CHAPTER 9

Nuclear Structure and Dynamics

T he nucleus houses the chromosomes together with This chapter describes what is known about the
the machinery for DNA replication and RNA transcrip- structure of the nucleus, the nuclear envelope, and the
tion and processing (Fig. 9.1). Immature RNAs must be transport of macromolecules into and out of the nucleus,
kept apart from the translational apparatus because and discusses their links to human diseases. Aspects of
eukaryotic genes are transcribed into RNAs containing nuclear structure and function that are discussed else-
noncoding intervening sequences that are removed by where include genome and chromosome organization
splicing to assemble mature RNA molecules with a (Chapter 7), chromatin structure (Chapter 8), DNA
continuous open reading frame. Sequestration of imma- replication (Chapter 42) and RNA transcription and
ture RNAs is one function of the nuclear envelope, two processing (Chapters 10 and 11).
concentric membrane bilayers that separate the nucleus
and cytoplasm. The nuclear envelope also regulates
the bidirectional transport of macromolecules in and
Overall Organization of the Nucleus
out of the nucleus, participates in chemical, protein Studies in which entire individual chromosomes are
and mechanical signaling pathways, contributes to labeled by in situ hybridization (chromosome painting;
genome organization, and provides mechanical stability see Fig. 8.10) reveal that chromosomes tend to occupy
to the nucleus. discrete regions within the nucleus called chromosome
territories. The boundaries of adjacent territories,
where more actively transcribed regions are generally
Nuclear located, overlap with one another such that approxi-
envelope mately 40% of each territory intermingles with adjacent
Nuclear territories. The chromatin of these overlapping regions
pores tends to be less compact than in the rest of the territory,
and is referred to as the interchromosomal domain.
Most RNA transcription and processing are thought to
occur within this domain. Although the nucleoplasm is
very crowded with chromosomes and ribonucleopro-
Nucleolus teins (RNPs), proteins can nonetheless diffuse surpris-
ingly rapidly through the nucleus, possibly by moving in
the interchromosomal domain. Evidence is accumulating
that actin is present in nuclei, presumably in the inter-
chromosomal domain. Although the role of this actin is
unknown, an attractive hypothesis is that it forms a scaf-
fold for other processes. Nuclear actin can influence the
positioning of nuclear subdomains.
Heterochromatin Specialized Subdomains of the Nucleus
FIGURE 9.1 ELECTRON MICROGRAPH OF A THIN SECTION Cell nuclei contain numerous discrete subdomains or
OF A CANCER CELL NUCLEUS WITH MAJOR FEATURES bodies with distinctive structural organizations and/
LABELED. (Courtesy Scott Kaufmann, Mayo Clinic, Rochester, MN.) or biochemical composition (Fig. 9.2 and Table 9.1).

143
144 SECTION III n Chromatin, Chromosomes, and the Cell Nucleus

A B C
Nucleoli

PML
bodies

Cajal
bodies Nucleoli
Nuclear Speckles
envelope Chromatin
Cajal bodies 5 µm

D
Speckles
PIKA

Nucleoli

FIGURE 9.2 EXAMPLES OF MAJOR SUBNUCLEAR STRUCTURES. A, Components involved in RNA processing are scattered throughout
the nucleus but concentrated in domains called speckles that are rich in interchromatin granules. Inhibition of RNA processing causes splicing
components to accumulate in enormous concentrations of interchromatin granule clusters. Several cells were injected with a short oligonucleotide
that disrupts the function of the U1 small nuclear ribonucleoprotein (snRNP) in RNA processing (see Fig. 11.15), and were then stained with an
antibody recognizing the Sm splicing components (green). The injected cells were marked by introducing an inert fluorescent dextran marker into
the cytoplasm (red). B, Nucleus with simultaneous staining of nucleoli (blue), PML (promyelocytic leukemia) nuclear bodies (red), Cajal bodies
(green), and the nuclear envelope (purple). C, Nucleus with simultaneous staining of chromatin (blue), nucleoli (red), speckles (green), and Cajal
bodies (white). D, Nucleus with simultaneous staining of DNA (blue) and the polymorphic interphase karyosomal association (PIKA)/53BP1 nuclear
body/OPT (Oct1/PTF/transcription) domain (red). Nucleoli appear as unstained areas. A number of proteins involved in the sensing and repair of
DNA damage concentrate in the PIKA. (A, Courtesy David Spector, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. B–C, Courtesy
Angus Lamond, University of Dundee, United Kingdom. D, Courtesy William S. Saunders and William C. Earnshaw.)

The most prominent of these is the nucleolus, dis- Perichromatin fibrils contain various splicing factors and
cussed in the next section. Although often referred to RNA-packaging proteins.
as organelles, nuclear subdomains, unlike cytoplasmic When factors involved in RNA processing are detected
organelles, are not membrane-bounded. In fact, many by fluorescence microscopy, 20 to 50 bright speckles
proteins that have been examined by the fluorescence are seen against a diffuse background of nucleoplasmic
recovery after photobleaching technique (see Fig. 6.3) staining (Fig. 9.2). The diffuse staining probably corre-
exchange relatively rapidly between a nuclear body sponds to splicing factors associated with perichromatin
and a nucleoplasmic pool. Therefore, these bodies fibrils at dispersed sites. Most speckles correspond to
represent highly dynamic associations of macromolecu- clusters of interchromatin granules, particles 20 to
lar complexes. By concentrating particular RNAs and 25 nm in diameter distributed throughout the interchro-
proteins with enzymes involved in their maturation, mosomal domain. Proteomic analysis reveals that isolated
they accelerate macromolecular assembly and matura- interchromatin granules contain more than 200 stably
tion processes. They may also concentrate components associated proteins, most involved with various aspects
involved in gene regulation or repair at particular of RNA processing. When tagged with green fluorescent
chromosomal loci. protein, components involved in pre–mRNA (messenger
Structures associated with RNA transcription and RNA) processing cycle between speckles and sites of
processing are found at up to 10,000 sites spread transcription in less than 1 minute in live cells.
throughout the typical mammalian nucleus as well as in Metabolic labeling experiments indicate that speckles
a few more prominent domains. The dispersed sites are not major sites of active transcription, although most
likely correspond to structures called perichromatin transcription sites are associated with the periphery of
fibrils, originally observed by electron microscopy speckles. Speckles are less prominent in cells that
on the surface of regions of condensed chromatin. transcribe RNA at high levels, and become strikingly
 CHAPTER 9 n Nuclear Structure and Dynamics 145

TABLE 9.1 Major Nuclear Subdomains

Structure Comments
Nucleolus The nucleolus (typically 1 to 5 structures of 0.5 to 5 µm diameter in mammalian cell nuclei) is the site of ribosomal
RNA (rRNA) transcription and processing, as well as of preribosomal assembly. It is also the site of processing of
several other noncoding RNAs, including the RNA component of the signal recognition particle (SRP; see Fig. 20.5).
It plays an important role in helping organize the genome during interphase, as well as in regulating the stability
of p53, a critical transcription factor that is involved in regulating the cell cycle, particularly when DNA damage
occurs.
Speckles Speckles are concentrations of components involved in RNA processing. They often correspond to clusters of
interchromatin granules seen by electron microscopy. They may serve as storage depots of splicing factors, or they
may play a more active role in splicing factor modification and/or assembly.
Cajal bodies Formerly known as coiled bodies. Approximately 0.2 to 1.0 µm in diameter, Cajal bodies have a coiled fibrous
substructure. First identified by electron microscopy, up to 10 of these structures are seen in transformed cells.
They are usually absent from nontransformed normal cells. They contain the human autoantigen p80-coilin and
survival of motor neurons (SMN) protein, which is encoded by the gene mutated in spinal muscular atrophy, a severe,
inherited, human, muscular wasting disease. They are involved in small nuclear ribonucleoprotein (snRNP) and small
nucleolar ribonucleoprotein (snoRNP) assembly and in maturation of telomerase (which also contains an RNA
component).
PML bodies Also known as PODs and ND10, 10 to 30 of these structures are scattered throughout the nucleus. They are thought to
enhance gene repression by serving as assembly sites for certain transcriptional corepresser complexes. They also
appear to be targeted during viral infections. Fusion of the marker protein PML to the α-retinoic acid receptor is often
found in acute promyelocytic leukemia (hence the name PML), in which the PML bodies appear highly fragmented.
Treatments that are effective against PML restore the normal morphology of PML bodies (see text).
Polycomb Concentrations of the PRC1 and PRC2 complexes (see Chapter 8) involved in the silencing of facultative
group bodies heterochromatin. One mechanism for gene inactivation may be translocation into these inactive domains.
53BP1 nuclear Defined as concentrations of the DNA repair-associated protein 53BP1. Also known as PIKA (polymorphic interphase
bodies karyosomal association) and OPT (Oct1/PTF/transcription) domain. These domains may be up to 5 µm in diameter
during G1 phase, but their morphology and number vary across the cell cycle. They appear to correspond to sites of
DNA damage during mitosis that arise as a result of incomplete DNA replication during S-phase.

prominent when RNA processing is inhibited (Fig. 9.2). Mammalian nuclei also contain approximately 10 to
Together these observations suggest that speckles may 30 bodies, varying in size from 0.3 to 1.0 µm, known as
be dynamic depots where RNA processing factors accu- promyelocytic leukemia (PML) bodies (Fig. 9.2B).
mulate then they are not active. They may also have a PML bodies were initially defined by the presence of a
role in rendering mRNAs competent for export to the protein called PML, an important regulator of cell growth
cytoplasm. and genome stability. PML has a RING-finger amino acid
Cajal bodies (formerly known as coiled bodies) are sequence motif and is therefore probably an E3 ligase for
compact structures approximately 0.3 to 1.0 µm in ubiquitin or ubiquitin-like proteins (see Fig. 23.2). Its
diameter (Fig. 9.2B) that resemble balls of tangled threads targets are unknown. In normal cells, PML bodies appar-
in the electron microscope. Nuclei of rapidly growing ently have a role in assembling corepressor complexes
transformed cells typically have one to 10 prominent that modify chromatin to repress transcription (see
Cajal bodies. These structures are absent from most Chapter 8). Their other functions are not known.
nontransformed (normal) cells. They contain an 80-kD The PML gene was identified by analysis of a chromo-
human autoantigen called p80-coilin and the survival of some translocation between chromosomes 15 and 17
motor neurons (SMN) protein, which is encoded by a found in patients with acute promyelocytic leukemia
gene mutated in spinal muscular atrophy, a severe inher- (APL). In many patients, this translocation produces a
ited human muscular wasting disease. The SMN protein gene fusion between PML and the retinoic acid receptor
participates in importing immature small nuclear ribonu- alpha (RARα). The fusion protein, PML-RARα, blocks
cleoproteins (snRNPs) into the nucleus after their differentiation of hematopoietic precursors and causes
assembly in the cytoplasm. p80-coilin recruits the SMN APL. In APL cells, PML-RARα is distributed in tiny punc-
complex to Cajal bodies, where the snRNPs are further tate foci scattered throughout the nucleus. When APL
processed to render them functional in RNA splicing cells are treated with arsenic trioxide, which is clinically
reactions (see Chapter 11). Cajal bodies also have a role effective in treating APL, PML-RARα aggregation causes
in the maturation of the RNP enzyme telomerase as well prominent PML bodies to reform. PML-RARα is ubiquity-
as other functions. lated within those bodies and subsequently degraded.
146 SECTION III n Chromatin, Chromosomes, and the Cell Nucleus

A Dense fibrillar B
component
Nucleoli Nucleus
Fibrillar center

Nucleolus
organizing regions

Granular Mitotic
component chromosomes

FIGURE 9.3 NUCLEOLUS AND NUCLEOLAR ORGANIZER REGION. A, Electron micrograph of a thin section of a typical nucleolus. The
fibrillar centers, dense fibrillar component, and granular component are indicated. B, Use of silver staining to visualize the nucleolus in interphase
nuclei and the nucleolar organizer regions on mitotic chromosomes of the rat kangaroo. (A, From Fawcett DW. The Cell. Philadelphia: WB
Saunders; 1981. B, From Robert-Fortel I, Junéra HR, Géraud G, et al. Three-dimensional organization of the ribosomal genes and Ag-NOR
proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy. Chromosoma. 1993;102:146–157.)

This allows the hematopoietic precursors to differentiate

A
and cures the cancer.

The Nucleolus: The Most Prominent Pre-bleach 0s 10 s 60 s 5 min 30 min

Nuclear Subdomain
The nucleolus, first described only 5 years after the
nucleus, in 1835, is the most conspicuous and best-
characterized nuclear subdomain (Figs. 9.1 and 9.3).
B
Most mammalian cells have one to five nucleoli, which
are specialized regions 0.5 to 5.0 µm in diameter sur-
rounding transcriptionally active ribosomal RNA (rRNA) Pre-bleach 0s 10 s 20 s 40 s 60 s

gene clusters. Nucleoli are the sites of most steps in

ribosome biogenesis, from the transcription and process-
ing of rRNA to the initial assembly of ribosomal subunits.
FIGURE 9.4 ANALYSIS OF DYNAMICS OF CHROMATIN AND
The ribosome is a complex macromolecular machine A MAJOR NUCLEOLAR COMPONENT. A, Fluorescence recovery
with four different structural rRNA molecules and after photobleaching (FRAP) of H2B-GFP (green fluorescent protein)
approximately 85 proteins that are assembled into two shows that chromatin is immobile within the cell nucleus. B, FRAP of
subunits (see Figs. 12.6 and 12.7). Transcription of rRNA fibrillarin-GFP shows that this major component of nucleoli is highly
dynamic. Scale bar: 5 µm. (A–B, Courtesy Tom Misteli. B, From Phair
by RNA polymerase I comprises nearly half of total cel-
RD, Misteli T. High mobility of proteins in the mammalian cell nucleus.
lular RNA synthesis in some cell types. This high level of Nature. 2000;404:604–609.)
synthesis is necessary to produce approximately 5
million ribosomes in each cell cycle, more than 30 every
second in budding yeast.
Nearly 700 proteins associate stably with human Other stable RNAs, including the RNA component of the
nucleoli. Many more may associate transiently, and this signal recognition particle (see Fig. 20.5), are also pro-
composition changes to reflect different metabolic states cessed in the nucleolus.
of the cell (Fig. 9.4). Many nucleolar proteins are involved Intriguingly, the nucleolus is involved in controlling
with either rRNA synthesis and modification or with the stability of the critical cell-cycle regulator protein
ribosome subunit assembly. The functions of many other p53 (see Fig. 41.5). Healthy cells keep p53 levels low by
nucleolar proteins remain unknown and may reflect the using ubiquitylation in the nucleolus to destabilize it.
involvement of nucleoli in other biological processes. Under certain types of stress, cells defend themselves by
 CHAPTER 9 n Nuclear Structure and Dynamics 147

activating p53. They do this by having nucleolar proteins what are termed nucleolus-organizing regions (NORs
bind and inactivate Mdm2, the key factor that ubiquity- [Fig. 9.3B]), which often form a prominent secondary
lates p53. constriction of the chromosome. (The primary con-
striction is the centromere.) Several nucleolar proteins
Ribosomal Biogenesis in Functionally Distinct and RNA polymerase I remain bound at NORs as cells
Regions of the Nucleolus enter and exit mitosis but most nucleolar proteins coat
Transmission electron micrographs of thin sections the surface of the mitotic chromosomes forming a peri-
show three morphologically distinct regions in the chromosomal layer or “skin.”
nucleolus (Fig. 9.3). Fibrillar centers contain concen- Nucleolar reformation begins in mitotic telophase as
trations of rRNA genes, together with RNA polymerase processing factors and unprocessed pre-RNA remaining
I and its associated transcription factors. Actively tran- from the previous cell cycle associate with NORs (10 in
scribed ribosomal genes are found near the border human), which then cluster into one to five foci. Next,
between the fibrillar centers and a dense fibrillar a wide variety of nucleolar components assemble into
component that surrounds them. The granular com- particles termed prenucleolar bodies that associate
ponent is the site for many steps in ribosome subunit with the NORs in a process requiring transcription of
assembly and is made up of densely packed clusters of the rRNA genes. Normally, nascent transcripts, rather
ribosomal precursors called preribosomal particles 15 to than ribosomal genes, nucleate assembly of the nucleolus
20 nm in diameter. in each cell cycle. If antibodies to RNA polymerase I
rRNA loci have a modular organization. Genes alter- are microinjected into mitotic cells, rRNA transcription
nate with spacer regions in large tandemly arranged is blocked, and nucleoli do not reform in the next
clusters (see Fig. 11.10). The repeat unit in this array G1 phase.
(gene plus spacer) is approximately 40,000 base pairs
in humans. Humans have approximately 300 to 400
copies of the ribosomal DNA (rDNA) repeat unit located
Structure of the Nuclear Envelope
in clusters on chromosomes 13, 14, 15, 21, and 22. The nuclear envelope provides a selective permeability
Usually, only a fraction of these genes is actively tran- barrier between the nuclear compartment and the cyto-
scribed. An additional rRNA, 5S RNA, is encoded by plasm and acts as a platform that helps organize the
distinct genes and transcribed by RNA polymerase III chromosomes in discrete functional domains (Fig. 9.5).
(see Fig. 10.8). The barrier keeps pre-mRNAs in the nucleus until they
A simple yet efficient mechanism guarantees a balance are fully processed and licensed for export so that only
between the RNA components of the two ribosomal mature mRNAs are delivered to ribosomes in the cyto-
subunits. The major rRNA components are encoded by plasm for translation into protein. It also provides an
a single precursor RNA molecule. In humans, this 13,000-
base precursor is commonly described by its sedimenta-
tion coefficient in sucrose gradients as 45S. Following its
Rough CYTOPLASM
transcription, the RNA precursor is processed in a series endoplasmic
of cleavages to yield the 18S, 5.8S, and 28S rRNA mol- reticulum
ecules (see Fig. 11.10). In addition to the cleavages, Ribosome
rRNA processing also involves extensive base and sugar
modifications, including approximately 100 2′-O-methyl
ribose and approximately 90 pseudouridine residues per
molecule. The earliest stages of rRNA processing prob- Nuclear pore
complex
ably occur in the dense fibrillar component of the
nucleolus. Later stages take place in the granular compo-
nent. Ribosomal protein synthesis occurs in the cyto-
plasm on free ribosomes. The newly synthesized proteins
are transported into the nucleus for assembly into ribo-
somes, predominantly in the granular component. PERINUCLEAR SPACE Outer
membrane

Disassembly of the Nucleolus During Mitosis Inner

The nucleolus disassembles during each mitotic cycle, membrane
starting with the dispersal of the dense fibrillar and Nuclear
lamina
granular components during prophase. This disassembly NUCLEAR INTERIOR Chromatin
is driven by specific phosphorylation of nucleolar
proteins. Ultimately, the fibrillar centers alone remain FIGURE 9.5 OVERVIEW OF NUCLEAR ENVELOPE
associated with the mitotic chromosomes, forming ORGANIZATION.
148 SECTION III n Chromatin, Chromosomes, and the Cell Nucleus

additional level of genetic protection and control since Structure and Assembly of the Nuclear Lamina
various chromosomal events, including DNA replication The nuclear lamina is a thin protein meshwork composed
and expression of certain genes, are regulated, at least of type V intermediate filament proteins called nuclear
in part, by changes in the ability of factors to move lamins (Figs. 9.6 and 9.7). Lamins can be divided into
between the cytoplasm and nucleus. two families. Lamin A is encoded by a gene that gives
The nuclear envelope is composed of two concentric rise to four major polypeptides (including lamin C) by
lipid bilayers termed the inner and outer nuclear alternative splicing (see Fig. 11.6). Members of the
membranes. The outer nuclear membrane is continu- lamin B family are the products of two distinct genes.
ous with the rough endoplasmic reticulum and shares The various families of lamin proteins assemble into
some of its functions, including the presence of ribo- distinct fibrous networks (Fig. 9.6), exhibit different
somes. It also has unique proteins and functions. For patterns of gene expression, and appear to have distinct
example, it contains proteins that help link the nuclear roles in nuclear structure.
interior with the cytoskeleton. A fibrous nuclear lamina The pattern of lamin gene expression depends on the
of intermediate filaments supports the inner nuclear cell type and stage of development. The lamina of embry-
membrane in many eukaryotes. These and other inner onic stem cells and early embryos is comprised of B-type
nuclear membrane proteins mediate interactions of the lamins. Lamins A and C typically appear later in develop-
envelope with chromatin. The inner and outer nuclear ment as cells begin to differentiate, and their expression
membranes are separated by an approximately 50-nm varies in different cell types. This variation in lamina
luminal space that is continuous with the lumen of the composition may contribute to different patterns of gene
endoplasmic reticulum. Nuclear pore complexes and expression and mechanical stability of the nucleus.
the associated pore membrane bridge both nuclear Lamins A/C promote nuclear stiffness, whereas nuclei
membranes and provide the primary route for commu- containing only B-type lamins are more elastic.
nication between the nucleus and cytoplasm during Like other intermediate filament proteins (see Fig.
interphase. 35.2), nuclear lamins have a central, rod-like domain that

A OL OL

Nuclear pore Nuclear

complexes lamina C D

LA LB1

E F

FIGURE 9.6 NUCLEAR LAMINA. A, Thin-section electron micrograph of a nuclear envelope with a prominent nuclear lamina and nuclear
pores. B, Field emission scanning electron micrograph of the inner surface of an amphibian oocyte nuclear envelope. The nuclear pores are
prominent, protruding above the underlying nuclear lamina. C–F, Visualization of lamins A and B1 in a HeLa (Henrietta Lacks) cell nucleus by
structured illumination superresolution microscopy. Both lamins form short filaments that mostly do not colocalize. OL, overlay. (A, For reference,
see Fawcett DW. The Cell. Philadelphia: WB Saunders; 1981, Fig. 156 [top]. B, From Zhang C, Jenkins H, Goldberg MW, et al. Nuclear lamina
and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract. J Cell Sci. 1996;109:2275–2286. C–F, From Shimi T,
Kittisopikul M, Tran J, et al. Structural organization of nuclear lamins A, C, B1, and B2 revealed by superresolution microscopy. Mol Biol Cell.
2015;26:4075–4086.)
 CHAPTER 9 n Nuclear Structure and Dynamics 149

further, leaving the farnesylated cysteine at the carboxyl

A
terminus.
In contrast, once it is at the nuclear membrane, pre–
lamin A is processed by a protease called Zmpste24 (zinc
metalloprotease similar to yeast Sterile 24) that clips off
15 additional amino acids from the C-terminus including
the farnesylated cysteine, thereby loosening its associa-
tion with the membrane. Possibly as a result of this, some
A-type lamins also distribute throughout the nucleo-
B. Human Lamin A fiber
plasm. These intranuclear lamins have been suggested to
have roles in cell-cycle regulation (see next section).
The assembled lamina is tethered to the inner nuclear
membrane both by the farnesyl group and by interac-
tions with integral membrane proteins (see next section).
The surface of the lamina facing the nuclear interior also
C. Human Lamin A
interacts with the chromosomes. Thus, the lamina and
NLS
Ig domain its associated proteins both serve as a structural support
for the nuclear envelope and influence chromosome
α-helical coiled- distribution and function within the nucleus (see later).
Head coil dimerization
N CaaX

5 nm
Lamin A: ZMPSTE24 cleavage site
Tail Proteins of the Inner Nuclear Membrane
Several hundred integral membrane proteins are associ-
FIGURE 9.7 LAMIN ORGANIZATION AND ASSEMBLY. A, Sev-
eral stages in the assembly of isolated lamin B dimers into filaments
ated with the inner nuclear membrane, often in in a
in vitro. The dimers at left have two globular heads at the C-terminal tissue specific manner. Of these, the lamin B receptor,
end of a rod that is 52 nm long. B–C, Diagram of the structural LAP2 (lamina-associated protein 2), emerin, MAN1,
organization of the nuclear lamins. The sequence CaaX (see text) is a SUN1, and SUN2 have been characterized in detail. Some
signal for the attachment of a farnesyl group. NLS, nuclear localization inner nuclear membrane proteins bind lamins to help
sequence. (A, From Heitlinger E, Peter M, Haner M, et al. Expres-
sion of chicken lamin B2 in Escherichia coli: characterization of its
anchor the lamina polymer to the membrane and many
structure, assembly, and molecular interactions. J Cell Biol. 1991;113: can interact with chromatin. The lamin B receptor binds
485–495.) heterochromatin protein HP1 (Fig. 9.8) and links the
envelope to condensed chromatin. Codisruption of the
lamin B receptor and lamin A releases most heterochro-
is largely α-helical (Fig. 9.7). The basic building block of matin from the nuclear periphery.
lamin assembly is a dimeric α-helical coiled-coil (see Fig. The LEM domain, a 40-amino-acid motif common to
3.10) of two identical parallel polypeptides. Lamin several nuclear proteins, including LAP2, emerin, and
dimers self-associate end to end to form protofilaments MAN1, binds to an abundant small protein called barrier-
that associate laterally in a process that is under active to-autointegration factor (BAF), so named for a separate
investigation. role facilitating viral genome integration for HIV. BAF
The coiled-coil is followed by a large C-terminal binds directly to DNA and to histones and functions in
domain with a central globular fold and containing a organizing chromatin across the cell cycle.
nuclear localization sequence (see later section) that LAP2 can affect chromatin organization in multiple
promotes the rapid import of newly synthesized lamin ways. Some of its several splice variants lack the trans-
precursors into the nucleus. In most lamins, the membrane region for inner nuclear membrane associa-
C-terminus acquires a lipid posttranslational modifica- tion and are soluble. Both soluble and transmembrane
tion that targets them to the nuclear membrane. This forms have the LEM domain and bind BAF, but the
involves enzymatic addition of the C15-isoprenoid transmembrane forms can also bind a histone deacety-
hydrocarbon tail farnesyl (see Figs. 13.10 and 20.15). lase. Interactions of intranuclear lamin A and a soluble
The farnesyl group is added to a cysteine side chain in splice variant of LAP2 are important for cell-cycle regula-
an amino acid motif called the CaaX box (Ca1a2X, where tion by forming a complex with the tumor suppressor
C is a cysteine located four amino acids from the carboxyl retinoblastoma protein (pRb; see Chapter 41). This,
terminus; a1 is any aliphatic amino acid; a2 is valine, in turn, regulates the transcription factor E2F (see
isoleucine or leucine; and X is usually methionine or Fig. 41.9), which is important for activating the G2-to-S
serine) at the carboxyl terminus of the protein. This transition. Several other inner nuclear membrane pro-
motif was first recognized in the Ras proteins (see teins also bind transcriptional activators, in some cases
Fig. 25.7). The aaX residues are removed after addition sequestering them at the nuclear periphery away from
of the farnesyl group. B-type lamins are not processed their gene targets.
150 SECTION III n Chromatin, Chromosomes, and the Cell Nucleus

CYTOPLASM
KASH TM domain
Outer Nuclear Membrane
KASH binding
LAP1 domain of SUN
LAP2 Man-1 Emerin
LBR (β–γ)
Lamin A SUN1/2
~45nm
Lamin B SUN3
HP1 BAF ~20nm
LEM

SUN4/5
~12nm
Heter
ochromatin
NUCLEOPLASM

FIGURE 9.8 INTEGRAL PROTEINS OF THE INNER NUCLEAR MEMBRANE. Lamin B receptor (LBR), lamina-associated protein 2 (LAP2),
Man-1, and emerin all bind lamin B. LBR associates with chromatin via HP1. The other three associate with chromatin via the barrier-to-
autointegration factor (BAF). Emerin and lamina-associated protein 1 (LAP1) also bind to lamin A. The α form of LAP2 is not membrane associated
and is not shown here. Three isoforms of SUN proteins link the inner nuclear membrane to KASH domain proteins of the outer nuclear membrane.
KASH proteins asssociate with the cytoskeleton. (SUN proteins from Sosa BA, Kutay U, Schwartz TU. Structural insights into LINC complexes.
Curr Opin Struct Biol. 2013;23[2]:285–291. For reference, see Protein Data Bank [www.rcsb.org] file 4DXT [ribbon diagram of SUN/KASH].)

The SUN proteins bind lamin A, and then connect at a distance from the nuclear periphery are either associ-
across the nuclear envelope lumen to huge KASH domain ated with a similar repressive compartment surrounding
proteins in the outer nuclear membrane that in turn bind nucleoli or are in the nuclear interior. Interestingly, asso-
to all three major cytoplasmic filament systems, actin fila- ciation of the chromosomes with the nuclear envelope is
ments, intermediate filaments, and microtubules (see perturbed in some nuclear envelope-associated diseases
Fig. 9.8 and Chapters 33 to 35). Thus, the lamina is (see the next section).
linked to the rest of the cytoskeleton. Deletions or muta- Chromatin interactions with nuclear pores can have
tions in several lamina proteins, including the SUN pro- both positive and negative effects on gene expression.
teins, reduce the mechanical stability of the cell and In mammalian cells, the chromatin near pores appears
interfere with cell migration. SUN proteins are also less condensed (less heterochromatic) than most chro-
important for maintaining the uniform spacing of the matin adjacent to the lamina. The significance of these
nuclear envelope lumen. Their disruption results in interactions is still under study.
uneven separation of the inner and outer nuclear Disassembly of the nuclear envelope during mitosis in
membranes. metazoa releases the chromosomes so that they can be
These diverse functions in genome organization and segregated to the daughter cells by the cytoplasmic
regulation, cell cycle regulation, signaling cascades and mitotic spindle (see Fig. 44.1). Mitotic segregation of
cell and nuclear mechanical stability could explain the chromosomes to daughter cells takes place within the
link between mutations in nuclear envelope proteins nucleus in many other eukaryotes, including yeasts.
and human disease (see later).
Nuclear Envelope Defects Lead to Human Diseases
Role of the Nuclear Envelope in In 1994, a gene mutated in patients with human X-linked
Genome Organization Emery-Dreifuss muscular dystrophy was found to encode
A high-throughput method revealed that the nuclear a protein of the inner nuclear envelope. The gene was
lamina has an important role in chromosome organiza- named emerin. This link between the nuclear envelope
tion within nuclei. The method uses a DNA-modifying and human disease was the tip of a huge iceberg. Genetic
enzyme fused to a lamin protein, so nearby DNA is modi- defects in nuclear envelope proteins cause at least 20
fied and can be mapped along the genome. In human disorders, including muscular dystrophies, lipodystro-
cells, approximately 40% of the genome is found in phies, and neuropathies (diseases of striated muscle,
1000 to 1500 LADs (lamina-associated domains; Fig. 9.9) fatty tissue, and the nervous system, respectively). The
ranging in size from 10 kb to approximately 10 Mb. most dramatic of these is Hutchinson-Gilford progeria
Analysis of single cells revealed that approximately syndrome (Fig. 9.10). Affected individuals are essentially
15% of LADs are associated with the lamina in most normal at birth, but they appear to age rapidly and
cells, with the remainder varying from cell to cell. The die in their early teens of symptoms (including athero-
constitutive LADs have low transcriptional activity. sclerosis and heart failure) that are typically associated
Indeed, heterochromatin-associated histone marks such with extreme age.
as H3K9me3 (see Fig. 8.7) promote association of par- More than 500 mutations scattered across the gene
ticular chromosomal regions with the lamina. The LADs encoding lamin A/C cause at least 15 different diseases,
 CHAPTER 9 n Nuclear Structure and Dynamics 151

Nuclear CYTOPLASM might be particularly sensitive to these mutations, owing

envelope to mechanical stress during contraction. However, this
M M
M M M M
M
Dam
methylase
mechanism cannot account for the link between lamin
Methyl
groups M
M
mutations and lipodystrophy—fat is not a force-generating
M M
M
Chromatin fiber
LAD M tissue—neuropathy, or progeria.
An alternative suggestion is that these mutations
Isolated DNA
change interactions between the inner nuclear mem-
M M MM M M M
M
M M M M
brane and chromatin and this alters gene expression
A patterns. Cells from patients with Hutchinson-Gilford
progeria syndrome show signs of aging in culture
X µm
that are accompanied by dramatic alterations in hetero-
Lamina chromatin (see Fig. 8.6), but changes in gene expression
are relatively small and vary between patients.

LADs Nuclear Pore Complexes

In a typical growing cell, nearly all traffic between the
B nucleus and cytoplasm passes through approximately
5
3000 channels, called nuclear pore complexes,
Cell 1
0 that bridge the inner and outer nuclear membranes
Observed/expected (OE)

5
0
Cell 2 (Fig. 9.11). Nuclear pore complexes have a scaffold
5 Cell 3 consisting of three stacked rings each with eightfold
0
5 Cell 4
symmetry. Cytoplasmic and nuclear rings flank a
0
prominent spoke ring that is intimately associated with
5 Cell n–1
0 the pore membrane linking the inner and outer nuclear
5 Cell n membranes. The nuclear ring is anchored to the nuclear
0
LADs lamina. A less-prominent fourth luminal ring surrounds
5 Average
0 the pore membrane in the NE lumen. The minimum
0 40 80
C Position on chromosome 17 (Mb) diameter of the central channel through the pore is
approximately 40 nm, and the channel is approximately
FIGURE 9.9 NUCLEAR LAMINA HELPS ORGANIZE THE
CHROMATIN INTO FUNCTIONAL DOMAINS. A, Dam methylase 50- to 70-nm long.
fused to a lamin protein methylates the DNA in chromatin that is closely Eight filaments project outward from both the nuclear
associated with the nuclear lamina. Isolation of the DNA allows the and cytoplasmic rings. These are involved in docking of
methylation sites to be mapped. B, Constitutive LADs associate with macromolecules to be transported through the pore.
the lamina even after the cell has gone through mitosis and the lamina
The nuclear filaments are linked at their outer ends by a
has been disassembled and reassembled. C, Sites of increased
methylation on chromosome 17 from six single human cells, plus an terminal ring, much like the wire that secures the cork
average of the entire population. Lamina-associated domains (LADs) on a champagne bottle. This structure is called the
are indicated. (B–C, Modified from Kind J, Pagie L, de Vries SS, et al. nuclear basket.
Genome-wide maps of nuclear lamina interactions in single human Vertebrate nuclear pore complexes are large struc-
cells. Cell. 2015;163:134–147.)
tures with a mass of approximately 90 to 120 million Da
as assessed by electron cryomicroscopy. Core com­
ponents identified by mass spectrometry account
for approximately 70 million Da of the mass. Yeast
collectively termed laminopathies, some of which are nuclear pores are similar in overall structure but
variants of the diseases mentioned above (Fig. 9.10). At about half the mass.
least two laminopathies are also linked to mutations The protein composition of nuclear pore complexes
in the Zmpste24 protease. Some of the symptoms of is remarkably conserved. Approximately 30 core pro-
laminopathies can be modeled in mice, where loss of teins, called nucleoporins (Fig. 9.12), are present in
lamin A causes nuclear envelope defects and leads to a multiples of eight copies. Mass differences between
type of muscular dystrophy. electron cryomicroscopy and mass spectrometry mea-
The most surprising aspect of the laminopathies is the surements may be accounted for by transport factors
fact that except for premature aging, the defects linked and other auxiliary subunits that do not have a key
to each mutation are limited to a few tissues such as structural role.
striated muscle, even though lamins A/C are ubiquitous Two multiprotein complexes, the 10-member Y
in differentiated cells throughout the body. Lamin muta- complex (named because of its shape) and the five-
tions appear to compromise the stability of the nuclear member Nup93 complex make up the scaffold of the
envelope, so it has been suggested that muscle nuclei pore. The cytoplasmic and nuclear rings are assembled