Most eukaryotic cells have a single nucleus in which a nuclear
envelope (NE) separates the chromosomes from the cytoplasm. The
NE (Fig. 1) is made of a double membrane that is perforated by
nuclear-pore complexes (NPCs). The outer nuclear membrane,
which is continuous with the ER, connects with the inner nuclear
membrane at the curved membrane regions that surround each NPC.
In metazoans, the NE also contains a meshwork of proteins, which
are collectively called the nuclear lamina, that underlies the inner
nuclear membrane and interacts with portions of the chromatin. The
nuclear lamina is made predominantly of intermediate filaments
called lamins, of which there are two main types: type A and type
B (for a review, see Dechat et al., 2008). In mammals, there are
two major A-type lamins, lamin A and lamin C, which are generated
by alternative splicing of the LMNA gene. There are also two major
B-type lamins, lamin B1 and lamin B2, each encoded by its own
gene. Lamin A and the two lamin Bs have a C-terminal domain
that is lipid modified (farnesylated), thereby promoting their
attachment to the inner nuclear membrane. This domain is missing
in lamin C and is normally removed by proteolytic cleavage in lamin
A. In addition to their location at the nuclear periphery, lamins are
also present in the nucleoplasm (Dechat et al., 2008).
The nuclear lamina also contains a variety of lamin-associated
proteins and other proteins that are embedded in the inner nuclear
membrane (reviewed by Wilhelmsen et al., 2006). The nuclear
lamina might also play a role in the internal organization of the
nucleus. For example, within the nucleus, chromosomes are
organized in chromosome territories: rather than being distributed
throughout the nucleus, each chromosome appears to occupy a
discrete region, or territory (for a review, see Cremer et al., 2006).
In addition, heterochromatic chromosomal regions, which are
chromosome domains that are transcriptionally inactive, tend to
localize at the nuclear periphery (Akhtar and Gasser, 2007). It is
likely that the nuclear lamina contributes to these types of
intranuclear chromosome organization. Unicellular eukaryotes and
plant cells do not have lamins, although they might have proteins
that function as a nuclear lamina.
During mitosis, a parent cell gives rise to two daughter cells,
each with its own nucleus. Two main strategies have evolved to
successfully carry out this task: open mitosis and closed mitosis
(Fig. 2). Open mitosis occurs in most eukaryotic cells, whereas
closed mitosis occurs in certain species of fungi. In open mitosis,
the NE disassembles early in mitosis, allowing microtubules that
emanate from cytoplasmic centrosomes to contact the chromosomes
and promote their segregation (reviewed by Prunuske and Ullman,
2006). At the end of open mitosis, the NE reassembles around the
two segregated DNA masses to form the two daughter nuclei. In
closed mitosis, the NE does not disassemble and chromosome
segregation takes place entirely within the confines of the nucleus.
This strategy works in cell types where the centrosome equivalents,
known as spindle-pole bodies, are embedded in the NE, allowing
microtubules to associate with chromosomes without the need for
NE disassembly. Some organisms, such as Aspergillus nidulans,
undergo semi-open mitosis, in which the partial disassembly of the
NPCs creates large holes in the NE, but the envelope itself does
not completely disassemble (for a review, see De Souza and Osmani,
In all forms of mitosis – open, closed or semi-open – the NE
undergoes dramatic structural changes. During open mitosis, the
NE has to reform around all of the chromosomes, rather than around
a subset of chromosomes, and it then must expand to attain its final
size and shape. During closed mitosis, the NE expands to
accommodate the movement of the segregating chromosomes, and
it must then get cleaved, resealed and restructured to form two round
daughter nuclei. In the past few years, numerous studies have shed
light on the molecular mechanisms that underlie these processes.
To understand how these nuclear gymnastics take place, we first
discuss the factors that contribute to nuclear shape and size. We
then examine how the NE reassembles to form a nucleus with the
proper attributes following mitosis.
The nucleus is one of the most prominent cellular organelles,
yet surprisingly little is known about how it is formed, what
determines its shape and what defines its size. As the nuclear
envelope (NE) disassembles in each and every cell cycle in
metazoans, the process of rebuilding the nucleus is crucial for
proper development and cell proliferation. In this Commentary,
we summarize what is known about the regulation of nuclear
shape and size, and highlight recent findings that shed light on
the process of building a nucleus, including new discoveries
related to NE assembly and the relationship between the NE
and the endoplasmic reticulum (ER). Throughout our
discussion, we note interesting aspects of nuclear structure that
have yet to be resolved. Finally, we present an idea – which we
refer to as ‘the limited flat membrane hypothesis’ – to explain
the formation of a single nucleus that encompasses of all of the
cell’s chromosomes following mitosis.
Key words: ER membrane, Reticulons, ER tubules, ER sheets,
Lamina, Nuclear envelope assembly, Nuclear pore complex, Lipin
Sizing up the nucleus: nuclear shape, size and
Micah Webster*, Keren L. Witkin* and Orna Cohen-Fix‡
The Laboratory of Cellular and Molecular Biology, NIDDK, NIH, Bethesda, MD 20892, USA
*These authors contributed equally to this work
‡Author for correspondence (e-mail: email@example.com)
Journal of Cell Science 122, 1477-1486 Published by The Company of Biologists 2009
Journal of Cell ScienceJournal of Cell Science
The nuclei of most cells are either round or oval. This, in itself, is
hardly remarkable except for the fact that various diseases, as well
as aging, are associated with alterations in nuclear shape (Fig. 3).
Moreover, in certain specialized cell types, altered nuclear shape
is important for cell function. But what determines nuclear shape,
and how does shape affect function? In many cell types, altered
nuclear shape is due to changes in the nuclear lamina. In some cases,
however, the shape of the nucleus is altered by forces that act from
the cytoplasm. In either case, it is still not entirely clear how nuclear
shape affects function, although two main hypotheses exist. The
first hypothesis posits that changes in nuclear shape alter the rigidity
of the nucleus; this could be beneficial for cells that need to squeeze
through tight spaces, but deleterious to cells that are under
mechanical duress. The second hypothesis proposes that changes
in nuclear shape result in chromatin reorganization and thereby
affect gene expression. It is important to note that these two
hypotheses are not mutually exclusive. In addition, because nuclear
shape changes are often accompanied by an altered nuclear lamina,
it is possible that the dramatic effect on cell function is due to
aberrant properties of the lamina rather than nuclear shape changes
per se. In this section, we examine some of the cell types and
conditions that are associated with irregular nuclear shape, and we
discuss, when known, the causes of these shape changes and how
they affect cell function.
Normal cells with abnormal nuclei
Of all the cell types that normally exhibit unusual nuclear shape,
neutrophils have been studied the most thoroughly. Neutrophils are
cells of the immune system that migrate through tissue towards sites
of infection. They are characterized by their multi-lobed nuclei,
typically exhibiting three or four lobes that are connected by thin
DNA-containing filaments (Fig. 3B) (reviewed by Hoffmann et al.,
2007). Neutrophils with hypolobulated nuclei are associated with
Pelger-Huet anomaly, caused by a mutation in the lamin B receptor
(Hoffmann et al., 2002). Hypolobulated neutrophils have
deficiencies in various cellular functions, including the ability to
migrate through small openings. This suggests that nuclear
lobulation is an adaptation to fit cell function (Hoffmann et al.,
2007). Nuclear lobulation is dependent not only on lamin B
receptor, but it is also associated with a paucity of various nuclear
lamina proteins (Olins et al., 2008), and it depends on microtubules:
treatment of differentiating neutrophils with microtubule-
depolymerizing drugs reduced the extent of lobulatons (Olins and
Olins, 2004). Thus, in neutrophils, both intranuclear and cytoplasmic
proteins affect nuclear shape.
The involvement of microtubules in altering nuclear shape can
also be seen during cellularization in the Drosophila embryo when
nuclei change shape from spherical to ellipsoid. This change is
dependent on both cytoplasmic microtubules and an inner nuclear
membrane protein called kugelkern or charleston (Brandt et al.,
2006; Pilot et al., 2006). Presumably, kugelkern/charleston is
necessary to generate chromatin-NE associations that transduce the
forces exerted by cytoplasmic microtubules into nuclear shape
changes. Whereas some cell types use microtubule-generated forces
to actively alter the shape of their nuclei, others must counteract
microtubule-generated forces to maintain their normal nuclear
shape. For example, during interphase in fission yeast, microtubule-
generated forces in the cytoplasm can alter nuclear shape if any
one of several inner nuclear membrane proteins is inactive (King
et al., 2008). In this case, NE proteins that normally make the nucleus
more rigid must resist nuclear distortions that are caused by
cytoplasmic microtubule-associated forces.
Pathologies and conditions associated with altered nuclear
It has long been known that reducing the levels of lamina proteins,
either by mutation or RNA interference (RNAi), leads to alteration
in nuclear shape (Furukawa et al., 2003; Lammerding et al., 2005;
Liu et al., 2000). Indeed, cells with abnormally shaped nuclei are
often seen in diseases in which lamina proteins are mutated
(collectively called laminopathies) (Capell and Collins, 2006) (see
Fig. 3C). Thus, the lamina has an active role in maintaining the
spherical shape of the nucleus. Mutations in lamina proteins cause
various types of lipodystrophies, resulting in abnormal fat tissue,
and myopathies, which affect muscle function. For the most part,
the link between the lamina defect and disease manifestation is not
known, although both an inability to withstand mechanical stress
and altered gene expression can be envisioned. A defect in nuclear
shape can result not only from reduced levels of lamina proteins,
but also from aberrant processing. One of the best-characterized
examples of such a case is the premature aging syndrome Hutchison-
Gilford progeria syndrome (HGPS). The mutation causing HGPS
was mapped to the gene encoding lamin A (De Sandre-Giovannoli
et al., 2003; Eriksson et al., 2003); in the mutated protein, the
activation of a cryptic splice site generates an aberrant form of lamin
A, called progerin, which is constitutively lipid-modified (Rusinol
and Sinensky, 2006). This presumably causes lamin A to remain
associated with the inner nuclear membrane. It is likely that this
membrane retention is the cause of the abnormal nuclear
morphology, because treating cells from HGPS patients with
compounds that inhibit this lipid modification reverses the
abnormalities in nuclear shape (Capell et al., 2005; Glynn and
Journal of Cell Science 122 (10)
Nuclear pore complex
Fig. 1. The nuclear envelope. The NE is an integral part of the ER-membrane
network (in blue-green). The inner nuclear membrane (INM) and outer nuclear
membrane (ONM) connect at sites of NPCs (green barrels) where the
membrane curves as it surrounds the NPC. The ONM is continuous with the
peripheral ER. The NE contains a variety of proteins that are embedded in the
INM (purple) or the ONM (light blue). Most ONM proteins are also found in
the peripheral ER. INM proteins can interact with the underlying nuclear
lamina (dark blue), with ONM proteins or with chromatin (red), often through
linker proteins (yellow). For a detailed description of the various proteins
associated with the NE see recent reviews (Crisp and Burke, 2008; Guttinger
et al., 2009).
Journal of Cell ScienceJournal of Cell Science
Building a nucleus
Glover, 2005; Mallampalli et al., 2005; Toth et al., 2005). Other
disease-causing mutations that lead to the retention of the lipid
modification of lamin A, such as inactivation of the lamin A protease
Zmpste24, also result in premature aging and aberrant nuclear
morphology (reviewed by Rusinol and Sinensky, 2006).
Similarly to premature aging, normal aging is also associated with
abnormal nuclear shape, both in humans (Fig. 3D) and in model
organisms (Brandt et al., 2008; Haithcock et al., 2005; Scaffidi and
Misteli, 2006). In humans, the aging-dependent change in nuclear
shape has been linked to the nuclear lamina, and in particular to
progerin, the altered form of lamin A seen in patients with HGPS
(Scaffidi and Misteli, 2006). Although the link between the membrane
retention of lamin A and aging remains to be discovered, recent studies
suggests that an altered nuclear lamina might lead to aging by affecting
the transcription profile of stem cells, thereby interfering with their
ability to retain an undifferentiated state and reducing the overall stem
cell pool and proliferation capacity (Espada et al., 2008; Scaffidi and
An abnormal nuclear shape is also associated with cancer (Zink
et al., 2004). In fact, altered nuclear shape is one of the key
diagnostic tools used in identifying cancerous cells, and it is the
basis for the Pap smear, which is widely used for the early detection
of cervical cancer. The functional relationship between altered
nuclear shape and cellular transformation – or even the underlying
cause of altered nuclear morphology – is often not known, although
it has been speculated that changes in nuclear shape lead to changes
in chromosome organization, which in turn can affect gene
expression (He et al., 2008). Others have proposed that the altered
nuclear shape in cancer cells facilitates the formation of metastases
because of reduced nuclear stiffness, which could increase the ability
of transformed cells to penetrate tissue (Dahl et al., 2008).
In addition to the studies described above, depletion studies and
the characterization of numerous mutations have linked other
proteins to abnormal nuclear morphology, but the mechanisms
involved are mostly unknown. It is plausible, at least in some cases,
that the relationship between protein inactivation and altered nuclear
shape is indirect. For example, studies in the past decade have shown
that the inactivation of proteins that are associated with the ER
affects nuclear shape (Higashio et al., 2000; Matynia et al., 2002).
These findings suggest that there is an intimate relationship between
the ER and the shape of the NE; this relationship will be further
explored when we discuss NE assembly (see below).
Finally, nuclear shape can be affected by lipid synthesis. This
has been shown in both yeast and C. elegans, where the inactivation
of a lipid phosphatase that is homologous to the mammalian lipin
(Reue and Zhang, 2008) was shown to cause expansion of the ER
membrane and alteration in NE shape (Campbell et al., 2006; Golden
et al., 2009; Gorjanacz and Mattaj, 2009; Siniossoglou et al., 1998;
Tange et al., 2002). Interestingly, at least in budding yeast, this
expansion was confined to the region of the NE adjacent to the
nucleolus, whereas the NE associated with the bulk of the DNA
remained unchanged (Campbell et al., 2006). This observation
suggests that some NE domains are more sensitive than others to
shape disruption caused by changes in lipid biosynthesis or
membrane composition. We will revisit the relationship between
lipid synthesis and the NE when we discuss how a single nucleus
is formed at the end of mitosis.
The nucleus increases in size from the time of its formation,
immediately following NE assembly, to when it reaches its final
size in interphase. This raises the question of what controls nuclear
A Open mitosis
B Closed mitosis
G2–MAnaphase Interphase Interphase
Spindle pole body
Fig. 2. Open and closed mitosis. (A) Open mitosis is so named because of the disassembly of the NE (green) during mitosis, which opens up the nucleus and
exposes the chromosomes (red) to the cytoplasm. The NE breaks down early in mitosis, as the chromosomes condense, allowing microtubules (purple filaments)
that emanate from centrosomes (purple structures) to associate with the chromosomes. During mitosis, the chromosomes congress to the metaphase plate, followed
by separation of sister chromatids in anaphase. The NE begins to reassemble shortly thereafter, in telophase. Once the NE is completely assembled, the nucleus
expands and the chromosomes return to their decondensed state in interphase. (B) Closed mitosis is so named because of the persistence of the NE throughout the
cell cycle, such that the nucleus never ‘opens’ to the cytoplasm. This type of mitosis occurs in certain fungi (such as budding yeast, shown here), in which the
centrosome equivalents, called the spindle-pole bodies (purple), are embedded in the NE. During closed mitosis, the spindle-pole bodies nucleate microtubules
within the nucleus, but as the DNA (red) begins to segregate, the nucleus has to elongate. Once segregation is completed, the nucleus divides and re-establishes a
spherical shape. Note that, in budding yeast, chromosome condensation and a metaphase plate are not visible by microscopy.
Journal of Cell Science Journal of Cell Science
size: is it a mere consequence of increase in volume over time, or
are there factors that regulate or limit nuclear expansion? Decades
of observations suggest that the latter is true, and that nuclear and
cytoplasmic volumes are somehow related to each other; this
phenomenon is referred to as the karyoplasmic ratio (Gregory,
2005). Moreover, in both budding and fission yeasts, the ratio of
nuclear to cellular volume remains constant throughout the cell
cycle, even as cell volume increases (Jorgensen et al., 2007;
Neumann and Nurse, 2007). This suggests the existence of a
mechanism that links nuclear and cellular volumes. If indeed such
a mechanism exists, does cellular volume dictate nuclear volume,
or does nuclear volume determine cell volume? What cellular factors
determine nuclear volume? And finally, why is nuclear volume
Factors that affect nuclear volume
There are conflicting reports regarding the dominant cellular factors
that determine nuclear volume. One idea, known as the
nucleoskeletal theory, is that DNA content influences the volume
of the nucleus, which in turn influences the size of the cell
(Cavalier-Smith, 2005; Gregory, 2005). Intuitively, DNA may
affect nuclear volume, because the size of the nucleus could be
directly proportional to amount of DNA it contains and the extent
to which that DNA is compacted. Simply comparing genome size
to nuclear and cell volume among species supports this theory,
because species with larger genomes generally have larger nuclear
and cellular volumes (Cavalier-Smith, 2005; Jovtchev et al., 2006).
Experiments in mice also give credence to the nucleoskeletal theory:
it has been shown that tetraploid mouse embryos have nuclei that
are twice as large as those in a diploid control (Henery et al., 1992;
Henery and Kaufman, 1992).
However, other data suggest that genome size per se is not the
determining factor of nuclear size. Rather, it is likely that there is
a nuclear-scaling mechanism whereby nuclear volume is
proportional to, and determined by, the levels of one or more cellular
factors. Indeed, nuclear transplant experiments support this claim:
implanting a small hen erythrocyte nucleus into a HeLa cell results
in expansion of the nucleus to the appropriate size for its new
environment, without affecting DNA content (Harris, 1967).
Moreover, the nucleoskeletal theory does not explain why cells from
different tissues in a given organism have the same amount of DNA
but varied nuclear sizes (Altman and Katz, 1976). Studies in yeast
also contradict the notion that DNA content dictates nuclear and
cellular volumes (Jorgensen et al., 2007; Neumann and Nurse,
2007). In neither fission yeast nor budding yeast does nuclear
volume increase sharply during S phase, as would be expected if
DNA content had a direct affect on nuclear size (Jorgensen et al.,
2007; Neumann and Nurse, 2007). Furthermore, even a 16-fold
increase in ploidy does not affect nuclear size in fission yeast
(Neumann and Nurse, 2007). Instead, the displacement of nuclei
by centrifugation in multi-nucleated fission yeast showed that
nuclear size adjusted in proportion to the amount of surrounding
cytoplasm (Neumann and Nurse, 2007). These studies support a
mechanism whereby nuclear size is determined by cytoplasmic
volume rather than DNA content.
Assuming that cytoplasmic factors determine nuclear size, what
might these be? In cell-free extracts of Xenopus oocytes, an
increase in nuclear volume after NE reassembly requires an intact
ER (Anderson and Hetzer, 2007). This suggests that the membrane
for the newly formed NE is supplied by the ER, and therefore
membrane availability could be a limiting factor in determining
nuclear size. The ER exists as a continuous meshwork of membrane
sheets and membrane tubules. Proteins known as reticulons cause
tubule formation in the ER (Voeltz et al., 2006), and high levels of
reticulons are inhibitory to nuclear growth, which suggests that the
availability of membrane in the form of sheets can put an upper
limit on nuclear size (Anderson and Hetzer, 2008; Kiseleva et al.,
2007). Work in the Xenopussystem has demonstrated a requirement
for NPCs and nuclear import in nuclear growth after NE assembly
(D’Angelo et al., 2006; Newport et al., 1990), which suggests that
the import of one or more nuclear proteins contributes to sizing the
nucleus. Indeed, several nuclear lamina proteins that are transported
into the nucleus through the NPCs have been found to affect
interphase nuclear growth (e.g. Brandt et al., 2006; Dittmer et al.,
2007; Newport et al., 1990). However, many questions remain. For
example, how do yeast – which lack lamins and lamin-associated
proteins – adjust nuclear volume in response to changes in
cytoplasmic volume? Also, what is the mechanism, in any organism,
that establishes the upper limit to nuclear growth?
Does size matter for nuclear function?
Although the mechanisms that control nuclear volume remain
unclear, the existence of a karyoplasmic ratio suggests that nuclear
size is important for cell function. Disturbance of this ratio is
associated with certain types of cancers (Slater et al., 2005; Zink
et al., 2004), suggesting that the ratio between nuclear and
cytoplasmic volumes is crucial for cell integrity. Moreover, it has
been proposed that cell-cycle progression depends on nuclear size
(Roca-Cusachs et al., 2008; Yen and Pardee, 1979), and that cells
monitor the ratio between cytoplasmic and nuclear volume to gauge
Journal of Cell Science 122 (10)
A C. elegans
9-year old 96-year old
Fig. 3. Variation in nuclear shape. The nuclei of most cells, such as those of
the C. elegans embryo (A), are either oval or round. However, various cell
types or conditions display non-round nuclei. Shown are the nuclei of
neutrophils (B), of cells from a patient with HGPS (C) and of cells from a 96-
year-old individual (D, right panel) compared with nuclei of cells from a 9-
year-old individual (D, left panel). Visualization of nuclei was performed with
a GFP-tagged NPC component, NPP-1 (A), an antibody specific for lamin B
(B), an antibody specific for emerin (a lamina-associated protein, C) and an
antibody specific for lamin A and lamin C (D). The image in B was reprinted
with permission from Ada Olins and Donald Olins (Olins and Olins, 2005).
The image in C was reprinted with permission from Goldman (Goldman et al.,
2004). The images in D were provided by Tom Misteli and Paola Scaffdi
(NCI, Bethesda MD) (see also Scaffidi and Misteli, 2006). Nuclei are not
shown to scale.
Journal of Cell Science Journal of Cell Science
Building a nucleus
the proper time to enter the cell cycle (Futcher, 1996). In addition,
a strong correlation between nuclear size, RNA transcription levels
and cell size has been found (e.g. Sato et al., 1994; Schmidt and
Schibler, 1995). It is therefore possible that larger nuclei facilitate
the increase in transcription that is required in larger cells.
Additionally, the volume of the nucleus might be important for
maintaining nuclear compartments, such as the nucleolus, and the
activity of enzymes such as DNA polymerase, which are sensitive
to macromolecular crowding (Hancock, 2004; Miyoshi and
Sugimoto, 2008; Sasaki et al., 2006). An increasingly popular view
of molecular dynamics within the nucleus favors self-organization
of complex structures – a process that depends on biochemical and
physical interactions between numerous proteins (Misteli, 2001).
A recent example is the assembly of Cajal bodies, which are nuclear
structures involved in the biogenesis of small nuclear
ribonucleoproteins (snRNPs). Cajal bodies assemble by self-
organization through stochastic interactions between the building
blocks of which they are composed (Kaiser et al., 2008; Misteli,
2008). Because self-organization may be acutely sensitive to the
concentration of the individual components, the regulation of
nuclear volume might have an important role in enabling this
For over two decades, NE assembly has been studied using an
invaluable in vitro system based on Xenopus egg extract (Lohka
and Masui, 1983; Newport, 1987). This extract contains all of the
components that are necessary for the assembly of a functional NE
when mixed with demembranated sperm chromatin. In this system,
the NE assembles from vesicles that contain NE proteins. It was
assumed that these vesicles form during NE breakdown earlier in
the cell cycle. Moreover, NE assembly in vitro was shown to require
the small GTPase Ran, GTP hydrolysis, and the AAA ATPase p97,
which is involved in membrane fusion (Hetzer et al., 2001). These
observations led to the idea that the NE is assembled by the fusion
of mitotic vesicles that contain NE proteins. However, more recent
studies raise the possibility that such vesicles are a consequence of
ER fragmentation during extract preparation(Collas and Courvalin,
2000). Indeed, experiments in mammalian cells strongly suggest
that transmembrane NE proteins are not sequestered into vesicles,
but rather are incorporated into the ER during mitosis (Daigle
etal., 2001; Ellenberg and Lippincott-Schwartz, 1999;
Ellenberg et al., 1997; Yang et al., 1997). It is currently unclear
whether NE components disperse completely within the ER network
or whether they cluster into specific ER subdomains. Intriguing
models propose that NE proteins remain in ER subcompartments
during mitosis, and that these subdomains serve as the precursors
for NE reassembly. The requirement for GTP hydrolysis in vitro
can be explained by the need to form an intact ER before NE
assembly (Anderson and Hetzer, 2007). In addition, the requirement
for p97 can be explained by its ability to extract proteins from
chromosomes: chromatin-bound aurora kinase inhibits NE
formation, and p97 relieves this inhibition by extracting aurora
kinase from chromosomes in telophase (Ramadan et al., 2007).
Nonetheless, at present, the possibility that some vesicles are
involved in NE assembly cannot be ruled out.
Nuclear-envelope formation: from ER tubules to membrane
During mitosis in mammalian cells, a large portion of the ER is
converted into tubules (Puhka et al., 2007). Given that the membrane
for the NE comes from the ER, how are ER tubules converted into
an intact NE, which is essentially a membrane sheet? Data from
both in vitro and mammalian systems argue that early in telophase,
the tips of ER tubules contact chromatin, initiating the process of
NE assembly (Anderson and Hetzer, 2007; Anderson and Hetzer,
2008) (Fig. 4). This interaction requires neither energy nor the
cytosol, and is made possible by the fact that the multiple ER-
associated inner nuclear membrane proteins have a high affinity for
DNA (Anderson and Hetzer, 2007; Antonin et al., 2005; Mansfeld
et al., 2006; Ulbert et al., 2006). These proteins bind DNA early in
NE assembly, thereby recruiting ER tubules to chromatin. Following
this initial binding, the membrane tubules flatten into sheets, which
spread across the chromatin and re-organize into a sealed NE
(Anderson and Hetzer, 2007; Anderson and Hetzer, 2008) (Fig. 4).
Inner nuclear membrane proteins are also required for the sealing
steps (Anderson and Hetzer, 2007; Antonin et al., 2008; Chi et al.,
2007; Gorjanacz et al., 2007; Mansfeld et al., 2006), although the
exact mechanism by which they are involved remains to be
unveiled. In mammalian cells, membrane flattening is inhibited by
the overexpression of reticulons and made more efficient by
reticulon depletion, suggesting that the equilibrium between flat and
curved membrane is vital for this step (Antonin et al., 2008).
Reticulon depletion has also been shown to interfere with NE
disassembly in Caenorhabditis elegans (Audhya et al., 2007),
underscoring the importance of ER-membrane structure in NE
dynamics. The final step in membrane sealing requires closing the
Chromatin ER tubules NE
Fig. 4. Nuclear-envelope assembly. (A) A general view of NE assembly. Initial
contacts with the chromatin (black and gray bars) are thought to be made by
tips of ER tubules (blue-green). These tubules then flatten to form an intact
NE, which then expands and the chromosomes decondense. (B) A closer view
of NE assembly. The ER tubules are decorated with chromatin-binding NE
proteins (shown in multiple colors), which are thought to mediate the
interaction between the membrane and the chromatin (black). These proteins
are eventually located on the inner nuclear membrane. As NE assembly
progresses, the membrane flattens onto the chromatin (note the progressive
accumulation of chromatin-binding proteins at the interface between
the membrane and chromatin). Because the ER membrane is one continuous
membrane, the gap between two adjacent tubules will be filled by
this membrane-flattening process.
Journal of Cell Science Journal of Cell Science
holes that remain after the membrane expands across the chromatin
surface. The mechanistic details of this step are still a mystery. The
completion of NE assembly also requires the insertion of NPCs.
Although it is tempting to speculate that NPC insertion serves to
‘plug’ the holes left over from membrane flattening (Anderson and
Hetzer, 2007), there is currently no evidence that this is the case;
in addition, it is clear that NPCs can be inserted into intact NE, for
example, in cells that undergo closed mitosis (D’Angelo and
Hetzer, 2008). So how are NPCs introduced into the NE?
NPC insertion into the NE
Post-mitotic NPC assembly occurs in a step-wise process that begins
early in anaphase, with soluble NPC proteins positioning on the
chromatin even before membrane reformation, followed by the later
recruitment of transmembrane nucleoporins (e.g. Bodoor et al.,
1999; Dultz et al., 2008; Rasala et al., 2008); for a detailed
discussion, the reader is directed to two excellent recent reviews
(Antonin et al., 2008; D’Angelo and Hetzer, 2008). But NPC
addition is not restricted to cells undergoing open mitosis. The
number of NPCs increases during interphase of dividing cells, well
after NE assembly, and during yeast closed mitosis, indicating that
another mechanism must exist for insertion of NPCs into a fully
formed NE (Maul et al., 1971; Maul et al., 1973; Winey et al., 1997).
Studies in the cell-free Xenopus system revealed that interphase
addition of NPCs requires the addition of nucleoporins from both
the cytoplasmic and nuclear side of the NE (D’Angelo et al., 2006).
More recently, reticulon proteins have been implicated in NPC
assembly in yeast and Xenopus systems, presumably by inducing
curved membranes around the inserted NPC (Dawson et al., 2009).
This role is strikingly separate from their function in tubular ER
formation, and might reflect a novel use of their membrane-shaping
properties to form or stabilize the pore membrane (Dawson et al.,
2009). Biochemical analysis of Xenopus extract capable of post-
mitotic NPC addition unexpectedly uncovered a role for the major
vault protein, suggesting that the vault ribonucleoprotein also
facilitates membrane distortions required for NPC assembly (Vollmar
et al., 2009). Finally, D’Angelo and co-workers reported that the
transmembrane nucleoporins, which serve as the NPC scaffold, are
inserted into the NE only in dividing cells, although other NPC
subunits are exchanged with newly synthesized ones in nondividing
cells (D’Angelo et al., 2009). This implies that the NPC scaffold in
nondividing cells, such as neurons, must remain functional for years.
Indeed, NPCs of cells from old individuals are more ‘leaky’ than
NPCs of young individuals, suggesting that the permeability barrier
of NPCs deteriorates over time (D’Angelo et al., 2009).
Organizing the nucleus after NE assembly
Once a sealed NE complete with functional NPCs is formed, the
NE expands to its final size and shape. An interesting but still
unanswered question is how soon after NE assembly do
chromosomes organize into their characteristic nuclear positioning,
including chromosome territories and the peripheral localization of
heterochromatin? At least two scenarios are possible: in the first,
the chromatin is organized nonrandomly in telophase (for example,
with regions of heterochromatin facing the exterior of the telophase
chromatin mass), such that the reassembling NE makes contacts
preferentially with chromatin that will be targeted to the nuclear
periphery in the newly formed nucleus. In the second scenario, the
chromatin in telophase is not in a particular arrangement, and NE
assembly begins with the indiscriminate binding of membranes and
lamina proteins to whatever chromatin they encounter. If the latter
scenario is correct, then as the NE expands, the nuclear lamina must
eventually detach from the chromatin to which it is initially bound
and reattach to chromatin that is destined to be at the nuclear
periphery. Consistent with this idea, Thomson and colleagues
(Thomson et al., 2004) found that peripheral chromatin localization
is established during early G1 phase, after NE formation, suggesting
that remodeling of NE-chromatin binding takes place early after
nuclear expansion. However, this observation does not exclude the
possibility that some specific NE-chromatin interaction occurs at
the time of NE assembly. Whether the NE assembly process has
an inherent specificity to it – either in chromosome configuration
or the regions of initial NE-chromatin contact – awaits further
The formation of a single nucleus at the end of mitosis
The goal of NE reassembly is the construction of a single nucleus
of the appropriate size and shape. Factors that influence nuclear
shape and size are discussed above, but what determines the
formation of a single NE around the entire chromatin mass? In fact,
some organisms undergo stages of development that are
characterized by the initial formation of multiple smaller nuclei.
For instance, during early embryonic divisions of Xenopus, sea
urchins and polychaetes, separate NEs form around the individual
chromosomes, creating structures called karyomeres (Montag et al.,
1988). Each karyomere is capable of DNA replication and has a
complete NE with a nuclear lamina and functional NPCs (Lemaitre
et al., 1998). Once the blastula stage is reached, the karyomeres
fuse into a single nucleus. Why karyomeres form remains a
mystery; perhaps they facilitate rapid entry into S phase during the
extremely short cell cycles of early embryonic development
(Lemaitre et al., 1998; Lenart and Ellenberg, 2003). Equally
fascinating is the question of what dictates separate
compartmentalization of the chromosomes in the early embryo. Is
there a mechanism that holds chromosomes apart in order to prevent
their capture inside a single envelope? Relevant factors might
include proteins that are involved in chromosome compaction, the
length of the mitotic spindle in these extremely large cells and
thecopious lipids and membrane proteins that are synthesized during
early embryogenesis (Lemaitre et al., 1998). It is tempting to
speculate that changes in ER membrane structure (i.e. the ratio of
tubules to sheets) during development could also contribute, as
Multiple nuclei within single cells are also observed in disease
states and commonly occur as micronuclei in cancer cells.
Micronuclei result from either chromosome breakage or imperfect
mitosis, when a chromosome fragment or an entire chromosome
gets separated from the bulk of the DNA (Fig. 5) (Ford et al., 1988;
Norppa and Falck, 2003). Upon NE reassembly, the lagging DNA
is excluded from the nucleus and becomes encapsulated in its own
NE, complete with a nuclear lamina and NPCs (Walker et al., 1996).
Micronuclei spontaneously accumulate in lymphocytes in an age-
dependent manner, but they can also be triggered by environmental
factors, exposure to genotoxic chemicals (Norppa and Falck, 2003)
or depletion of factors required for chromosome segregation and
congression to the metaphase plate (e.g. Goshima et al., 2003; Salina
et al., 2003). Thus, the physical distance between chromosomes in
telophase is clearly important for the encapsulation of all of the
chromatin into a single NE.
Recent studies provide insight into how chromosomes might
achieve the tight packing that ensures formation of a single nucleus.
The highest degree of chromosome axial compaction occurs in late
Journal of Cell Science 122 (10)
Journal of Cell ScienceJournal of Cell Science
Building a nucleus
anaphase and requires dynamic microtubules and Aurora kinase.
Interfering with this compaction by inhibiting either of these factors
causes nuclear morphology defects, including the formation of
multi-lobed nuclei (Mora-Bermudez et al., 2007). Interestingly, this
defect in compaction did not result in the formation of multiple
nuclei, suggesting that other factors are involved in either
maintaining chromosome proximity or otherwise limiting the
formation of multiple nuclei. More recently, the chromokinesin Kid
was reported to be required for maximum compaction of anaphase
chromosomes. When Kid was depleted from HeLa cells,
chromosome compaction was altered, leading to the formation
multi-lobed, wrinkled nuclei. The phenotype was even more severe
in Kid–/–mouse zygotes, including the formation of multiple
micronuclei (Ohsugi et al., 2008). Whereas Kid affects chromosome
compaction in each cell division, it is required to prevent the
formation of micronuclei only in oocyte meiosis and in the first
few mitotic divisions. This is curious, and might suggest that
extreme chromosome compaction is uniquely required to prevent
the formation of multiple nuclei during the first few cell cycles.
This further suggests that there are other, yet unidentified, factors
involved in ensuring that only a single nucleus is formed at the end
The formation of multiple nuclei is also seen in C. elegans after
depletion of the nucleoporin gp210, cyclin B, the GTPase Rab-5
or reticulon proteins (Audhya et al., 2007; Galy et al., 2008;
Sonnichsen et al., 2005). Interestingly, a subset of these conditions
also disrupts ER structure. Multiple nuclei are also formed after
depletion of LPIN-1, the C. elegans homolog of lipin, an enzyme
that is involved in fat metabolism and lipid synthesis (Golden et al.,
2009). Similarly to the effect of Rab-5 or reticulon depletion,
downregulation of LPIN-1 expression disrupts ER structure (Golden
et al., 2009; Gorjanacz and Mattaj, 2009). Thus, although the
distance between chromosomes clearly constitutes an important
consideration in the formation of a single nucleus during telophase,
the amount of membrane available, and its potential to adopt proper
sheet and tubule structures, might also have an important role.
The limited flat membrane hypothesis
The above observations regarding NE assembly led us to propose
the ‘limited flat membrane hypothesis’ (Fig. 6). According to this
hypothesis, the limiting factor for the surface area of the NE is the
amount of ER membrane that can be converted into membrane
sheets, and it is this requirement that contributes to the formation
of a single nucleus at the end of open mitosis, or the formation of
a round nucleus after closed mitosis. This hypothesis is based on
the following suppositions: first, that there is a constant ratio
between nuclear size and cell size; second, that the membrane used
for NE formation originates in the ER; and third, that only a fraction
of the ER membrane – that which is not captured in specialized
structures such as tubules – is available for NE formation. There
is good evidence in support of the first two suppositions, whereas
the third is more speculative and is based on the observed effects
of altered reticulon levels and altered lipid synthesis on NE
formation (described above).
In the case of closed mitosis, altered lipid synthesis due to the
inactivation of the lipin pathway results in extensive ER membrane
sheets and the inability to form a spherical nucleus after nuclear
division (Campbell et al., 2006; Siniossoglou et al., 1998; Tange
etal., 2002). Because lipid synthesis is decreased as cells exit mitosis
(Santos-Rosa et al., 2005), we propose that, in closed mitosis, limited
lipid synthesis, and specifically limiting amounts of membrane in
the form of sheets (i.e. ‘flat’ membrane), drives the nuclear shape
change from elongated to round. When the amount of flat membrane
is not limiting, such as when lipin is inactive, this transition does
not occur (Fig. 6). To see how this hypothesis applies to open
mitosis, let us assume that, at the time of NE reassembly, a
membrane can form around all of the chromosomes, leading to the
formation of a single nucleus, or it can form around a subset of
chromosomes, leading to the generation of multiple nuclei that
together can expand to the same volume as a single nucleus.
Importantly, in the latter case, the combined surface area of the
multiple nuclei would be greater than the surface area of a single
nucleus with the same volume. If the amount of flat membrane is
limited and much of the ER is captured in the form of tubules, the
NE assembly reaction will be pushed towards the formation of a
single nucleus. If this were the case, it would explain the observation
that multiple nuclei are formed under a variety of conditions in
which more flat membrane is available. Additionally, it is tempting
to speculate that in cancer cells the ratio of ER sheets to tubules is
altered, or that the rules that link nuclear volume to cell size are
relaxed, thereby facilitating the formation of multiple nuclei.
Further research into the relationship between ER structure, lipid
synthesis and NE dynamics will be useful for testing the validity
of this hypothesis.
Conclusions and Perspectives
The existence of diseases associated with altered nuclear shape and
size underscores the importance of uncovering the mechanisms that
control NE dynamics. In the past few years, the field of nuclear
architecture has witnessed an explosion of knowledge, from the
understanding of how nuclear lamina proteins function to the basic
principles of NE assembly. We still need to determine the link
between nuclear shape and nuclear function, and to distinguish
between cases where an altered cellular function is a direct
consequence of altered nuclear shape and those where both altered
function and shape are independent consequences of defects in a
particular structure (e.g. the nuclear lamina). Equally interesting is
how nuclear size is determined; although a link between nuclear
size and cytoplasmic volume has been suspected for many years,
recent studies in yeast have introduced a tractable genetic system
in which this question could be answered. Likewise, mutant and
RNAi screens in other organisms could uncover proteins that are
involved in nuclear size determination. Finally, the finding that NE
assembly begins with ER tubules, rather than vesicles, has opened
a new avenue of investigation that is focused on understanding the
properties of the ER and of the proteins that contribute to ER-
membrane dynamics. In the coming years, it is likely that we will
learn more about the relationship between the ER and NE dynamics;
specifically, how the balance between membrane tubules and
membrane sheets is maintained and regulated, and how lipid
synthesis contributes to both ER and NE structure.
We thank Will Prinz, Paula Fearon, Daphna Joseph-Strauss (National
Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda,
MD), and Donald Olins and Ada Olins (Bowdoin College, Brunswick
ME) for comments on the manuscript. We also thank Tom Misteli and
Paola Scaffidi (National Cancer Institute, Bethesda, MD) for generously
providing the images in Fig. 3D, and Ada Olins and Donald Olins for
allowing us to use the image in Fig. 3B. M.W., K.L.W. and O.C.F. are
funded by an intramural grant from the National Institute of Diabetes
and Digestive and Kidney diseases.
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Commentaries and Cell Science at a Glance
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Mammalian macroautophagy at a glance David Rubinsztein
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WASP and SCAR/WAVE proteins – the drivers of actin assembly Robert Insall
The ESCRT machinery at a glance – Jim Hurley
DUBs at a glance Keith Wilkinson
Podosomes and invadopodia at a glance Stefan Linder
Collective cell Migration at a glance Peter Friedl
Establishment and maintenance of DNA Methylation Patterns Tim Bestor
mTOR signalling at a glance David Sabatini
Molecular mechanisms of clathrin-independent endocytosis Ben Nichols
Ena/VASP function – a pointed controversy at the barbed end Frank Gertler
Semaphorin signalling Luca Tamagnone
How do ESCRT proteins control autophagy? – Harald Stenmark
How peroxisomes multiply Ewald Hettema
An update on nulcear calcium signalling Martin Bootman
Mechanisms for transcellular diapedesis Chris Carmen
The Crumbs complex Elisabeth Knust
ABL-family kinases Tony Kolesky
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