non-directional Brownian motion between the
ER and INM. In mitosis, the binding to chromatin is switched off by
phosphorylation of the membrane protein, which then quickly equilibrates with
the ER. At the end of mitosis, dephosphorylation activates the binding again, and
selective retention leads to the attachment of ER cisternae that contain the
chromatin-binding protein to chromosomes.
FLUORESCENCE RESONANCE
ENERGY TRANSFER
(FRET). A technique to measure
the proximity of two
fluorophores, a donor and an
acceptor. If these are within
2–10 nm of each other and if the
emission spectrum of the donor
overlaps with the excitation
spectrum of the acceptor, energy
can be transferred nonradiatively from acceptor to
donor by dipole–dipole coupling
or ‘resonance’. The efficiency of
the transfer is extremely distance
sensitive, so FRET is often
referred to as a molecular ruler.
DYNEIN
A large, cytoplasmic
microtubule-dependent motor
protein that converts the energy
of ATP into motion towards the
minus end of microtubules.
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| JULY 2002 | VOLUME 3
the fate of nuclear membranes in vivo. But studies in
intact mammalian cells have shown that nuclear membrane proteins reside in the ER in metaphase19,30,44, and
that the ER itself remains an intact network in dividing
somatic cells19,47.
This apparent contradiction between vesiculation
and ER dispersion has been proposed to reflect the difference between embryonic (Xenopus oocytes) and
somatic (mammalian culture cells) systems. However,
evidence for vesiculation that was obtained in fractionated HeLa cells48, and for intact ER in echinoderm
embryos49, argues against cell-type-specific differences
alone. A more plausible explanation is that the extraction procedures that are required to obtain pure membrane fractions in vitro inevitably result in vesiculation
of the ER network that is present in the intact cell. This
experimentally induced fragmentation of a continuous
membrane network would cause transmembrane proteins and lipids with different properties to partition
into distinct membrane fragments. This probably
reflects their organization into ER subdomains in
intact cells — a concept that is quite well accepted for
the plasma membrane50. The idea that a membrane
network is the precursor for NE assembly is strengthened by the recent finding that, even in Xenopus
extracts, membrane homogenates form networks
before NE formation51–53.
To gain more insight into mitotic and meiotic ER
organization, it will be interesting to investigate the
properties of INM proteins in the mitotic ER using
biophysical methods such as FRAP54 and FLUORESCENCE
55
RESONANCE ENERGY TRANSFER (FRET) . It would also be
useful to immunolocalize nuclear membrane proteins
in M-phase cells by immunoelectron microscopy,
especially in Xenopus eggs. So, in summary, nuclearmembrane vesiculation does not seem to be involved
in NEBD in the intact cell systems that have been
examined so far.
Does NPC disassembly trigger NEBD? NEBD has
recently been studied by imaging germinal vesicle breakdown in live starfish oocytes34, and by electron
microscopy of prophase nuclei from syncytial
Drosophila melanogaster embryos35. In starfish, there are
two phases of NE permeabilization, as measured by the
influx of inert fluorescent cytoplasmic molecules: a slow
gradual increase followed by a sharp and localized entry
wave34. The gradual increase in the permeability coefficient of the NE would be consistent with the slowly progressing disassembly of a pore complex, such as by
removal of the central plug. This would make each individual pore leaky, yet leave the NE intact. However, the
wave-like entry of material can be explained only by
large discontinuities in the NE, such as a fenestration
that would result from the complete removal of NPCs.
The crescent shape of the wave that enters the nucleus
further argues for a rapid expansion or spreading of
such fenestrations once they are formed. Curiously, this
occurs at only a few sites on the NE surface34.
In Drosophila, scanning and transmission electron
microscopy of prophase nuclei showed that NPCs lose
their cytoplasmic fibrils in prophase (this is possibly preceded by removal of the central plug), whereas the
nuclear membrane remains intact35. The data from
echinoderms and flies indicate that disassembly of the
pore complex could be an initial step and serve as a trigger of NEBD. It will be interesting to see whether a similar process occurs in vertebrate cells, in addition to the
dramatic mechanical effects that are observed in this
system (see below).
Microtubules tear the nuclear lamina. The structure of
the NE is affected by mitotic microtubules, which are
nucleated by the separating centrosomes in early
prophase. Early electron-microscopy studies observed
an intimate connection of spindle microtubules and the
NE in HeLa cells56,57 and plant cells58, as well as deep,
pocket-like distortions of the NE in the centrosomal
regions before NEBD 57 (FIG. 2b). A pioneering correlation of video light microscopy and electron microscopy
in lily cells58 proposed that pulling and pushing of the
spindle apparatus on the NE led to ‘undulations’. A more
recent reinvestigation of these structures in mammalian
cells found microtubule bundles deep within NE invaginations, and proposed piercing of the NE by microtubules as the mechanism for NEBD37.
From studies of microtubule motors, we know that
cytoplasmic DYNEIN localizes to the NE of mammalian
cells in prophase 59, and that it is required to attach centrosomes to the NE in Caenorhabditis elegans and
Drosophila 60–62. The NE–dynein interaction has been
interpreted in the context of centrosome positioning
and separation, as well as nuclear movement.
Regarding the latter, it has been shown that dynein is
responsible for nuclear movements in Xenopus
extracts, which could be equivalent to female pronuclear movements after fertilization in many oocytes63.
Two recent studies have now clarified the involvement
of both dynein and microtubules in NEBD32,36. These
studies, which used either a combination of live-cell
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DYNACTIN
A complex of several proteins
that are associated with dynein,
which links the motor to cargo
and regulates its activity.
MINUS END
The slower growing end of
microtubules.
a
imaging and photobleaching, or immunofluorescence
and electron microscopy, showed that microtubules
facilitate NEBD by literally tearing the NE open (FIG. 3).
This mechanism proceeds by the binding of dynein
and DYNACTIN to the outer face of the NE in prophase,
and subsequent massive distortion of the NE near the
centrosomes that are dependent on dynein and microtubules. The MINUS-END-directed force of the
dynein–dynactin complex, which is crosslinked to the
NE but moves along microtubules towards the spindle
poles, generates tension in the NE. As a result, it is
d
b
e
c
f
Figure 2 | Nuclear envelope, microtubules and chromosomes through the cell cycle.
Immunofluorescence images of normal rat kidney cells stained for DNA (blue), microtubules
(red) and the nuclear envelope (NE) marker POM121 (green). a | Interphase with smooth
nuclear rim. b | Prophase with dramatic invaginations of the NE around the centrosomes.
c | Prometaphase with fragmented NE. d | Metaphase with dispersed NE. e | Late
anaphase with partially assembled NE. f | Cytokinesis with mature daughter nuclei. Scale
bar in f, 10 µm.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
folded massively at the centrosomes and stretched further away from the ASTERS, where it finally tears when
the tension reaches a critical point (FIGS 3b,c). This tearing site represents a physical discontinuity where NPCs,
INM proteins and lamins are absent, and through
which cytoplasmic molecules can now rush unhindered into the nucleus.
The dispersion of most NE proteins starts only after
this tearing event in a stepwise disassembly, with B-type
lamins one of the last components to be completely solubilized in late prometaphase (FIGS 3d,e). Interestingly,
the interactions between microtubules and the NE do
not stop after the tearing, but continue to pull NE fragments away from chromosomes towards the centrosome. This virtually clears the chromosome surface for
spindle attachment.
In conclusion, recent findings in mammalian cells
have arrived at a model that elegantly explains NE
deformations and emphasizes that spindle formation
and NEBD are by no means separate. Instead, they are
intimately related processes that work hand in hand to
transfer the safeguarding of chromosomes from membranes to microtubules4 (FIG. 3).
What lies ahead? In the future, it will be interesting to
see whether — and how — NPC disassembly is coordinated with NE tearing in mammalian cells. In addition,
it will be important to investigate how widespread the
tearing mechanism of NEBD is in other species. It is
interesting to speculate that a microtubule-based
mechanical system might also be involved in separating
other organelles, such as the ER, mitochondria and the
Golgi apparatus64. Interestingly, NEBD is not always
complete, and there are different degrees of ‘openness’
of mitosis in metazoans. In Drosophila, the NE is broken
down only partially35,65, whereas NEBD in C. elegans is
delayed until anaphase, but is eventually completed66.
By contrast, in sea urchin49 and mammalian cells32,36,
NEBD is complete and occurs early in prometaphase.
At the same time, the complexity of the NE protein
composition increases systematically through
evolution67. This could point to more extensive and
‘antimitotic’ connections between NE and chromosomes in vertebrate cells, and might explain the need for
a completely open mitosis by mechanical rupture and
pulling of the NE to clear the chromosomes and make
them available for microtubule attachment. In cells in
which the chromosomes are easily detached from the
NE by condensation, partial disassembly of the NE
would be enough to allow the spindle microtubules efficient access to chromosomes. It is conceivable that, in
this way, a mechanism for microtubule–NE interaction,
based on dynein–dynactin that initially functioned in
centrosome positioning and separation using the NE as
a traction surface, evolved into a means of disrupting
the NE barrier as it became increasingly rigid with the
complex organization of vertebrate genomes.
The dynamics of nuclear envelope biogenesis
Until the early 1980s, studies on NE re-formation had
focused largely on the ultrastructure of mitotic cells.
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However, in 1983, Lohka and Masui published a landmark paper68 that described the in vitro assembly of
authentic NEs around chromatin templates in Xenopus
egg extracts. Nuclei that are formed in this way are
transport competent and will replicate DNA68–70. These
studies spurred the development of other in vitro
assembly systems that are based on various cell
types71–74, and laid the foundations for a biochemical
dissection of assembly.
ASTERS
Many microtubules that are
nucleated from one point (such
as a centrosome) in a radial
manner.
a
b
Tearing
c
d
e
Figure 3 | Model of nuclear envelope breakdown in
mammalian cells. The schematic shows a ‘half-cut-open’
nucleus cleared from most of the surrounding endoplasmic
reticulum (ER) for clarity. Depicted are chromosomes (purple),
lamina (green), nuclear membranes (yellow), microtubules (red)
and centrosomes (orange). Nuclear pores are omitted for
simplicity. a | Interphase. Decondensed chromosomes are
surrounded by the smooth and elastic lamina and the double
membrane layer of the nuclear envelope (NE). Connections to
the ER are truncated. Centrosomes nucleate long and stable
interphase microtubules. b | Prophase. About 30 minutes
before NE breakdown, microtubule-filled folds appear in the NE
close to the centrosomes, followed by the appearance of
condensed chromosomes along the NE. As prophase
proceeds, folds in the NE increase in size. At this time, the NE
becomes stretched on the side that is opposite to the
centrosomes due to pulling forces of minus-end-directed
microtubule motor proteins on its surface. c | NE breakdown
occurs when the tension has reached a critical point and tears
the lamina and the associated membranes, which produces a
hole in the NE. Hole formation is followed by nuclear collapse
as the tension is released. d | Prometaphase. The initial hole
spreads rapidly and fragments the NE. However, large pieces
of NE are still attached to chromosomes. e | Metaphase.
NE-associated minus-end-directed motors remove NE
remnants from the chromosomes towards the centrosomes.
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Forming a sphere from a network. Fractionation of
Xenopus extracts has uncovered two biochemically distinct vesicle populations that together are required for
nuclear formation46,75,76. Related fractionation studies of
mammalian cells also show a heterogeneous microsomal population that is enriched in NE components
over ER-specific proteins48. We would argue that these
vesicles arise during extract preparation, through fragmentation of an ER network in which NE- and ER-specific proteins might be distributed in a mosaic of
microdomains. Nevertheless, they provide a valuable
means of examining the contributions of different
membrane components to the NE assembly process.
Indeed, Vigers and Lohka46 altered the frequency of
NPC formation simply by changing the relative concentrations of different membrane fractions in their in
vitro reactions.
Observations on both fixed and live mammalian
mitotic cells indicate that nuclear membrane re-formation involves the coating of newly segregated chromatids
with ER-like cisternae — a process that begins during
anaphase19,57. The emerging consensus is that membrane attachment is mediated by INM proteins77. In this
way, increasing availability of chromatin and/or laminbinding sites leads to the immobilization of more INM
proteins and establishment of the INM domain.
Differences in the relative timing of binding of various
nuclear membrane components, although often cited as
evidence for NE-specific vesicles in vivo, would simply
reflect the asynchronous appearance of different INMprotein-specific binding sites at the nuclear periphery. In
addition, differential affinities of INM proteins for their
specific receptors on chromatin, as well as differences in
the ability of INM proteins to diffuse rapidly across the
ER network, could underlie the ordered assembly. It is
important to point out that this view of nuclear re-formation is simply another manifestation of the selectiveretention mechanism for INM protein localization during interphase (BOX 2).
Focus on fusion. Even in the absence of NE precursor
vesicles in vivo, nuclear re-formation from a membrane
network at the end of mitosis must involve extensive
membrane fusion (BOX 3). Recent studies from Mattaj,
Warren and colleagues53,78 indicate the involvement of
several fusion mechanisms. By following NE formation
around demembranated sperm chromatin in Xenopus
egg extracts, they documented several biochemically
separable phases in the establishment of sealed nuclear
membranes. Early stages in NE formation involve the
appearance of an ER-like membrane network across the
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AAA ATPASE
A family of enzymes that
hydrolyse ATP and have a
common ATPase module. They
typically form ring-shaped
oligomers and are involved in
diverse cellular functions, such
as membrane fusion (for
example, p97 and NSF), DNA
replication and proteolysis.
surface of the sperm chromatin. This finding is consistent with the idea that, in vivo, the ER forms a mitotic
reservoir of nuclear membrane components.
The appearance of this network depends on GTP
hydrolysis by Ran52,53, a small Ras-related protein that
has an important regulatory function in nucleo-cytoplasmic transport, as well as in mitotic spindle assembly. The precise function of Ran in the formation of
the chromatin-associated membrane network is not
clear. However, it is intriguing that Ran-coated beads
can substitute for chromatin as a template for NE
assembly in vitro 79.
After the appearance of the membrane network, two
further steps can be recognized53. The first of these is the
formation of a sealed NE; the second involves NE
expansion to accommodate chromatin decondensation.
Both steps are mediated by an AAA ATPASE called p97. This
is closely related to the N-ethyl-maleimide-sensitive
fusion protein (NSF), which regulates membranefusion events and is involved in many aspects of vesicular transport80. Originally identified as the product of
the CDC48 gene in yeast, p97 and an associated protein,
p47, mediate re-formation of the Golgi apparatus at the
end of mitosis81, and are also involved in the establishment of transitional ER82,83.
Hetzer et al.53 have shown that the final expansion
phase of nuclear re-formation in vitro is mediated by
p97/47, presumably in association with a receptor protein on the NE. However, for the initial closure or sealing of the NE, the fusion event is mediated by p97 in
association with two other proteins, called Ufd1 and
Npl4 (REFS 53,78). In yeast, Ufd1, in combination with
cdc48, is involved in ubiquitin-dependent protein
degradation84,85. Together with Npl4, which was originally identified as an NPC component that is essential
for nuclear protein import86, these proteins have been
implicated in the ubiquitin-dependent cleavage and
activation of membrane-associated transcription
factors 87,88. Whether p97–Ufd1–Npl4-mediated nuclear
membrane fusion occurs through interactions with
receptor molecules on the target membrane or through
ubiquitin-dependent activation of another fusion molecule remains to be seen.
Why use two separate fusion systems in sealing and
expansion of the NE? This question has yet to be
resolved, but one suggestion is that the geometry of the
fusion reactions dictates two different mechanisms89.
To form a sealed NE, it is necessary to close holes or
gaps in the double nuclear membranes — a process
that demands annular or circular fusion events. At
some point, this must involve constriction of holes
that span the nuclear membranes, and it is conceptually quite different from the familiar NSF model of
vesicle fusion in membrane transport80 (BOX 3). By contrast, during NE expansion in vitro, new membrane
components must be incorporated, and it is easy to see
how this could be accomplished by p97/47-mediated
homotypic fusion of ER elements with the ONM. In
this way, p97/47 would catalyse what are basically
point fusion events that are equivalent to those that
occur in re-formation of the Golgi apparatus81. It is less
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
Box 3 | Membrane fusion
a Point fusion
b Annular fusion
Membrane fusion is central to many cellular activities. It
is a tightly regulated process, and it is easy to see why —
promiscuous fusion would lead to an intermixing of
cellular membranes that would ultimately result in loss
of organelle identities.
Most intracellular fusion events are controlled by the
AAA ATPase family members N-ethyl-maleimidesensitive fusion protein (NSF) or p97, and mainly involve
point fusion. This would be exemplified by the fusion of
a vesicle with a target organelle, as occurs in the secretory
and endocytic pathways. Point fusion could also
represent the mechanism of nuclear membrane
expansion during nuclear re-formation in vitro (figure,
part a).
A second type of fusion process — annular fusion — is
required for the closure of gaps in the double nuclear
membranes. This is fundamentally different to point
fusion — it requires the constriction of a membrane ring
followed by resolution of the inner nuclear membrane
and outer nuclear membrane, as the ring is eliminated by
fusion at its cytoplasmic surface (figure, part b). Annular
fusion also probably occurs in the fenestrated
membranes of the endoplasmic reticulum and Golgi
apparatus, as well as in the final step of cytokinesis.
Although molecules that are involved in point fusion
have been extensively characterized, little is known of the
mechanisms of annular fusion.
clear whether this expansion mechanism would operate in vivo. Given their interconnections, the ER could,
in principle, feed membrane components directly into
the ONM, and thereby eliminate the requirement for
p97/p47 in nuclear growth. In addition, the situation is
more complex in vivo than the simple assembly of NEs
around sperm heads. In frog oocytes and embyros
(but also in Drosophila and sea urchin), NEs initially
assemble and seal around individual chromosomes to
form a set of ‘mini’ nuclei90. These karyomeres are
already import and replication competent91; that is,
they contain functional nuclear pores. Karyomeres
then fuse together in a second step to form one homogeneous nucleus that contains all the chromosomes. It
will be interesting to see whether karyomere fusion is
VOLUME 3 | JULY 2002 | 4 9 3
REVIEWS
a Seeding
and that it must involve fusion between the INM and
ONM94–96. Although the identity of the fusion apparatus is still unknown, the large NPC membrane protein
gp210 is one candidate97. This is because of its large
lumenal domain, and the presence of hydrophobic
sequences that are similar to the fusogenic peptides that
are seen in certain viral-envelope glycoproteins.
b Fusion
NPC seed assembly
Membrane recruitment
Intralumenal fusion
NPC assembly
Figure 4 | Different modes of nuclear pore assembly. a | Nucleoporin-seeded assembly on
the chromatin surface (blue) followed by membrane recruitment. b | Pore insertion into a
continuous double membrane. Seeding can be explained simply by binding interactions;
however, it cannot easily explain the pore insertion into an intact NE, as it occurs in S phase.
Insertion requires a sophisticated, as-yet-unidentified fusion machinery. The two models are not
mutually exclusive, and both mechanisms could conceivably function simultaneously during
nuclear assembly. Yellow denotes endoplasmic reticulum; light/dark purple denotes components
of the nuclear pore complex.
recapitulated in the in vitro assembly systems, or
whether it requires its own specific fusion mechanism.
How to make a channel through two membranes. NPC
reassembly within the double nuclear membranes represents an intriguing topological problem (FIG. 4). There
are two main ways in which this reassembly might be
accomplished. The simplest would be to assemble the
central region of the spoke-ring complex on the chromatin surface, then surround this by flattened membrane cisternae. These would then form the INM and
ONM, as well as the POM. All that would be required is
fusion to form sealed membranes around each NPC
(FIG. 4a). This mechanism could operate during nuclear
assembly before the chromatin surface is completely
sealed by a continuous double membrane.
The alternative approach would be to create a
largely continuous double membrane using the appropriate p97-fusion apparatus, then to insert the NPC, or
NPC subunits (FIG. 4b). Certainly, during S phase, when
NPC numbers double92,93, de novo NPC assembly
must occur in intact membranes. This mechanism
demands a further fusion event between the lumenal
faces of the INM and ONM to create an aqueous
channel between the nucleus and cytoplasm, as well as
to form the POM (FIG. 4b).
Studies on artificial nuclei in Xenopus extracts used
inhibitors of NPC assembly to show that NPC re-formation can indeed occur in regions of intact membranes,
494
| JULY 2002 | VOLUME 3
Which comes first? Sequence of nuclear assembly.
Detailed observations in mammalian systems show that
binding of proteins to the nuclear periphery after chromatid segregation occurs in a stepwise fashion19,30,45,98–100
(FIG. 5). The earliest events that have been recognized in
NE reassembly begin in mid-anaphase, and involve the
association of certain soluble NPC proteins with the
chromatid surfaces. These include Nup153, which is a
component of the nuclear basket, and Nup133, which is
associated with the NPC spoke-ring complex30,45,98.
Soon after the association of these soluble nucleoporins, membranes begin to associate with the lateral
and polar margins of the chromatids. At this time,
INM proteins such as LAP2, LBR and emerin concentrate at the membrane–chromatin interface19,98,100. This
presumably reflects the appearance of specific chromatin-associated binding sites for these proteins. The
NPC membrane protein POM121 is also bound during this period30. Surprisingly, the other vertebrate
NPC membrane protein, gp210, does not begin to
concentrate at the nuclear periphery until late
telophase or early G1, when the NE is sealed98,99. The
implication is that POM121, rather than gp210, is
required for the establishment of early membraneassociated NPC assembly intermediates. This is consistent with findings that, in interphase, POM121 is an
extremely stable component of the NPC (once incorporated into an NPC, it essentially never leaves)30,
whereas gp210 seems to exchange between NPCs
within minutes based on photobleaching experiments
and heterokaryon analyses (B.B. and K. Bodoor; J.E.
and G. Rabut, unpublished observations).
The late enrichment of gp210 at the nuclear
periphery is, at first sight, inconsistent with a role in
fusion between the INM and ONM. However, given
the continuity of the ER-membrane system, some
gp210 will be present at the nuclear periphery, even at
the earliest times during NE assembly (albeit at a low
concentration). This, combined with its ability to
exchange between NPCs, might allow gp210 to carry
out early functions in NPC formation. Taking this
thought one step further, gp210 might function more
as an NPC assembly factor than as a bona fide structural nucleoporin.
The early association of soluble nucleoporins with
chromatin is reminiscent of the ‘pre-pores’ that
Sheehan et al. observed101 on sperm chromatin after
extended incubation in Xenopus egg extracts. A reasonable view is that these proteins could function in the
immobilization of POM121 after the association of
membranes with the chromatin surfaces. In this way,
chromatin-attached nucleoporins would define assembly sites for NPCs. Indeed, Nup153 is required for the
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Anaphase
Nup153
Nup98
Nup133
Telophase
Nup93
Cytokinesis/G1
Nup214
Nup358
Tpr
Nucleoporins
Nup62
Pom121
LAP2
LBR
Emerin
Nuclear membrane
gp210
Lamin B
Lamin A
Lamins
Steen and Collas107 have devised a method of interfering with the binding of PP1 to AKAP149 in mitotic
cells in vivo, which inhibits the reassembly of B-type
lamins. Remarkably, the cells could still complete
mitosis, but they underwent apoptosis about 6 h later.
So it seems that NE assembly in vivo can occur independently of lamins. Consistent with this, B-type
lamins can assemble only after the formation of
closed NEs30,99 — although there is some debate about
their timing31 and lamin fragments can interfere with
the assembly of artificial nuclei in Xenopus extracts108.
In mature nuclei, reduced lamin content is associated
with extreme mechanical fragility of the NEs109,110, as
well as mislocalization of certain INM proteins such
as emerin21. Ultimately, this is not compatible with
cell survival.
Conclusions
Figure 5 | The sequence of events during nuclear envelope assembly. The time of nuclear
envelope (NE) protein localization to the surface of post-mitotic chromatin is indicated by arrows
for soluble nucleoporins (purple arrow), integral membrane proteins (yellow arrow) of the inner
nuclear membrane (INM; yellow) and nuclear pore complex (NPC; purple), as well as for lamins
(green arrow). The lower panel presents a schematic view of the re-forming NE by filling in the
four structural units of the NE at the appropriate location when a component is bound (compare
with FIG. 1). The first NE proteins bind to the chromatin surface during anaphase. Early NE
proteins include nucleoporins and INM proteins, and both soluble cytoplasmic and
transmembrane proteins that are dispersed in the ER. Early proteins, such as the Nup133
complex and Nup153, could potentially form the core of the NPC, which then sequentially
recruits other nucleoporins. As can be seen by the localization mapping in the bottom panel, this
core probably corresponds to part of the spoke-ring complex, to which the central plug and the
cytoplasmic and nuclear filaments are assembled in subsequent steps. Compared with
nucleoporins and INM proteins, lamins (green) are late components; they are imported through
the already sealed NE.
immobilization of NPCs in the NE, and it is essential
for the assembly of other nucleoporins, including
Nup98 and Nup93 (REF. 102).
Last but not least: the lamina. There is broad agreement
that the nuclear lamina is essential in the maintenance
of NE integrity. However, the function of individual
lamins in NE reassembly is less clear. Certainly, A-type
lamins cannot be required for nuclear re-formation,
given that not all cell types express these proteins103,104.
Furthermore, in cells in which A-type lamins are
expressed, the bulk of these are re-imported into the
nucleus in early G1, after a sealed NE has been
formed31,105 (FIG. 5). The reassembly of B-type lamins in
mammalian cells has recently been shown to be under
the control of protein phosphatase 1 (PP1)106. During
mitosis, PP1 is associated with chromatin, but it is targeted to sites of nuclear membrane formation by interaction with AKAP149, an ER-membrane protein. PP1
can then dephosphorylate B-type lamins, which allows
them to associate to form the nuclear lamina.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
Although our understanding of NE dynamics during
the cell cycle is still incomplete, important themes have
been outlined. The finding that the ER functions as a
mitotic reservoir for NE-membrane components has
forced us to reconsider the mechanisms by which the
NE is dispersed during prometaphase, and later how an
NE-specific membrane domain is re-established at the
end of mitosis.
Regarding NEBD, it is clear that mechanical
processes that link the mitotic spindle to the stable NE
protein network are instrumental. However, the role of
NPCs still raises many questions. For instance, do
vacated NPCs form the epicentre for microtubuleinduced nuclear membrane tearing? Do NPCs contain a
dynein–dynactin binding site that could permit the
transmission of tensile forces to the INM? If so, how is
this binding regulated?
For the better-understood process of NE assembly,
interactions of chromatin and INM proteins seem to
be the key determinants of nuclear membrane re-formation and drive the segregation of the INM domain
from the ONM and bulk ER by selective retention of
membrane proteins. Likewise, chromatin-associated
nucleoporins might define assembly sites for NPCs by
binding and retaining POM121 early during NE
assembly. More chromatin proteins than identified so
far will probably be involved in the complex orchestration of rebuilding nuclear architecture. The lamins,
although essential for the maintenance of nuclear
architecture, turn out to be dispensable with respect to
the formation of a minimal NE that contains ONM
and INM domains and NPCs.
There are still gaps in our understanding of the
essential mechanisms behind NE assembly. It is not
clear what machinery underlies NPC reassembly, especially with respect to fusion of the INM and ONM to
create aqueous channels between the nucleus and cytoplasm. Similarly, the role of Ran in the formation of the
early chromatin-associated membrane network and the
function of p97–Ufd1–Npl4 in the sealing of the NE
remain largely obscure. From the pace of recent
progress, the answers to these questions could be forthcoming in the not-too-distant future.
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Acknowledgements
The authors would like to thank P. Lénárt for preparing the illustrations and K. Ribbeck for preparing FIG. 2.
Online links
DATABASES
The following terms in this article are linked online to:
Interpro: http://www.ebi.ac.uk/interpro/
LEM domain
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink
AKAP149 | MAN1 | RanBP2
Saccharomyces Genome Database: http://genomewww.stanford.edu/Saccharomyces/
CDC48 | Npl4 | Ufd1
Swiss-Prot: http://www.expasy.ch/
cdc2 | emerin | GFP | gp210 | Nup98 | Nup133 | Nup153 | p34cdc2 |
POM121 | Ran
Access to this interactive links box is free online.
VOLUME 3 | JULY 2002 | 4 9 7