Nuclear Organization (Nuclear Structure and Dynamics)

Y Mimura and N Imamoto, RIKEN, Saitama, Japan

r 2016 Elsevier Inc. All rights reserved.

Introduction daughter chromosomes with the aid of protein–protein and

protein–DNA interactions.
In eukaryotic cells, unlike prokaryotic cells that contain naked In this section, we briefly describe chromatin organization,
DNA in their cytoplasm, chromosomes composed primarily of the structure and function of the NE, and the mitotic dynamics
DNA and histones are wrapped with double lipid bilayers and of the nucleus.
are thus separated from the cytoplasm. This structure con-
taining the genetic information is called the nucleus, and the
Nuclear Structure and Function
double lipid bilayer wrapping the chromosomes is called the
nuclear envelope (NE). The nucleus, in which essential cellular
Chromatin
processes such as replication, transcription, and ribosome
biogenesis are constantly occurring, is essential for basic cel- All of the cells that compose our body contain the same base
lular activities and for maintaining cellular homeostasis. sequences of DNA in their nuclei. In humans, out of the entire
The NE is mainly composed of four components: the nu- genome, approximately 20 000 genes encode proteins. To
clear membrane, the nuclear pore complex (NPC), the nuclear generate the various organs such as brain or muscle from the
lamina, and inner nuclear membrane (INM) proteins. The NE fertilized egg that is a large single cell, gene expression must be
separates the nuclear contents from the cytoplasmic activities, regulated spatially and temporally through the developmental
which necessitates their mutual communication through the processes to build up specific gene expression patterns. To this
continuous exchange of molecules. The NPC, embedded in the end, cells potentially have many highly ordered and hier-
NE, mediates nucleocytoplasmic molecular transport. The NE archical mechanisms generating the diverse tissues via the
also plays a role in protecting genomic DNA from toxic me- structural alterations of the chromatin through histone ex-
tabolites produced in the cytoplasm, such as reactive oxygen change, posttranslational modifications of histones, and DNA
species produced in mitochondria. Along with these functions, modifications.
the NE is considered to regulate gene expression, DNA repli- Eukaryotic DNA, whose length is approximately 2 m in
cation, and chromatin organization. human, is wrapped around cores of histone octamers that are
The nuclear interior is a well-ordered structure in which composed of histone H2A, H2B, H3, and H4 (Luger et al.,
individual chromosomes are nonrandomly arranged into the 1997). In this way, all eukaryotic DNA is highly compacted in
particular nuclear space, termed as the chromosomal territory. the nucleus. Histone H2A exclusively forms a heterodimer
Although the mechanism for the establishment of the with histone H2B, while Histone H3 forms a heterodimer with
chromosome territory remains elusive, the interaction between Histone H4. A core histone octamer is assembled from two
the chromosome and the NE is considered to play an essential pairs of these heterodimers. The approximately 150 base pairs
role in the establishment of the chromosome territory. In of DNA wrapped the core histone octamer is called a nucleo-
addition, specialized domain structures, such as the nucleolus, some, and it is the minimum module of chromatin structure.
Cajal body, and promyelocytic-leukemia nuclear body (PML- The nucleosome is formed in a stepwise manner. First, a pair
NB) are formed in the nuclear interior and dynamically of histone H3–H4 dimers wraps the DNA, and this inter-
interact with each other to coordinate nuclear functions. mediate is called a ‘tetrasome.’ Next, a pair of histone
Finally, it is also important to note that the nucleus is a H2A–H2B dimers is further incorporated into the tetrasome
dynamic structure. During the cell cycle, DNA in chromo- and consequently matured into a complete nucleosome
somes must be replicated, and the replicated chromosomes structure. These nucleosome assembly steps require the aid of
must be precisely separated to transmit genomic information a specific set of histone chaperones (De Koning et al., 2007;
into daughter cells. Chromosome separation occurs during Kurumizaka et al., 2013).
mitosis. In multicellular organisms, the nuclear structure is Core histones without histone H4 contain many variants
broken down at the mitotic entry. The NE–chromosome that are structurally distinct. Depending on the several
interaction is lost, and the NE is retracted into the endoplasmic cellular contexts (i.e., developmental processes, transcription,
reticulum (ER). The chromosomes are highly condensed, and and DNA damage response), the core histones are dynamically
a kinetochore is assembled on the centromere, which is a and appropriately exchanged for other histone variants via
specific region in the chromosome. Centrosomes are dupli- specific histone chaperone activity. As a result of alterations of
cated in interphase, and mitotic microtubules form con- nucleosome structure via histone exchange, the cell can flexibly
nections between the centrosome and kinetochore. Together respond to internal and external stimuli (Volle and Dalal,
with spatially and temporally well-orchestrated mitotic activ- 2014).
ities, such as kinases, phosphatases, nucleocytoplasmic trans- In addition to histone replacement, another important
port machineries, and motor proteins, the chromosomes are regulatory mechanism of chromatin for the response to many
segregated with the sophisticated machinery termed the mi- cellular processes is posttranslational modifications described
totic spindle into the daughter cells. In late mitosis, the NE as ‘epigenetics.’ Histone proteins can be structurally separated
that was retracted into the ER is reassembled on the surface of into the tail and core regions, and both regions contain many

Encyclopedia of Cell Biology, Volume 2 doi:10.1016/B978-0-12-394447-4.20027-8 311

312 Organelles: Structure and Function: Nuclear Organization (Nuclear Structure and Dynamics)

Cytoplasm

Nuclear pore complex Cytoskeleton

Cytoplasmic filaments

Cytoplasmic ring
Nesprin
Spoke ring ONM

SUN−KASH LBR
MAN1 LAP2 LEM2
Emerin interaction
INM

BAF
SUN1/2
Nuclear
ring LEM domain proteins Linc complex
Heterochromatin
Central Nuclear lamin
channel

Nuclear basket

Nucleoplasma

Figure 1 Structure of metazoan nuclear envelope (NE). Schematic representation of the overview of NE. The nuclear pore complex is embedded
in the NE composed of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). INM proteins containing LEM proteins, SUN1/
2 and Lamin B receptor (LBR) exclusively localized to the INM. SUN1/2 and Nesprin form linker of nucleoskeleton and cytoskeleton (LINC)
complex via SUN–KASH interaction, and connect the nucleoskelton to cytoskeleton through their interaction. Heterochromatin is accumulated
underneath the INM via the interaction with INM proteins and the nuclear lamina.

sites that accept various posttranslational modifications, par- proteins, either directly or through association with lamins,
ticularly phosphorylation, acetylation, and methylation. DNA, interact with chromatin. The ONM is fused with the INM at sites
especially cytosine residues, is also methylated via DNA termed pore membrane at which the NPC is present. The NE
methyltransferase activities. Many of these modifications of functions as the physical barrier that protects the nuclear interior
histones are reversible, but it remains unknown how methyl- from the cytoplasmic activities (Figure 1).
ated cytosine is eliminated from DNA (Cantone and Fisher,
2013). Epigenetic regulation through the posttranslational Nuclear lamin
modifications of histones and DNA are considered to affect In metazoans, the nuclear lamina, which is a meshwork
gene expression by altering the interactions between histones, structure, is composed of two types of lamin proteins termed
DNA, nucleosomes, and other proteins, including DNA- A-type and B-type lamins. Both A-type and B-type lamins be-
binding proteins. Accommodative functions with both pro- long to the type V intermediate filaments (IFs), and the nuclear
cesses, histone exchange and DNA/histone modifications, are lamina confers physical strength to the NE. Interestingly,
required mechanisms for development, homeostasis and in- lamins are highly evolutionarily conserved in metazoans such
heritance (Sasaki and Matsui, 2008). Conversely, misregula- as vertebrates but are not conserved in plants and yeast. Lamin
tion of these mechanisms is closely associated with several A, Lamin C, Lamin C2, and Lamin AD10 belong to the A-type
critical diseases, such as cancer and leukemia, as well as with lamins and are encoded by the single LMNA gene (Burke and
aging (Greer and Shi, 2012; Chi et al., 2010). Stewart, 2013), while Lamin B1 and Lamin B2 belong to the
B-type lamins and are encoded by separate genes, LMNB1 and
LMNB2, respectively. Lamin C is a C-terminus truncated form
of Lamin A from an alternative splicing of the LMNA product.
NE
At least one form of B-type lamin is expressed in each cell,
Nuclear membrane whereas the expression of A-type lamins is regulated in tissue-
The nuclear membrane is comprised of two phospholipid specific and development-dependent manners.
bilayers. The membrane facing the cytoplasm is termed the Both A-type and B-type lamins share the same structural
outer nuclear membrane (ONM), and the membrane facing the features, including head and α-helical rod domains in the
nucleoplasm is termed the INM. The ONM continuously con- N-terminus. Although all tail regions in Lamin proteins con-
nects to the ER, and its surface, like that of the ER, is decorated tain the nuclear localization signal (NLS), their tail regions
with ribosomes. The INM accumulates specific sets of proteins show structural differences. After translation, Lamin proteins
collectively termed INM proteins, and A-type and B-type lamins mature through a series of posttranslational processes. The
construct the nuclear lamina underneath the INM. Many INM carboxy-terminals of Lamin A, Lamin B1, and Lamin B2, but
 Organelles: Structure and Function: Nuclear Organization (Nuclear Structure and Dynamics) 313

not Lamin C, contain the CAAX motif that is also conserved in accumulation of DNA damage, and premature senescence. It is
the Ras superfamily (C: Cysteine, A: aliphatic amino acid, X: considered that these cellular defects are caused by the ex-
any amino acid). Initially, cysteine residues in these motifs of pression of ‘farnesylated progerin’ because FTase inhibitors can
pre-Lamins are posttranslationally modified by farnesyl- inhibit these cellular defects in HGPS patients.
transferase (FTase) that transfer the 15-carbon farnesyl lipid to
cysteine, and the isoprenyl groups are transferred to its side NPC
chain (Roskoski, 2003). These posttranslational modifications The NPC, one of the largest protein complexes in the cell, has
require the presence of Zn2 þ and Mg2 þ ions. Following the structurally and functionally well-conserved architecture in
isoprenylation, pre-Lamin A is cleaved at the carboxy-terminal eukaryotic cells from yeast to mammals. The NPC is assembled
end of the isoprenylcysteine residue in this motif by zinc from approximately 30 distinct proteins termed nucleoporins
metalloprotease ZMPSTE24, which is embedded in the NE/ER (Nups), and it has an eight-rotational asymmetric structure
membrane via its 7-transmembrane regions and specifically forming a tunnel between the cytoplasm and the nucleoplasm
recognizes the isoprenylcysteine residue (Quigley et al., 2013; (Figure 2). The hollow space in the NPC structure is filled with
Pryor et al., 2013). Meanwhile, pre-Lamin B is cleaved via many disordered/unstructured polypeptides containing
farnesylated proteins-converting enzyme2 (FACE2) at the hydrophobic amino acids, collectively called FG repeats
same site as pre-Lamin A. After removing the AAX tripeptide, (phenylalanine glycine repeats). This hollow is called the
isoprenylcysteine residues in the carboxyl terminal of a pre- central channel, which is a site of the nucleocytoplasmic traf-
Lamins becomes methylated via isoprenylcysteine carboxyl ficking of molecules. Relatively small molecules of less than
methyltransferase (ICMT) that also contain the multi-trans- 50 kDa can freely pass through the NPC, while larger mol-
membrane regions and is embedded in the ER membrane ecules above 50 kDa cannot do so. This size-dependent ex-
(Winter-Vann and Casey, 2005). It is hypothesized that this clusion mechanism of the NPC is called a ‘permeability
isoprenyl group on Lamin B1 and B2 aids the Lamin barrier,’ and the hydrophobicity in the central channel plays
B-phospholipid membrane attachment via a hydrophobic an essential role in the molecular transport that is a funda-
interaction. Only pre-Lamin A undergoes secondary digestion mental function of the NPC. In general, macromolecules
via ZMPSTE24 on the INM, leading to the removal of the 15 containing specific amino-acid signal sequences, the NLS and
polypeptides in its carboxy terminal, including the iso- the nuclear export signal (NES), can pass through the NPC in a
prenylated cysteine. This series of processes produces mature nuclear transport receptor (NTR)-dependent manner.
Lamin A. Nups can be roughly categorized into four broad types by
Both A-type and B-type lamins form homodimers as min- their structural roles: scaffold Nups, transmembrane Nups,
imum modules for the construction of the nuclear lamina via central Nups, and peripheral Nups. Several Nups comprises
α-helical coiled-coil regions within a rod domain. Similar to subcomplexes throughout the cell cycle. For example, in
other IF family proteins, filament-like structures are formed by mammalian cells, one of these subcomplexes, the NUP107-
head-to-tail self-association of these homodimers both in vivo 160 subcomplex, is composed of NUP107, NUP160, NUP133,
and in vitro. The precise overall structure and organization of NUP96, NUP75, NUP43, NUP37, Seh1, and Sec13, and the
the nuclear lamina is, however, an ongoing debate (Burke and other, the NUP93-205 subcomplex, is composed of NUP93,
Stewart, 2013). The nuclear lamina certainly provides the NUP205, NUP188, NUP155, and NUP53. The NUP107-160
platform for a specific set of integral-membrane proteins, subcomplex has a Y-shaped structure, and it forms ring-like
termed INM proteins, and specific DNA domains, termed substructures at both the nucleoplasmic and cytoplasmic sides
lamin-associated domains (LAD). (termed the nucleoplasmic ring and the cytoplasmic ring, re-
Several human diseases associated with NE components, spectively). The NUP93-205 subcomplex is also assembled in
including both lamins and INM proteins, are collectively a ring-like substructure, termed a spoke–ring complex, that
called ‘laminopathies’ (see also INM proteins section). Com- adjoins the cytoplasmic and nucleoplasmic rings. The
pared with LMNB1 and LMNB2, a huge number of mutations spoke–ring complex anchors the central Nups to form the
in LMNA have been reported to be associated with lamino- central channel (Grossman et al., 2012).
pathies. Depending on the specific mutation, patients har- Transmembrane Nups, NDC1, POM121, and GP210 in
boring mutations in LMNA associated with laminopathies mammalian cells, contain transmembrane regions and are
show various symptoms, including cardiomyopathy, muscular embedded into the pore membrane. Because transmembrane
dystrophy, lipodystrophy, mandibulofacial dysplasia, and Nups have an affinity for both NUP107-160 and NUP93-205
progeria syndrome. One of the most famous and characterized scaffold subcomplexes, this group of Nups is considered to
LMNA-associated laminopathies is Hutchinson-Gilford pro- anchor scaffold substructures to the pore membrane.
geria syndrome (HGPS) (Butin-Israeli et al., 2012). Patients Central Nups, which consist of NUP54, NUP58, and
with HGPS show various clinical manifestations including NUP62, form a subcomplex and play a critical role in the
premature aging, loss of subcutaneous fat, and atherosclerosis. formation of the central channel. These three Nups contain FG
Cells derived from HGPS patients express an abnormal Lamin repeats and confer the relatively hydrophobic environment to
A protein called progerin, which is an immature Lamin A the central channel. This hydrophobicity of the central channel
protein containing an uncleaved farnesyl group in its C-ter- plays the central role in the establishment of the permeability
minus, due to a point mutation in the LMNA gene (C1828T). barrier and macromolecular transport.
Although the progerin protein shows normal localization to Other Nups are categorized as peripheral Nups. This group
the INM, cells from HGPS patients show nuclear blebbing and of Nups is also associated with the establishment of the per-
biological activity defects such as the loss of heterochromatin, meability barrier and molecular transport because of FG-repeat
314 Organelles: Structure and Function: Nuclear Organization (Nuclear Structure and Dynamics)

Cytoplasmic filament
NUP358
hGC1
Aladin
NUP214 NUP98 Permeability barrier Peripheral nups
NUP88 Rae1
NUP107 NUP160
NUP133 NUP96 Scaffold nups
NUP85 NUP43 Cytoplasmic ring
ONM NUP37 Sec13 Seh1

POM121 NUP93 NUP62 Scaffold Transmembrane

NUP205 NUP54 nups
NDC1 NUP188 NUP58 Nups
NUP155 Central Spoke
GP210 NUP53 nups ring
INM NUP107 NUP160
ELYS

NUP133 NUP96 Scaffold nups

NUP85 NUP43 Nuclear ring
NUP37 Sec13 Seh1
NUP153 NUP98
Rae1
NUP50

t
ske
ba
TP

ar
R

cle
Nu

Figure 2 Metazoan nuclear pore complex (NPC) components. Schematic representation of the distribution of Nups whitin the NPC. Scaffold Nups
and transmembrane Nups are indicated with red and green colors, respectively. Central Nups and peripheral Nups are indicated with blue and gray
colors, respectively. The peripheral Nups that associate with cytoplasmic filament and nuclear basket are indicated with brown color. Note that this
Nups distribution within the NPC is experimentally predicted model so far.

containing peripheral Nups. These Nups form two types of ‘post-mitotic NPC assembly’ (Wandke and Kutay, 2013). By
substructures on both the cytoplasmic and nucleoplasmic contrast, during interphase, NPCs are embedded in the grow-
sides: cytoplasmic fibrils and nuclear baskets. Cytoplasmic fi- ing and closed NE. For this reason, the INM and ONM must be
brils are filamentous structures that are mainly composed of fused via unknown mechanisms to form the pore membrane
NUP358/RanBP2. Nuclear baskets are basket-like structures (Rothballer and Kutay, 2013).
that are mainly composed of NUP153 and TPR. Both sub-
structures participate in the regulation of transport receptor- INM proteins
dependent macromolecular exchange (Hoelz et al., 2011). INM proteins (also called as nuclear envelope transmembrane
In proliferating cells, NPCs are constantly assembling (NET) proteins) are defined as preferentially INM-localized
throughout the cell cycle. At the mitotic entry, NPCs are highly proteins, and they contain several transmembrane regions. The
phosphorylated via several mitotic kinases, and they are con- INM proteins construct the nuclear lamina together with the
sequently broken down into subcomplexes (see section NE nuclear lamins. The total number of INM proteins is reported
disassembly and reassembly in Mitosis). After the onset of as nearly 200 proteins, and the number of identified INM
anaphase, Nups are dephosphorylated via protein phos- proteins continues to increase (Wilson and Berk, 2010; Berk
phatases (PPases), and they then assemble into NPCs on the et al., 2013). Most INM proteins remain uncharacterized, and
surface of daughter chromosomes. This process is termed only a small number of INM proteins have been thoroughly
 Organelles: Structure and Function: Nuclear Organization (Nuclear Structure and Dynamics) 315

investigated. The INM proteins that have been investigated interact with cytoplasmic actin, microtubules, centrosomes, or
interact with either A-type lamin, B-type lamin or both, and IFs via the long N-terminal cytoplasmic region that contains a
they regulate processes including heterochromatin pos- large number of spectrin repeats (Rajgor and Shanahan, 2013).
itioning, signal transduction, and the nucleo-cytoskeleton via After the spectrin repeats, all Nesprin proteins contain a single
protein–protein and protein–DNA interactions (Wilson and transmembrane region and a luminal KASH domain in their
Foisner, 2010). The interaction between INM proteins and the C-termini. In the luminal region in the NE, the SUN domain
nuclear lamina contribute, at least in part, to the retention of rigidly and stably binds with KASH domain, thereby forming
INM proteins at the INM. the LINC complex (Horn, 2014).
The lamina-associated polypeptide (LAP) family was ori- Laminopathies are also caused by the mutation of genes
ginally defined as integral-membrane proteins that localize to encoding lamin-associated proteins. Patients harboring mu-
the INM and bind to the nuclear lamina (Senior and Gerace, tations in the Emerin gene show X-linked Emery-Dreifuss
1988). Presently, LAP family proteins also include nuclear muscular dystrophy (X-EDMD), and mutations in SYNE1 or
lamina-binding proteins that do not contain transmembrane SYNE2, encoding Nesprin1 and Nesprin2, respectively, also
regions, such as LAP2α (Wilson and Foisner, 2010), but all induce EDMD (Horn, 2014). Other mutations in the Emerin
LAP family proteins are localized to the INM. gene cause limb-girdle muscular dystrophy, cardiomyopathy,
Other characterized nuclear lamina-binding proteins con- or familial atrial fibrillation (Wilson and Foisner, 2010). The
tain LEM (LAP2, Emerin, MAN1) domains, which directly LEM domain-binding protein BAF causes Nestor-Guillermo
interact with a protein called Barrier to Autointegration Factor progeria syndrome (Berk et al., 2013), and LBR mutations are
(BAF). BAF binds with DNA with high affinity, and it also associated with hydrops-ectopic calcification-moth-eaten/
binds with core histones, linker histones, nuclear lamina Greenberg skeletal dysplasia and Pelger–Huët anomaly.
proteins, and BAF itself (Margalit et al., 2007). LEM domain
proteins therefore interact with chromatin through BAF, and
they also interact with chromatin directly. Because of these NE Disassembly and Reassembly in Mitosis
properties, LEM domain-containing proteins play an import-
‘Open’ and ‘closed’ mitosis
ant role in anchoring the INM to chromatin. Another chro-
Mitosis is the most important cellular process for dividing
matin-anchoring INM protein is Lamin B receptor (LBR). LBR
cells. The genetic information in the eukaryotic nuclei must be
is a transmembrane protein that contains an N-terminal
accurately transmitted to daughter cells by the mitotic spindle,
nucleoplasmic region and eight membrane-spanning regions
which is mainly composed of microtubules and centrosomes.
(Olins et al., 2010). LBR constantly associates with B-type
The mitotic spindle is generally assembled such that the
Lamins via its N-terminal nucleoplasmic region. In addition to
microtubules, extending from the centrosomes and other
its interaction with B-type Lamin, the N-terminal nucleo-
microtubule-organizing centers, capture the kinetochores
plasmic region also interacts with components of hetero-
formed at the centromeres of mitotic chromosomes
chromatin such as core histones, heterochromatin protein 1
(O’Connell and Khodjakov, 2007).
(HP1), and heterochromatin methyl-binding protein 1
Eukaryotic cells have two general styles of mitosis. One is
(MECP1).
‘closed mitosis,’ in which the NE is sustained through mitosis,
Importantly, the nuclear lamina is linked with micro-
and the other is ‘open mitosis,’ in which the NE is completely
tubules and actin filaments present in the cytoplasm. The
broken down (Fernandez-Martinez and Rout, 2009; Sazer
skeletal structures of the nucleoplasm (nucleoskeleton) and
et al., 2014). Yeast cells exhibit closed mitosis. They embed the
cytoplasm (cytoskeleton) are linked through a protein com-
spindle pole body (SPB), the microtubule-organizing center,
plex called linker of nucleoskeleton and cytoskeleton (LINC)
in the NE. This allows mitotic spindle assembly in yeast
complex. This connection between the nucleoskeleton and the
without NE breakdown (NEBD). By contrast, in multicellular
cytoskeleton is important for the intracellular positioning of
organisms that exhibit open mitosis, the microtubule-organ-
the nucleus, centrosome tethering, the regulation of the dis-
izing center, the centromere, resides in the cytoplasm. In these
tance between the INM and ONM, and the transduction of
cells, the NE must be broken down at the onset of mitosis so
extracellular mechanical stress. The LINC complex is com-
that the kinetochores can be captured by the microtubules
posed of SUN (Sad1 and UNC-84) domain-containing pro-
extending from the centromeres. In late mitosis, the NE that
teins on the INM and KASH (Klarsicht, ANC-1, Syne
was broken down must be reassembled on the surface of
homology) domain-containing proteins on the ONM. In
segregated daughter chromosomes (Anderson and Hetzer,
mammals, at least five SUN domain-containing proteins have
2008; Wandke and Kutay, 2013).
been discovered: SUN1, SUN2, SUN3, SUN4/SPAG4, and
SUN5/SPAG4L. Among these proteins, SUN3 and SUN4/
SPAG4 and SUN5/SPAG4L show tissue-specific expression in RanGTP gradient in interphase and mitosis
the testis (Sosa et al., 2013). SUN1 and SUN2 each contain a During interphase, large numbers of macromolecules are
single transmembrane domain and a C-terminal SUN domain, bidirectionally transported between the cytoplasm and the
which is located in the luminal region of the NE. SUN1 and nucleus by NTRs such as the importin β superfamily (Clarke
SUN2 are exclusively localized to the INM, and they directly and Zhang, 2008). This bidirectional macromolecular trans-
bind to Lamin A at the nucleoplasmic side. In mammals, six port is driven by the small GTPase Ran. The GTP-bound form
KASH domain-containing proteins have been reported; among of Ran (RanGTP) is concentrated at the nuclear interior, where
them, NE spectrin-repeat proteins 1–4 (Nesprin1-4) are well the nucleotide exchange factor regulator of chromosome
characterized for LINC complex formation. Nesprin proteins condensation 1 (RCC1) exclusively binds to chromatin and
316 Organelles: Structure and Function: Nuclear Organization (Nuclear Structure and Dynamics)

(1) Cargo recognition

Mitosis Interphase

(1) Cargo recognition 
Microtubule

Spindle binding
inhibition

 (5) Nucleotide
GTP GTP
hydrolysis

(2) Cargo (6) RanGTP (4) NTR (2) Cargo

(4) Microtubule GTP inport
transport inport export
nucleation
Cytoplasm
and
organization

RCC1 GTP (3) Cargo

(3) Release RanGEF release GTP
and GTP
GTP
Mitotic chromosome deposition RCC1 GTP GTP GTP
GTP RanGEF GDP GTP

Nucleotide Nucleotide
exchange exchange

Nucleoplasm

(a) (b)

SAFs
Importin  Importin  CAS Ran NTF2 RanGAP1 NPC
cargo

Low High
RanGTP gradient

Figure 3 Importin α/β and Ran system in Interphase and Mitosis. (a) RanGTP and NTRs spatially regulate the spindle microtubule assembly via
binding of spindle assembly factors (SAFs) to chromosome. (1) SAF harboring the nuclear localization signal (NLS) is recognized with nuclear
transport receptor (NTR) such as Importin α/β, and they form a transport complex. The interaction of SAF (in this case, Tahara, K., Takagi, M.,
Ohsugi, M., et al., 2008. Importin-β and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.
Journal of Cell Biology 180, 493–506) with NTR inhibits SAF’s chromatin binding activity. (2) and (3), When the transport complex reaches mitotic
chromosome, it disassembly is triggered by GTP-bound form of Ran (RanGTP) bound to NTR. (4) SAF released from transport complex
accumulate on the chromosome, and promote the spindle formation. RanGTP is constitutively generated via RCC1 residing on chromosomes, thus
high concentration of RanGTP exist at the vicinity of chromosomes. (b) RanGTP and NTRs regulate the nuclear import of cargo proteins harboring
the NLS. (1) By the same rule as (a), the cargo protein is recognized by NTRs, and they form a transport complex. (2) The transport complex pass
through the nuclear pore complex (NPC), and enters the nuclear interior. (3) At the nuclear interior, RanGTP binds to the NTR and triggers
disassembly of the transport complex. As a result, the cargo protein is released from the complex. (4) After releasing the cargo, the NTR binds to
RanGTP and exit the nucleus through NPC. (5) In cytoplasm, GTPase activity of RanGTP bound to NTR is activated by the aid of RanGAP.
Consequently, GTP on Ran is immediately hydrolyzed into GDP. (6) RanGDP generated by RanGAP in the cytoplasm is recognized by NTF2, and is
imported into the nucleus. At the nuclear interior, GDP on Ran is replaced to GTP by RCC1 activity.

constantly replaces GDP with GTP on Ran. In the cytoplasm, GTP on Ran to GDP in the cytoplasm, and the export complex
RanGAP1 (RanGTPase-activating protein 1), which is ex- consequently disassembles (Guttler and Gorlich, 2011;
clusively localized to the cytoplasmic side of the NPC, facili- Figure 3).
tates the GTPase activity of Ran, and GTP on Ran is hydrolyzed During mitosis, although the NE, including the nuclear
to GDP. The GDP-bound form of Ran (RanGDP) in the lamina and the NPC, is broken down, RCC1 remains to bind
cytoplasm is imported into the nucleus by nuclear transport mitotic chromosomes. The action of RCC1 produces a high
factor 2 (NTF2). As a result, the concentration of RanGTP is concentration of RanGTP around the mitotic chromosomes.
higher in the nucleus than in the cytoplasm. When a cargo The concentration of RanGTP gradually decreases toward the
macromolecule containing an NLS is imported into the nu- plasma membrane (Kalab et al., 2006). As in interphase, the
cleus, it binds to a nuclear import receptor and then passes RanGTP gradient drives cargo release and binding to NTR
through the NPC. At the nuclear interior, RanGTP binds to the during mitosis. Therefore, NLS-containing spindle assembly
cargo-bound nuclear import receptor, thereby inducing cargo factors (SAFs) such as TPX2 and KID bind to nuclear import
release into the nucleoplasm. Conversely, when a cargo receptors in the mitotic cytoplasm. In the vicinity of mitotic
macromolecule containing an NES is transported from the chromosomes, SAFs are released from nuclear import receptors
nucleus into the cytoplasm, the cargo interacts with the nuclear and become loaded onto mitotic chromosomes because of the
export receptor and RanGTP in the nucleus, forming the export high concentration of RanGTP. Many SAFs are associated with
complex. After passing through the NPC, RanGAP1 hydrolyzes microtubule nucleation, stabilization and elongation; therefore,
 Organelles: Structure and Function: Nuclear Organization (Nuclear Structure and Dynamics) 317

the RanGTP gradient around mitotic chromosomes during cycle, the chromatin structure is dynamically altered via post-
mitosis forms a spatial context for regulating mitotic spindle translational modifications and histone exchange depending
assembly (Fuller, 2010; Kalab and Heald, 2008). In addition to on both internal and external stimuli. In eukaryotic cells
mitotic spindle formation, the mitotic RanGTP gradient is also during interphase, the NE is the physical barrier protecting
associated with the NE reassembly during late mitosis. chromatin against numerous cytoplasmic toxic metabolites
such as reactive oxygen species. The NE is also critical for the
NE disassembly and reassembly establishment of the macromolecular nucleocytoplasmic
When the cell enters open mitosis, the entire NE structure, in- transport system that is operated by the NPC and diverse
cluding the nuclear lamina, NPC, and nuclear membrane, must NTRs. The INM provides the sites in which heterochromatin is
be disrupted for proper formation of the mitotic spindle. In this anchored via the interaction with the nuclear lamina, which is
context, it is considered that phosphorylation by mitotic kinases composed of lamins and INM proteins. By contrast, in cells
is at least one of the key processes to disassemble the NE. The demonstrating open mitosis, the NE is disassembled at the
proteins forming the NE are directly phosphorylated via mitotic mitotic entry through mitotic kinase activities that allow
kinases including cyclin-dependent kinase1 (CDK1)-CyclinB, chromatin segregation via microtubules. These cells re-
Polo-like kinase1 (PLK1), Protein kinase C (PKC), Aurora A and assemble the NE at late mitosis by the coordinated in-
Aurora B. As initial phosphorylation targets in late prometa- activation of mitotic kinases and activation of PPases. RanGTP
phase, peripheral FG repeat-containing Nups such as NUP98 and Importin β, which mediate nucleocytoplasmic transport in
become phosphorylated, before the phosphorylation of scaffold interphase, are required for assembling the mitotic spindle,
Nups, by multiple mitotic kinases including CDK1-CyclinB, chromosome segregation and NE formation, processes im-
disrupting the permeability barrier of NPCs (Laurell et al., portant for the inheritance of the mass of chromatin by
2011). Consequently, cytoplasmic proteins leak into the nuclear daughter cells.
interior and accelerate the NEBD. The lamins are also phos- During the past decade, a number of NE proteins have
phorylated and depolymerized via CDK1-CyclinB activity at the been identified, along with their modifications and
mitotic entry. The LEM domain-binding protein BAF is phos- interactions (protein–protein, protein–DNA, protein–RNA),
phorylated via Vaccinia-related kinase (VRK) during mitosis. advancing our understanding of the structure and function of
Phosphorylation of BAF by VRK causes the loss of its DNA- the NE itself. However, because the NE is tightly related to
binding activity and reduces its interaction with Emerin (Mar- many cell biological processes involving cellular metabolism,
galit et al., 2007). LBR is phosphorylated by mitotic kinases in cytoskeletons, and the nuclear genome, we require further
its N-terminal region and loses its heterochromatin-binding clues to understand NE organization and dynamics in the
activity. For proper progression of the NEBD, such phos- context of its connections to a variety of cellular physiological
phorylation-dependent attenuation of the interactions between processes.
INM proteins–chromatin might be essential. Furthermore, the
cytoplasmic microtubules also support the destruction of the
NE through their pulling force (Guttinger et al., 2009).
After the onset of anaphase, PPases such as PP1α/β/γ and See also: Organelles: Structure and Function: Nuclear Pores
PP2A/B are relatively active, whereas net kinase activities grad-
ually decrease (Wurzenberger and Gerlich, 2011). The activated
PP1 dephosphorylates the mitotically phosphorylated NE References
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