amphipathic analogue forbes

Transcription

CHAPTER
Fifty Years of Nuclear Pores
and Nucleocytoplasmic
Transport Studies: Multiple
Tools Revealing Complex
Rules
1
Aurélie G. Floch*,{, Benoit Palancade*, and Valérie Doye*
*
Institut Jacques Monod, CNRS, UMR 7592, Univ. Paris Diderot, Sorbonne Paris Cite´, F-75205
Paris, France
{
Ecole Doctorale Ge`nes Ge´nomes Cellules, Universite´ Paris Sud-11, Orsay, France
CHAPTER OUTLINE
Introduction ................................................................................................................ 2
1.1 The NPCs: A Modular Macromolecular Assembly ................................................... 3
1.1.1 Toward a Refined View of the NPC Structure......................................... 3
1.1.2 Nucleoporins: The Building Blocks of NPCs .......................................... 6
1.1.3 Integrating the Nups Into the 3D Architecture of the NPC ...................... 8
1.1.3.1 Nup Localization...........................................................................8
1.1.3.2 Interactions Among Nups .............................................................9
1.1.3.3 Nup Stoichiometry ......................................................................10
1.1.3.4 Toward a Detailed Map of NPCs..................................................10
1.1.4 Nups are Composed of a Limited Set of Structural Domains ................. 10
1.2 Nucleocytoplasmic Trafficking: The Rules of the Road ......................................... 12
1.2.1 Investigating Nucleocytoplasmic Transport ......................................... 12
1.2.1.1 Historical Overview .....................................................................12
1.2.1.2 Expanding the Toolbox................................................................14
1.2.2 The Signals for Nucleocytoplasmic Exchanges .................................... 15
1.2.3 A Family of Protein Transport Receptors: The Karyopherins .................. 16
1.2.4 The Ran GTPase: A Key to Transport Directionality.............................. 17
1.2.5 The Case of INM Targeting ................................................................ 19
1.2.6 Distinct Pathways Contribute to RNA Export ....................................... 20
1.2.7 Translocation Across the NPCs: A Dual Function for
FG-Nups as Barrier and Gate ............................................................. 21
1.2.8 Noncanonical Transport Pathways through the NE ............................... 23
Methods in Cell Biology, Volume 122
Copyright © 2014 Elsevier Inc. All rights reserved.
ISSN 0091-679X
http://dx.doi.org/10.1016/B978-0-12-417160-2.00001-1
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CHAPTER 1 Introduction to NPC and Nuclear Transport
1.3 The Nuclear Transport Machinery: A Dynamic and Versatile Device ...................... 23
1.3.1 NPC Biogenesis Throughout the Cell Cycle ......................................... 24
1.3.1.1 NPC Disassembly .......................................................................24
1.3.1.2 Post-mitotic NPC Assembly.........................................................24
1.3.1.3 De novo NPC Assembly ..............................................................26
1.3.2 Multiple Functions of the Nuclear Transport Machinery
During the Cell Cycle ........................................................................ 27
1.3.3 NPCs, Nuclear Organization, and Gene Expression .............................. 29
1.3.4 NPCs and Genetic Stability ............................................................... 30
Concluding Remarks ................................................................................................. 31
Acknowledgments ..................................................................................................... 31
References ............................................................................................................... 32
Abstract
Nuclear pore complexes (NPCs) are multiprotein assemblies embedded within the nuclear envelope and involved in the control of the bidirectional transport of proteins and
ribonucleoparticles between the nucleus and the cytoplasm. Since their discovery
more than 50 years ago, NPCs and nucleocytoplasmic transport have been the focus
of intense research. Here, we review how the use of a multiplicity of structural, biochemical, genetic, and cell biology approaches have permitted the deciphering of the
main features of this macromolecular complex, its mode of assembly as well as the
rules governing nucleocytoplasmic exchanges. We first present the current knowledge
of the ultrastructure of NPCs, which reveals that they are modular and repetitive assemblies of subunits referred to as nucleoporins, associated into stable subcomplexes
and composed of a limited set of protein domains, including phenylalanine-glycine
(FG) repeats and membrane-interacting domains. The outcome of investigations on
nucleocytoplasmic trafficking will then be detailed, showing how it involves a limited
number of molecular factors and common mechanisms, namely (i) indirect association of cargos with nuclear pores through receptors in the donor compartment,
(ii) progression within the channel through dynamic hydrophobic interactions with
FG-Nups, and (iii) NTPase-driven remodeling of transport complexes in the target
compartment. Finally, we also discuss the outcome of more recent studies, which indicate that NPCs and the transport machinery are dynamic and versatile devices,
whose biogenesis is tightly coordinated with the cell cycle, and which carry nonconventional duties, in particular, in mitosis, gene expression, and genetic stability.
INTRODUCTION
One of the major evolutionary steps that occurred at the cellular level was the acquisition of internal compartments, enclosed by lipid membranes. Membrane internalization and organelle formation provided a major evolutionary advantage: by
1.1 The NPCs: A Modular Macromolecular Assembly
simultaneously carrying out different functions within these distinct compartments,
cells increased their robustness and complexity. The nucleus, observed for the first
time by Antonie van Leeuwenhoek in the seventeenth century, is the defining organelle that distinguishes eukaryotic from prokaryotic cells. Its boundary, the nuclear
envelope (NE), sequesters the genetic material from the cytoplasm. This separation
notably enables eukaryotic cells to spatially and temporally regulate distinct stages
of genome expression (mRNA transcription, maturation, translation, and decay).
However, the nuclear content is not totally isolated from the cytoplasm, thanks
to a gating system called nuclear pore complex (or NPC). These structures, localized
at the points of fusion between the outer and inner nuclear membrane of the NE,
connect the nucleoplasm to the cytoplasm and allow the bidirectional trafficking
of a flow of cellular components. While small molecules (water, sugars, and
ions) can freely translocate though the NPCs, large macromolecules (proteins and
nucleic acids) and even megadalton-sized macromolecular complexes such as ribosomal subunits or viral particles undergo highly selective nuclear import and/or export processes (Fig. 1.1). Regulating this transport is a crucial issue for the cell, both
for the maintenance of nuclear identity and for the control of gene expression. It requires the assembly and maintenance of stable NPCs as well as finely tuned cargo/
transport receptor systems that organize bidirectional transport and provide
selectivity.
Over the past 50 years, the combination of a huge variety of approaches, performed in a broad range of model organisms, has provided the main rules governing
nuclear pore assembly and nucleocytoplasmic transport. In this chapter, we give an
overview of the main approaches that have been used in the field to elucidate the
fundamental principles that govern NPC organization and nucleocytoplasmic traffic,
and to demonstrate the dynamic and versatile nature of the nuclear transport apparatus that also exerts duties beyond transport.
1.1 THE NPCS: A MODULAR MACROMOLECULAR ASSEMBLY
1.1.1 Toward a refined view of the NPC structure
In 1950, Callan and Tomlin, who observed the giant nuclei of amphibian oocytes by
electron microscopy (EM), were the first to report that pores, organized in annular
structures, pierced the NE. Franke (1966) and Gall (1967) subsequently described in
various species a macromolecular structure, embedded at fusion points of the NE
bilayer, with a eightfold rotational symmetry (Fig. 1.2A). Since then, NPCs have
been imaged in multiple organisms (see introduction to Chapter 2 and references
therein; Wente & Rout, 2010). The density of NPCs on the NE is most frequently
in the range of 3–15 per mm2 of NE, leading to 1000–5000 NPCs per mammalian
nucleus, and 100–200 in the much smaller yeast nuclei. In contrast, the large nuclei
from amphibian oocytes (0.4 mm diameter) are characterized by a very high
NPC density (60 NPCs/mm2) and contain about 50 million pores. Xenopus laevis
has thus been an extensively studied model system to uncover NPC structure
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FIGURE 1.1 An Overview of Nucleocytoplasmic Exchanges
The main nuclear processes involving nucleocytoplasmic transport of proteins and RNA–
protein complexes are represented: chromatin assembly; DNA metabolism; RNA synthesis/
processing; and ribosome biogenesis. Note that viral genomes can enter the nucleus through
NPCs either as intact viral particles or upon disassembly of the viral capsids on the
cytoplasmic side. ONM, outer nuclear membrane; INM, inner nuclear membrane; and ER,
endoplasmic reticulum.
(see Chapters 2 and 4 and references therein). Although their overall architecture
appears to be conserved with little change during evolution, NPC diameter varies
between 100 and 150 nm, and thickness by 50–70 nm. Reflecting this variation,
the total mass of NPCs was initially estimated to be 125 MDa in vertebrates,
but only 60 MDa in yeast (reviewed in Stoffler, Fahrenkrog, & Aebi, 1999).
The overall NPC architecture is composed of three rings: the nuclear and cytoplasmic rings that sandwich a central spoke ring delineating a central channel of 40 nm.
Anchored to this membrane-embedded central framework, peripheral NPC components extend into the cytoplasm and the nucleoplasm. These filamentous structures,
although detectable by thin-section EM (see Chapter 4), are best visualized by
FIGURE 1.2 The Ultrastructure of Nuclear Pore Complexes
(A) a. Pioneer EM image of a nuclear pore from a negatively stained preparation of the oocyte
envelope from a newt, Triturus, revealing its eightfold rotational symmetry. b. Original schematic
representations including the sizes and a three-dimensional view of a nuclear pore in the
double-layered nuclear envelope. (B) Scanning EM micrographs of nuclear pores from diverse
eukaryotes revealing cytoplasmic (top panels) and nucleoplasmic (lower panels) extensions.
Scale bar, 100 nm. (C) Top and lateral views of the cryo-EM structure of human NPCs at 3.2 nm
resolution. The cytoplasmic (CR), spoke (SR), and nuclear rings (NR) are indicated and the
pore membrane appears in yellow. An additional inner density likely corresponding to
molecules with high structural plasticity is colored in purple. Dotted lines highlight shapes
similar to the structure of some members of the Nup93 complex. The diameters and thickness
of NPCs are indicated.
(A) Reproduced from Gall (1967) # (1967), with permission from the author and The Rockefeller University
Press. (B) Images provided by Elena Kiseleva and reproduced from Brohawn, Partridge, Whittle, and
Schwartz (2009), with permission from Elsevier. (C) Reproduced from Bui et al. (2013) # (2013),
with permission from Elsevier.
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scanning electron microscopy (SEM) (Fig. 1.2B; see also Chapter 2). The eight
cytoplasmic filaments appear as 50-nm-long disordered structures, whereas the
so-called “nuclear basket” is composed of eight filaments (95 nm long in yeast
and 120 nm in vertebrates) that join to form a nuclear ring (reviewed in Stoffler
et al., 1999).
The complexity and size of NPCs have so far precluded the determination of their
whole structure at atomic resolution. However, introduction of cryo-electron tomography (cryo-ET) combined with in silico subtomogram averaging procedures has led
to substantial progress by refining the 3D architecture of the NPCs to 6 nm resolution (Beck, Lucic, Forster, Baumeister, & Medalia, 2007; Frenkiel-Krispin, Maco,
Aebi, & Medalia, 2010; Maimon, Elad, Dahan, & Medalia, 2012) and more recently
to 3.2 nm for NPCs imaged from purified human NEs (see Chapter 6; Bui et al.,
2013; Fig. 1.2C). These studies validated the dimensions of the NPCs and their overall architecture. They also confirmed that, in addition to the 40-nm-wide central
channel, the cytoplasmic and nuclear rings are not entirely apposed to the outer nuclear membrane (ONM) and inner nuclear membrane (INM), but leave peripheral
openings of 10 nm which traverse the spoke ring complex and could allow passage
of globular particles up to a size of 5 nm.
1.1.2 Nucleoporins: The building blocks of NPCs
The composition of NPCs has been elucidated by the combination of multiple biochemicals and genetic approaches. The molecular characterizations of the pore constituents (called nucleoporins or Nups) started in the 1980s by the production
of antibodies, notably monoclonal antibodies that bind in a polyspecific manner
to multiple Nups. For instance, Günter Blobel’s lab developed the widely used mouse
mAb414 antibody that first led to the characterization of mammalian Nup62
(Davis & Blobel, 1986). However, this antibody in fact recognizes a subset of Nups
called “FG-Nups” (see below) and presents a wide interspecies cross-reactivity in
eukaryotes. In budding yeast, genetic screens and total genome sequencing also
led to the description of new sets of Nups (for review, see Doye & Hurt, 1997).
Another important step was the development of approaches based on biochemical
purification of Nups subcomplexes, coupled with mass spectrometry analysis.
These techniques led to the characterization of multiple vertebrates and yeast Nups.
This culminated with the purifications of yeast and mammalian NPCs and their analysis by mass spectrometry (Cronshaw, Krutchinsky, Zhang, Chait, & Matunis, 2002;
Rout et al., 2000 and references therein). In these studies, bona fide Nups were validated through observation, upon tagging with GFP, of a typical punctate NE labeling
in live cells. Since then, only a few novel Nups have been identified (Fig. 1.3A).
Together, these studies have revealed that, despite their huge size, NPCs are composed
of only 30 Nups (Fig. 1.3A). However, due to the eightfold symmetry of the assembly,
and the additional twofold symmetry of the main scaffold relative to the axis of the NE,
each Nup is present in multiple copies leading to an assembly of 500–1000 proteins.
FIGURE 1.3 The Molecular Composition of NPCs
(A) Model of the organization of nucleoporins within the NPC framework in vertebrates (left)
and budding yeast (right). FG repeats containing nucleoporins are circled in red. Proteins
carrying enzymatic activities appear in bold. NPC-associated proteins other than bona fide
nucleoporins are circled with dotted lines. The Y-complex appears in blue. The hNup214/
yNup159–Nup82 subcomplexes appear in yellow. (B) a. Structural model of the human
Y-complex. Available crystal structures were fitted within the EM structure of the Y-complex
vertex. The arrows and dashed ellipses indicate the position of flexible hinges and static
connectors. b, c. Inner and outer Y-complexes segmented from the cryo-EM structure of
human NPCs. (C) Positioning of the Y-complex (in blue) and Nup82 (in yellow) within the
computational model of yeast NPC. (D) Positioning of two Y-complex rings (in blue and green)
and of the Nup214 complex (in yellow) within the cryo-EM structure of the human NPC.
(A) Modified from Wozniak, Burke, and Doye (2010) # (2010), with permission from Springer. (B) Reproduced
from Bui et al. (2013) # (2013), with permission from Elsevier. (C) Modified from Alber et al. (2007b)
# (2007), with permission from Nature Publishing Group. (D) Reproduced from Bui et al. (2013) # (2013),
with permission from Elsevier.
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1.1.3 Integrating the Nups into the 3D architecture of the NPC
Having a list of constituents in hand, a prerequisite for a detailed understanding of the
transport mechanism is to precisely integrate these Nups into the complex NPC
architecture.
1.1.3.1 Nup localization
A first requirement is to determine the localization of each Nup within the assembly.
With the exception of integral membrane Nups, which are evidently positioned within
the pore membrane, the localization of most Nups cannot be inferred from their primary
sequence. Thin-section EM in combination with immunogold labeling has been the main
approach used to assess their localization within the NPCs. These studies were performed
on isolated Xenopus nuclei, but also in mammalian or yeast cells using specific antibodies, or tagged Nups (see Chapters 3 and 4 and references therein). In yeast, nearly
all Nups were systematically localized by immuno-EM using strains carrying a
C-terminal protein A tag (Alber et al., 2007a; Rout et al., 2000). For specific Nups, immunogold labeling performed on SEM samples can further provide a combined view of
their localization with surface topology (see Chapter 2). Unlike EM, the resolution of
fluorescence microscopy has long impaired the refined localization of Nups within
the NPCs. However, the use of low concentrations of digitonin, that permeabilizes the
plasma membrane but not the NE, can provide clues on the restricted localization of specific Nups on the nuclear side of the NPCs (see for instance, Bastos, Lin, Enarson, &
Burke, 1996). In addition, discrimination of nuclear versus cytoplasmic localizations
could be achieved for a few peripheral Nups using confocal microscopy (see for instance,
Zhang, Saitoh, & Matunis, 2002).
Together, these studies have revealed that although most Nups present a symmetrical localization, some are only localized on the cytoplasmic or nucleoplasmic face,
or biased toward one side, conferring a global asymmetry to NPCs (Fig. 1.3A). Recently, super-resolution imaging, combined with single-particle averaging, has permitted the mapping of the average radial positions of individual fluorescent labels on
Nups with nanometer precision, thus providing another approach for the precise
mapping of Nups within the NPC framework (see Chapter 10).
Importantly, this huge molecular assembly is not static, and a systematic analysis
of multiple Nups using photobleaching experiments has revealed that in interphase
mammalian cells, NPC components exhibit a wide range of residence times from
seconds to days. Despite some exceptions, the central parts of the NPC appear to
be very stable, consistent with a function as a structural scaffold, whereas peripheral
components exhibit more dynamic behavior, suggesting adaptor as well as regulatory
functions (Rabut, Doye, & Ellenberg, 2004). Of note, these studies were facilitated in
vertebrate cells by the fact that NPCs are stably anchored within the nuclear lamina
(Daigle et al., 2001). In contrast, the whole NPCs can diffuse within the plane of the
NE in yeasts that lack a bona fide lamina (Belgareh & Doye, 1997; Bucci &
Wente, 1997).
1.1.3.2 Interactions among Nups
Information on the interactions between Nups is also crucial to build an accurate picture of the NPC. Biochemical studies, frequently combined with functional assays,
have revealed the assembly of subsets of Nups into stable heterooligomeric subcomplexes amenable to affinity purification experiments. These subcomplexes serve in
turn as modular building blocks to form larger NPC structures. Despite considerable
differences between the primary sequences of orthologous Nups, most yeast and
mammalian modules have been well conserved throughout evolution. The best currently characterized modules are the so-called metazoan Nup107–160 (yeast Nup84)
and Nup53–Nup93 (yeast Nup53–Nic96) subcomplexes which are essential architectural elements of the NPC scaffold, and the Nup62 (yeast Nsp1) subcomplex that
constitutes a major transporter module (reviewed in Brohawn et al., 2009; Wente &
Rout, 2010; see Fig. 1.3A for the positioning of the various NPC subcomplexes in
vertebrates and budding yeast).
The Y-shaped yeast Nup84/metazoan Nup107–160 complex is the best characterized NPC building block, as reflected by the extensive set of genetic, biochemical,
structural, and functional data accumulated over the years (reviewed in GonzalezAguilera & Askjaer, 2012). Pioneering single-particle negative stain EM studies
from the Hurt lab, using both isolated and in vitro-reconstituted complexes, revealed
the characteristic Y-shaped assembly formed by the seven Nups that constitute the
yeast Nup84 complex (Nup133, Nup120, Nup145C, Nup85, Nup84, Seh1, and
Sec13). The computational integration of biochemical and structural data subsequently enabled the determination of its structure to a precision of 1.5 nm
(Fernandez-Martinez et al., 2012 and references therein). In many eukaryotes, notably excluding Saccharomyces cerevisiae, the Y-complex contains two additional
proteins, Nup37 and Nup43 (Loiodice et al., 2004). In addition, because of its stable
interaction with the Nup107–160 complex, Elys/MEL-28, although solely localized
to the nuclear side of the NPCs (see Fig. 1.3A; Bui et al., 2013 and references
therein), is sometimes considered as a tenth member of this complex. Recently, a
structural model of the hNup107–160 subcomplex was obtained by combining electron tomograms of affinity-purified subcomplex particles, spatial constraints
obtained by cross-linking mass spectrometry, and previously available crystal structures or homology models (Bui et al., 2013, see Fig. 1.3B).
The metazoan Nup93 complex (that comprises Nup93, Nup188, Nup205,
Nup155, and Nup53—also termed Nup35 in vertebrates) forms the central spoke ring
of the NPCs. Its constituents are also conserved and display similar interactions in
budding yeast (see Fig. 1.3A). Two-hybrid screens and genetic analyses in yeast,
combined with in vitro assembly of the orthologous Nups from the fungus Chaetomium thermophilum, enabled reconstitution of this NPC module in vitro (Amlacher
et al., 2011). In this study, the structural DID-Dyn2 label, which is easily visible in
the electron microscope, was used to precisely position specific Nups within this
NPC subcomplex (see Chapter 5).
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1.1.3.3 Nup stoichiometry
In addition to their approximate position and specific partners, the copy number of
each Nup per NPC is another important parameter required to understand the molecular architecture of NPCs. The first insights toward Nups respective stoichiometry
were provided by semiquantitative investigations of their relative abundance in budding yeast (based on immunoblot signals of Nups tagged with the same protein
A moiety; Rout et al., 2000) and vertebrates (using quantifications of zinc or
Coomassie-stained gels loaded with NPC-enriched fractions; Cronshaw et al.,
2002). More recently, the combination of integrated targeted proteomics (see
Chapter 6) and super-resolution microscopy approaches have enabled the determination of the absolute stoichiometry of the human NPC. This analysis revealed a stable stoichiometry of most NPC scaffold Nups in distinct human cell types whereas
significant variations could be observed for peripheral Nups (Ori et al., 2013). This
confirms earlier observations that NPC composition could vary between different
tissues or during development (D’Angelo, Gomez-Cavazos, Mei, Lackner, &
Hetzer, 2012 and references therein).
1.1.3.4 Toward a detailed map of NPCs
With such parameters in hand, integrative approaches have been developed to resolve the molecular architecture of NPCs. A first approach involved the translation
of multiple datasets into spatial restraints that were then used to generate an ensemble
of structures consistent with the data. Analysis of this ensemble produced the first
approximation of the molecular architecture of the S. cerevisiae NPC (Alber
et al., 2007a, 2007b; Fig. 1.3C). More recently, by combining single-particle EM
and cross-linking mass spectrometry, the Y-complex, as well as a few additional
NPC constituents, could be precisely positioned within the refined cryo-ET structure
of the human NPC scaffold (Bui et al., 2013). This revealed that each human NPC
comprises 32 copies of the Y-shaped Nup107 complex that assemble into two reticulated rings (Fig. 1.3D). Of note, this organization differs from the yeast NPC model,
in which only 16 copies of the Nup84 complex were previously integrated (Alber
et al., 2007a,2007b). In the future, extending such a refined approach to additional
NPC subcomplexes will be required to bridge length scales from overall molecular
architecture down to atomic resolution. In addition, comparison of NPC architecture
between distant species or distinct tissues/cell types should shed light on its adaptability and specialization.
1.1.4 Nups are composed of a limited set of structural domains
Protein structure prediction analyses of Nups obtained in multiple species, including
the divergent eukaryote Trypanosoma brucei, as well as atomic resolution of the
structure of an increasing number of Nups, revealed that they are composed of a
few repetitive structural domains that likely evolved from the duplication of a small
set of precursors genes (reviewed in Aitchison & Rout, 2012; Brohawn et al., 2009;
Hoelz, Debler, & Blobel, 2011).
Although NPCs are anchored to the pore membrane, only a few Nups exhibit
transmembrane domains. There are three pore membrane proteins (Poms) in mammals (Pom121, gp210, and Ndc1) and four in S. cerevisiae (Pom34, Pom152,
Ndc1, and Pom33, of which the three first ones are associated to form the transmembrane ring; Onischenko, Stanton, Madrid, Kieselbach, & Weis, 2009) (Fig. 1.3A).
There is apparently no strong selective pressure for their conservation, as among
them, only Ndc1 and Pom33/TMEM33 are conserved throughout evolution (note that
the NPC localization of vertebrate TMEM33 remains to be demonstrated). In addition, the deletion of the three known integral membrane Nups in Aspergillus nidulans
is not lethal (Liu, De Souza, Osmani, & Osmani, 2009). While this may reflect the
existence of yet unidentified Poms, there are also alternative modes of interaction between the NPC and the pore membrane. Indeed, non-integral membrane-binding
modules have also been identified in several Nups. In particular, two distinct domains
with membrane-binding properties were functionally characterized in mammalian
Nup53, a constituent of the Nup93 complex that also interacts with Ndc1. The
C-terminal domain of Nup53, predicted to form an amphipathic helix, was further
shown to have membrane-deforming capabilities (Vollmer et al., 2012). In addition,
ALPS motifs (amphipathic lipid-packing sensor, first described in the COPI-coat assembly protein ArfGAP1) were found by in silico analysis in several human and yeast
Nups. The ALPS motif within human Nup133 (that belongs to the Y-shaped Nup107
complex) was demonstrated to effectively bind to curved membranes in vitro (Drin
et al., 2007).
These integral membrane proteins and membrane-binding domains anchor the
NPCs within the NE, thanks to their interaction with additional core scaffold proteins. Noteworthy, most Nups of the core scaffold are built of a-solenoid folds (antiparallel pairs of a-helices organized to form a coil), b-propellers folds (6–7 bladeshaped b-sheet subunits arranged around a central axis), or a combination of a
N-terminal b-propeller followed by a carboxy-terminal a-solenoid fold (reviewed
in Brohawn et al., 2009). Despite primary sequence divergences, these domains
are well conserved throughout evolution. As the combination of these two protein
fold types is also found in coated vesicle components (COPI, COPII, and clathrin),
it was proposed that they derive from an ancestral “protocoatomer,” that would have
had the capacity to interact with and stabilize highly curved membrane surfaces (see
DeGrasse et al., 2009 and references therein).
Another protein fold present in a few Nups corresponds to a-helices with heptad
repeat patterns (hxxhcxc, with h for hydrophobic and c for charged residues) that
allow homologous or heterologous “coiled-coil” interactions. Extended coiled-coil
domains, found in the vertebrate Nup Tpr and in its S. cerevisiae orthologs, Mlp1
and Mlp2, constitute the central element of the nuclear basket (Krull, Thyberg,
Bjorkroth, Rackwitz, & Cordes, 2004). In addition, more restrained coiled-coil
domains contribute to complex formation and NPC anchoring of “FG-Nups” (see
Brohawn et al., 2009; Devos et al., 2006).
Besides a domain contributing to their anchoring to NPCs, “FG-Nups” bear one
of the most prevalent structural motifs found within Nups. The so-called “FG
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domains” correspond to 5–50 repeats of phenylalanine-glycine (FG), Phe-X-Phe-Gly
(FXFG), or Gly-Leu-Phe-Gly (GLFG) residues, separated by variable hydrophilic or
charged spacer sequences (for a review on FG-Nups, see Terry & Wente, 2009). Such
repeats are found in 11 vertebrate “FG-Nups” and 13 S. cerevisiae Nups that localize
within the NPC central channel, the cytoplasmic filaments, or the nuclear basket
(Fig. 1.3A). FG domains are largely natively unfolded and form flexible filaments
able to take a large scale of dynamic conformations as notably described for
Nup153 (see Chapter 4 and references therein). This flexibility, along with the hydrophobic properties of these repeats, allows rapid association and dissociation of
FG-Nups with a large range of partners giving to these Nups a key function in nucleocytoplasmic transport (detailed below).
In metazoans, several of the FG-containing domains are modified by O-linked
N-acetylglucosamine (O-GlcNAc) addition on serine or threonine residues (Holt
et al., 1987). This modification leads to the recognition of several FG-Nups by wheat
germ agglutinin (WGA), a lectin that was demonstrated in early studies to inhibit
nuclear transport, possibly by generating steric hindrance (Finlay, Newmeyer,
Price, & Forbes, 1987; see also Chapter 8). Because of the enrichment for this modification within Nups, WGA also provides a convenient tool to label NPCs in fixed or
live cells (Hanover, Cohen, Willingham, & Park, 1987; Onischenko, Gubanova,
Kiseleva, & Hallberg, 2005). While the biological significance of nuclear pore
glycosylation has remained largely unknown, recent studies indicate that GlcNAc
may modulate associations between specific Nups, Nup stability, and possibly
NPC permeability (reviewed in Li & Kohler, 2014).
Other post-translational modifications described in multiple FG and non-FGNups include phosphorylations (notably mediated by cell cycle-dependent kinases,
see Section 1.3), sumoylation (Palancade & Doye, 2008), or ubiquitination
(Hayakawa, Babour, Sengmanivong, & Dargemont, 2012).
1.2 NUCLEOCYTOPLASMIC TRAFFICKING: THE RULES
OF THE ROAD
1.2.1 Investigating nucleocytoplasmic transport
1.2.1.1 Historical overview
The first EM observations of NPCs were accompanied by the evidence of material
in the process of translocating through the nuclear pores (Anderson & Beams,
1956 and references therein). EM studies in Chironomus salivary glands subsequently enabled the visualization of the nuclear export of Balbiani ring granules
that correspond to huge ribonucleoprotein particles (RNPs) (Stevens & Swift,
1966). Independently, pulse chase experiments combined with subcellular fractionation, as well as nuclear transplantation experiments, have revealed the transfer of proteins from the cytoplasm to the nucleus in HeLa cells (Byers, Platt, &
Goldstein, 1963; Speer & Zimmerman, 1968). Binucleated cells mainly obtained
1.2 Nucleocytoplasmic Trafficking: The Rules of the Road
by cell-fusion/heterokaryon assays further enabled the characterization of the
shuttling of proteins between the cytoplasm and the nucleus (see methods and references in Chapter 11 for mammalian cells and in Chapter 20 for budding yeast).
Other early transport studies were based on thin-section EM observations or localization of radiolabeled molecules by autoradiography following microinjections, notably into Xenopus oocytes (Feldherr, 1965, 1969). Thanks to their
large size, the nucleus and cytoplasm of amphibian oocytes can be manually separated at various times after injection to determine the fate of the microinjected
molecules (see Bonner, 1975 and references therein). In the past, cytoplasmic or
nuclear microinjections of colloidal gold particles, amorphous dextrans of various
sizes, recombinant proteins, or various RNA species have been successfully
employed (for reviews and references related to these techniques, see Chapters
4 and 18). Together, these early studies have revealed the main features of nucleocytoplasmic transport, namely the behavior of NPCs as sieve-like barriers, freely
permeable for small molecules, but able to selectively import or export molecules
larger than the passive diffusion limit (30–50 kDa, or 5 nm in size) in an
energy-dependent manner.
In vitro nuclear protein import assays were first performed on isolated or in vitroassembled nuclei incubated with Xenopus extracts and a fluorescently labeled nuclear
protein (Newmeyer, Finlay, & Forbes, 1986). However, one of the major advances in
the field has been the development of an in vitro transport assay based on digitoninpermeabilized vertebrate cells (Adam, Marr, & Gerace, 1990). In this assay, the
plasma membrane of tissue culture cells is specifically permeabilized with digitonin
and the system is then supplied with cell extracts or purified transport factors to reconstitute nucleocytoplasmic transport in a semi cell-free environment. Coupled with
biochemical fractionations and analyses, these experiments have led to the identification of soluble transport factors required for NPC targeting and nuclear translocation of distinct types of proteins, namely karyopherins/importins and the small
GTPase Ran (detailed below) (see Chapters 15 and 16 and references therein).
Genetic screens of yeast mutant collections (mainly S. cerevisiae) have also
enabled the identification of multiple factors contributing to nucleocytoplasmic transport. Among them, a screen for mutants that missorted a chimeric NLS-cytochrome c1
protein to the mitochondria, allowing growth on glycerol, led to the characterization of
several npl (nuclear protein localization) mutants (Sadler et al., 1989). Temperaturesensitive mtr (mRNA transport defective, Kadowaki, Zhao, & Tartakoff, 1992) mutants were initially identified based on their survival at restrictive temperature in
the presence of toxic amino acid. Their contribution to mRNA export was then validated using fluorescence in situ hybridization (FISH) using oligo-dT probes. In addition, FISH-based screens identified multiple rat (ribonucleic acid trafficking, Amberg,
Goldstein, & Cole, 1992) and brr (bad response to refrigeration, de Bruyn Kops &
Guthrie, 2001) mutants impaired for mRNA export. Besides export factors, these
screens also uncovered multiple Nups (notably members of the yeast Nup84 complex),
as well as factors involved at various stages of mRNA metabolism. Visualization of
GFP-tagged ribosomal protein localization (detailed in Chapter 20) and an in vivo
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nuclear tRNA export assay combined with overexpression screens (see Chapter 19)
also contributed to identify additional export factors. In addition, synthetic lethal or
high-copy suppressor screens based on Nup or nuclear transport mutant yeast strains
uncovered multiple players including Gle1 and Gle2 (GLFG lethal), and key RNA export factors such as Mex67 and Los1 (reviewed in Doye & Hurt, 1997).
Biochemical approaches, as well as yeast two-hybrid and three-hybrid interaction
screens, have also uncovered multiple RNA-binding proteins of which some were
subsequently demonstrated to contribute to the export of specific RNA species
(Anderson, Wilson, Datar, & Swanson, 1993; see Chapters 19 and 20).
Finally, because viruses often exploit nucleocytoplasmic transport pathways to
facilitate viral replication or escape the host antiviral response, the study of viral
interactions with the host cellular machinery also provided key tools that notably
contributed to the discovery of nuclear localization sequences and several mRNA
export pathways in vertebrates (reviewed in Yarbrough, Mata, Sakthivel, &
Fontoura, 2013).
1.2.1.2 Expanding the toolbox
Once identified, the contribution of specific factors in defined yeast nuclear transport
pathways can be further assessed using adequate functional studies. These notably
include the use of fluorescently tagged cargos or reporters (see Leslie, Timney,
Rout, & Aitchison, 2006; Chapters 14 and 20 and references therein) or FISH approaches (see Chapters 19 and 20; and see also Rahman & Zenklusen, 2013 for
single-molecule resolution FISH).
Likewise, multiple tools are nowadays available to study bidirectional transport
of macromolecules in metazoans cells (for specific tools available in Caenorhabditis
elegans, Drosophila melanogaqster, Arabidopsis thaliana, and Aspergillus nidulans, see Chapter 13; Mason & Goldfarb, 2009; Meier & Brkljacic, 2010; and
Markina-Inarrairaegui et al., 2011, respectively). Besides the widely used GFP,
the photoswitchable fluorescent protein Dronpa can be used to assess NE permeability, and, when fused to reporter proteins, to study the kinetics of active import/export
through the NPC (Ando, Mizuno, & Miyawaki, 2004; see also Chapter 10,
Section 10.2). Various export assays for RNAs have been developed (see
Chapter 18), and thanks to improved light microscopy approaches, single messenger
ribonucleoprotein (mRNP) imaging and tracking in Chironomus tentans salivary
gland cells or mammalian cells can now be used to monitor their export kinetics
(Kalo, Kafri, & Shav-Tal, 2013; Kaminski, Spille, Nietzel, Siebrasse, &
Kubitscheck, 2013).
Structural studies of transport factors have also provided considerable insight
into the elaborate protein–protein interactions that orchestrate nucleocytoplasmic
transport (for reviews, see Chook & Suel, 2011; Conti, Muller, & Stewart, 2006;
Lott & Cingolani, 2011; Stewart, 2010). To better characterize the selectivity
and permeability properties of the NPC and the contribution of FG-Nups in this
process, biophysical tools have been developed, including FG repeat hydrogels that
display permeability properties very similar to authentic NPCs (Labokha et al.,
2013 and references therein) and NPC mimics (see Chapter 17 by and references
therein).
This vast diversity of experimental model organisms and approaches has now facilitated the uncovering of the main molecular players and mechanisms contributing
to the fine-tuning of multiple nucleocytoplasmic transport pathways. As detailed below, active nuclear transport of macromolecules requires specific signals, shuttling
nuclear transport receptors recognizing these signals, transport-associated NTPases,
which account for the energy requirement of the process, and specific Nups.
1.2.2 The signals for nucleocytoplasmic exchanges
As a letter needs an address to reach its destination, cargos need specific signaling
sequences to either enter or exit the nucleus. Such amino acid sequences, named NLS
(nuclear localization signal) and NES (nuclear export signal), are defined as being
both necessary and sufficient to target proteins into and out of the nucleus, respectively. The first “classical” NLSs (cNLSs), initially identified in simian virus (SV40)
large-T antigen (Kalderon, Roberts, Richardson, & Smith, 1984), are short stretches
of positively charged residues (lysine and arginine). While these monopartite cNLSs
are found in 20–30% of the nuclear proteins, another 12–30% of them contain a
more complex “bipartite” cNLS, first characterized in nucleoplasmin (Dingwall,
Robbins, Dilworth, Roberts, & Richardson, 1988) and composed of two clusters
of charged residues separated by a spacer of 10 amino acids (for review, see
Marfori et al., 2011). However, multiple non-classical NLSs have also been identified (for review, see Chook & Suel, 2011; Chapters 14 and 16). Note that unlike
N-terminal sorting signals, NLS sequences are not cleaved and do not display any
specific position within the protein sequence. Such potential NLS can frequently
be predicted (see Marfori et al., 2011 and references therein). However, a substantial
number of proteins that are imported into the nucleus feature signals that are not
detected by NLS-based models. In addition, functional identification of a NLS is
sometimes further complicated by the coexistence of multiple distinct NLSs within
a given protein.
Characterization of NESs has lagged considerably behind the analysis of NLSs.
Typical NES sequences, also termed “leucine-rich NESs,” were first described in the
protein kinase inhibitor and human immunodeficiency virus type 1 (HIV-l) Rev protein (Fischer, Huber, Boelens, Mattaj, & Luhrmann, 1995; Wen, Meinkoth, Tsien, &
Taylor, 1995). These are short stretch of hydrophobic amino acids with variable
spacing (typically Fx2Fx3Fx2–3FxF or FPxFx2FxF, where F is an hydrophobic
residue, most frequently a leucine; for reviews, see Guttler & Gorlich, 2011;
Kutay & Guttinger, 2005). Although algorithms can be used to predict such NESs,
the fact that they share sequence similarity to regions that form the hydrophobic
cores of many proteins impairs their identification (see Chapter 16 and references
therein). In addition, as for NLSs, non-canonical export sequences have also been
characterized (reviewed in Hutten & Kehlenbach, 2007).
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16
1.2.3 A family of protein transport receptors: The karyopherins
As mentioned previously, in vitro transport assays combined with biochemical analyses have led to the characterization of soluble factors necessary for efficient transport, notably the karyopherins (or Kaps, from the greek karyon, nucleus and pherein,
to bring). Karyopherins-b, also termed importins, exportins, or transportins, are all
involved in nuclear import or export and share a similar architecture: multiple HEAT
repeats (helix-loop-helix), that contribute to substrate recognition and binding to FG
repeats present in Nups, and a N-terminal RanGTP-binding domain (see below). Despite their low sequence identity (20%), their organization and similar molecular
weights (95–145 kDa) suggest that the Kaps-b probably evolved from a common ancestor. So far, 14 Kaps-b have been identified in yeast (including 3 exportins and 1
bidirectional karyopherin) and 19 have been characterized in mammalian cells
(including 6 exportins and 2 bidirectional karyopherins) (for a comprehensive table,
see Tran, Bolger, & Wente, 2007; see also Chook & Suel, 2011; Guttler & Gorlich,
2011; Chapters 14 and 16).
There are two ways for karyopherins to recognize their cargos: a direct binding
or an indirect binding via an adaptor protein. The latter mechanism was the one initially described. Indeed, both monopartite and bipartite cNLSs are recognized by
adaptors of the importin-a family (Kap-a). While there is only one importin-a in
budding yeast (Kap60), multiple subtypes exist in most metazoans, thus providing
an additional means to finely regulate nuclear import of distinct substrates
(reviewed in Goldfarb, Corbett, Mason, Harreman, & Adam, 2004; Yasuhara,
Oka, & Yoneda, 2009). Importins-a bind cNLSs via three a-helix repeats called
armadillo (ARM) domains. They also contain an autoinhibitory N-terminal domain,
named IBB (importin-b binding), whose binding to the first described karyopherinb protein (importin-b/Kapb1; Kap95 in budding yeast) releases the ARM domains
for cargo binding. A similar IBB domain is also found in a few other adaptor proteins including snurportin (that contributes to the reimport of U snRNAs, see
Chapter 18). The IBB thus functions as a specialized NLS that evolved to transport
cargos together with importin-b (reviewed in Lott & Cingolani, 2011). However,
importin-b, as most other importins, can also bind directly to numerous cargos.
The identification of distinct binding sites and conformations further indicates that
Kapb1 may possibly interact simultaneously with distinct cargos (reviewed in
Chook & Suel, 2011). Conversely, there is a strong redundancy between the importins, especially for cargos with essential functions (Tran et al., 2007; for recent strategies to identify importin-specific cargos, see Chapter 16). This leads to a complex
system, where a discrete number of transporters are able to import a huge number of
nuclear proteins, thereby possibly providing an additional level of physiological
regulation.
A major breakthrough in the field of protein export was the demonstration that
export of “leucine-rich” NES-bearing proteins is sensitive to a cytotoxic drug, leptomycin B (LMB) (Wolff, Sanglier, & Wang, 1997). Indeed, a mutant allele of Schizosaccharomyces pombe crm1 (Chromosome Region Maintenance/Exportin-1,
Xpo1 in budding yeast) had been independently identified in a genetic screen for
LMB resistance (Nishi et al., 1994). Subsequently, several studies demonstrated that
Crm1, which belongs to the Kap-b family, is the export receptor for “leucine-rich”
NESs. LMB covalent attachment to a cysteine residue within Crm1 interferes with
NES binding in most cells (but not in S. cerevisiae, see Chapter 14; reviewed in
Guttler & Gorlich, 2011; Kutay & Guttinger, 2005; see also Chapter 16 and references therein). Subsequently, other exportins have been identified, that unlike
Crm1, export a narrow range of cargos (reviewed in Guttler & Gorlich, 2011).
Among them CAS (also called Exportin-2/Xpo2, Cse1 in S. cerevisiae) plays a
key function in nucleocytoplasmic transport as it recycles importin-a, its only known
export cargo, back to the cytoplasm. As detailed below, some exportins contribute to
RNA export. In addition, a few karyopherins (one in budding yeast and two in vertebrates) can translocate distinct sets of cargos in opposite directions.
1.2.4 The Ran GTPase: A key to transport directionality
Following the observation that translocation through NPCs requires energy, the
in vitro import assay enabled the identification of the small GTPase Ran (Ras-related
nuclear protein/TC4). Along with the observation that non-hydrolyzable analogues
of GTP inhibit the rate of in vitro nuclear import, this suggested that the metabolic
energy supplied by the RanGTPase system could provide the driving force for directional transport (Melchior, Paschal, Evans, & Gerace, 1993). As other small
GTPases, Ran (termed Gsp1/2 in budding yeast; Belhumeur et al., 1993) requires
specific cofactors to switch between its GTP and its GDP-bound forms. The RanGEF
(guanine nucleotide exchange factor, RCC1 in metazoans and its homologue Prp20
in yeast) is localized to the nucleus by virtue of its association with chromatin. Conversely, the GTPase-activating protein1 (RanGAP1, Rna1 in budding yeast) is localized in the cytoplasm, and even anchored on the cytoplasmic filaments of the NPC in
metazoans. The asymmetrical localization of these two enzymes, along with the existence of a conserved transport receptor specific for RanGDP (Ntf2), leads to the
nuclear accumulation of RanGTP. This Ran gradient is the key to establish the transport directionality (Fig. 1.4C). Indeed, all karyopherins preferentially interact with
RanGTP (as first demonstrated by Rexach & Blobel, 1995), but this interaction
has opposite consequences for importins versus exportins/cargos interactions. Indeed, RanGTP binding in the nucleus provokes the dissociation of the importin/
cargo complexes, thus leading to the nuclear release of the import substrates. The
importin/RanGTP complexes then translocate back to the cytoplasm where
RanGAP-mediated hydrolysis of GTP regenerates free importins, able to bind
new substrates (Fig. 1.4A). Conversely, RanGTP binding enhances the affinity of
exportins to their cargos inside the nucleus. Following translocation through the
NPCs, hydrolysis of RanGTP in the cytoplasm leads to the dissociation of the
Ran/exportin/cargo(s) complexes, allowing the recycling of exportins (Fig. 1.4B;
for reviews, see Conti et al., 2006; Guttler & Gorlich, 2011). These unique properties
17
FIGURE 1.4 The Molecular Mechanisms of Nucleocytoplasmic Transport
(A, B) Schematic representation of the different components of the protein nuclear import
and export machineries including karyopherins (importins and exportins) and their cargos
(import or export substrates containing NLS or NES sequences). (C) Factors required for
the establishment of the RanGTP/GDP nucleocytoplasmic gradient. (D) Schematic
representation of the mRNA export process. Note that other cellular RNAs are exported out
of the nucleus by a Ran/exportin-dependent mechanism as in (B) (see text for details).
Modified from Alves, Palancade, & Doye (2005). The nuclear pore: A control station at the nucleus
cytoplasm frontier. Biofutur, 254, 41–45 # (2005).
of Ran and Kaps thus contribute to the directionality, but also to the irreversibility of
the transport process.
1.2.5 The case of INM targeting
As INM proteins are synthesized in the endoplasmic reticulum (ER), which is continuous with the ONM (Fig. 1.1), it has initially been assumed that INM proteins
could diffuse within the pore membrane and freely reach the INM, where they would
be retained. But this diffusion–retention model was challenged by the observation
that targeting of INM proteins in HeLa cells is energy and temperature dependent
(Ohba, Schirmer, Nishimoto, & Gerace, 2004). Subsequently, an INM-sorting motif
(a region containing positively charged amino acids adjacent to the hydrophobic
transmembrane sequence) was identified. This motif is recognized by a
membrane-associated truncated form of importin-a (importin-a16 in insect cells,
Kpna-4–16 in vertebrates) that lacks the importin-b-binding domain and is required
for the concentration of some INM proteins in the vicinity of the pore membrane
(Braunagel, Williamson, Ding, Wu, & Summers, 2007 and references therein). In
both diffusion–retention and sorting motif-dependent models, translocation is anticipated to take place through the 10-nm-wide channels located at the periphery of
the NPCs, that could accommodate the diffusion or transport of INM proteins, assuming that they have a small extralumenal domain. This restriction is consistent
with early observations revealing that there is a 60-kDa extraluminal domain size
limitation for INM translocation (Soullam & Worman, 1995). More recently, however, NLS sequences that bind classical importin-a and mediate their translocation
through the NPC in an importin-b- and RanGTP-dependant manner have been
identified in several INM proteins (reviewed in Katta, Smoyer, & Jaspersen,
2013; Lusk, Blobel, & King, 2007). In this case, the peripheral NPC channels would
not accommodate the size of the INM protein–importin-a/b complexes that instead
would pass through the central channel of the NPC by using long unstructured
linkers. These unfolded linkers would slice through the NPC scaffold to enable binding between the transport factors and the FG domains in the center of the NPC
(Meinema et al., 2011). Finally, the observation that the lumenal domain of
SUN2 supports NE localization highlights the existence of INM targeting pathways
other than diffusion–retention and transport factor-mediated trafficking (Turgay
et al., 2010).
Genetic studies in yeast and RNAi experiments in mammals have further
revealed the involvement of transmembrane core scaffold Nups in INM targeting.
As these NPC alterations did not affect the kinetics of nuclear import of soluble cargos, this suggests that membrane bound and soluble cargos likely follow different
pathways through the NPC (reviewed in Antonin, Ungricht, & Kutay, 2011).
In the future, additional studies, of which some might take advantage of a recently
developed in vitro assay based on Xenopus nuclei (detailed in Chapter 9), will be
required to further clarify the various and redundant paths taken by INM proteins
to reach their final destination.
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1.2.6 Distinct pathways contribute to RNA export
With the exception of most mRNAs (detailed below), nuclear export of the various
classes of RNAs also involve Ran- and exportin-dependent pathways (for a general
review see Kohler & Hurt, 2007). Some RNAs directly interact with exportins,
thanks to specific secondary structures. This is notably the case for tRNAs whose
export, following their maturation and aminoacylation, involve Exportin-t (Xpo-t,
Los1 in S. cerevisiae) and Exportin-5 (Xpo-5, Msn5p in S. cerevisiae) (as well as
additional, factors, as detailed in Chapter 19). Likewise, highly structured small
RNAs, including microRNA precursors (pre-miRNAs), adenovirus VA1 RNA,
and human Y1 RNA, but also Dicer mRNA are exported by Exportin-5 (see
Bennasser et al., 2011 and references therein).
In addition, the Exportin1/Crm1-dependent export pathway indirectly contributes (e.g., via NES-containing adaptor proteins) to the nuclear export of several
RNAs. For instance, the export of unspliced HIV mRNAs involves the binding of
Rev, a NES-containing HIV-1-encoded protein, to the Rev-responsive element present within these viral RNAs (Fischer et al., 1995). Likewise, export of spliceosomal
uridine-rich small nuclear RNAs (U snRNAs) involves the recruitment of a NEScontaining adaptor, PHAX (PHosphorylated Adaptor for RNA eXport) that binds
to the U snRNA and its associated cap-binding complex (Ohno, Segref, Bachi,
Wilm, & Mattaj, 2000 and references therein; see also Chapter 18). Finally, ribosomal RNAs (rRNA) are exported within two distinct export competent preribosomal particles: the pre-60S subunit that contains the mature 25S, 5.8S, and 5S rRNAs,
and the pre-40s subunit that contains the 20S/18S rRNA (reviewed in Zemp & Kutay,
2007; see also Chapters 7 and 20). Export of both subunits was shown to be Crm1/
Xpo1 dependent, and a NES-dependent adaptor, Nmd3, was demonstrated to be
essential for the export of pre-60S subunits. In addition, pre-60S subunit export
requires additional export receptors, including the Mex67–Mtr2 heterodimer
(a key mRNA export factor, see below) in yeast, Exportin-5 in vertebrates, as well
as several auxiliary factors (references cited in Chapter 20; see also Wild et al., 2010
and references therein). While all the above-listed factors interact with the FG-rich
domains of Nups, a recent study has revealed the contribution in 60S export of yeast
Gle2 that interact with the non-FG domain of Nup116 (Occhipinti et al., 2013). Ribosomal subunits, likely because of their huge size, thus need to mobilize several
export pathways for an efficient passage through the NPC.
A common feature of these various RNA export processes is their tight link with
RNA maturation, which provides a quality-control mechanism ensuring that only
properly processed RNAs or assembled RNPs are exported. Likewise, mRNAs associate with multiple adaptor proteins and are exported as mRNPs, whose assembly
is closely linked with many aspects of mRNA biogenesis, including transcription,
processing, and quality control (for reviews, see Natalizio & Wente, 2013;
Oeffinger & Zenklusen, 2012; Stewart, 2010; see also Chapter 18). However,
unlike other RNA species, export of most mRNAs does not rely on exportinand Ran-dependent pathways (see, however, Natalizio & Wente, 2013 for
Crm1-dependent export of specific mRNPs). Instead, a conserved export dimer
(termed Mex67/Mtr2 in yeast and TAP/p15 or NXF1/NXT1 in vertebrates) is in
charge of mRNP export. NXF1 was initially demonstrated to directly interact with
a structured RNA sequence, termed constitutive transport element (CTE), required
for the export of specific viral mRNAs (Kang & Cullen, 1999 and references therein).
However, NXF1/yMex67 does not directly bind to cellular mRNAs. Instead, multiple trans-acting adaptor proteins that directly bind the mRNA and couple the recruitment of the export receptor with mRNA transcription, processing, and genome
organization, have been identified (Fig. 1.4D; reviewed in Natalizio & Wente,
2013; Nino, Herissant, Babour, & Dargemont, 2013; Oeffinger & Zenklusen, 2012).
Like karyopherin-mediated transport processes, this mRNA export pathway also
relies on the interaction between the export dimer and FG repeats. However, the main
difference is that directionality of mRNA export does not depend on the Ran system.
Instead, this process is driven by a DEAD box helicase (yDbp5 in budding yeast,
Ddx18 in vertebrates). This protein is positioned at the cytoplasmic filaments, thanks
to its binding to yNup159 (vertebrate Nup214). yDbp5, along with additional players
localized on the cytoplasmic side of the NPCs, notably yNup159, the mRNA export
factor yGle1, and its cofactor inositol hexakisphosphate (IP6), remodels the mRNP,
leading to mRNA release and allowing the recycling of the export factors (for review,
see Ledoux & Guthrie, 2011).
Finally, it is noteworthy that the core scaffold Y-complex (yeast Nup84/
metazoan Nup107–160) is also implicated in mRNA export (Doye & Hurt, 1997
and references therein; Vasu et al., 2001). While these Nups could act by virtue
of their function in maintaining NPC integrity, their reported physical interactions
with components of the mRNA export machinery (Resendes, Rasala, & Forbes,
2008; Yao, Lutzmann, & Hurt, 2008) suggest that they may provide additional binding sites contributing to mRNP translocation.
1.2.7 Translocation across the NPCs: A dual function
for FG-Nups as barrier and gate
Although the translocation across NPCs is at the heart of the transport process, its
precise mechanism is currently still a rather controversial issue. One thing for sure
is the key role played by the unstructured and hydrophobic FG repeated sequences
present in multiple Nups for both the establishment of the diffusion barrier and the
active transport through the pore channel. Several models have been proposed to explain the molecular mechanisms underlying the contribution of the FG-Nups in the
transport process. A common feature of all these models relies on the fact that all nuclear transport factors carry multiple low-affinity binding sites that can interact with
the thousands of FG sequences that are provided by the 200 FG-Nups present in
each NPC (for detailed reviews, see Aitchison & Rout, 2012; Terry & Wente, 2009).
A first set of models might be grouped as “virtual gating” models. The “Brownian
affinity gating” argues for a random (Brownian) movement of molecules within the
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pore channel. In this model, the FG domains set an entropic barrier that would be
overcome by the affinity of transported cargo-carrier complexes for FG-Nups
(Rout, Aitchison, Magnasco, & Chait, 2003). It was later proposed that FG-Nups
are organized in a layer anchored and closely apposed to the walls of the transport
channel. In this case, transporters are not free to move in 3-dimensions (as in the
Brownian movement) but only in 2-dimensions all along this surface, hence the name
of reduction of dimensionality, “ROD” model (Peters, 2005). In this model, passive
diffusion would be permitted through a 8–10 nm FG-free central channel. Alternatively, the unfolded FG domain might act as a “polymer brush” that would sweep
away macromolecules from their vicinity. In this context, it was proposed that transport factor binding would cause the participating FG domain(s) to collapse locally
toward their anchoring sites in the NPC (as notably observed using atomic force
microscopy), thereby reversibly releasing the entropic barrier (Lim et al., 2007).
An alternative model is based on the observation that the FG domains can interact
with each other by virtue of hydrophobic (via the phenylalanines residues) as well as
hydrophilic interactions, leading to the formation of a sieve-like meshwork. This
“hydrogel barrier” would prevent the passage of large molecules (the size of the
FG-mesh fixing the upper limit for diffusion) whereas selective translocation of
transporters would be achieved by a local dissolving of the FG–FG network.
A recent study refined this “selective phase” model by demonstrating that hydrogels
formed by distinct FG domains display distinct sieving effects, suggesting that NPCs
could contain several gel layers of distinct mesh sizes and capacities for selective
transport (Labokha et al., 2013 and references therein).
The “virtual gating” and “selective phase” models are not mutually exclusive,
and another hybrid model has been proposed, that is based on the existence of distinct
types of FG domains: FG domains (mainly FXFG) with a dynamic extended coil conformation and FG domains (mainly GLFG types) with a globular collapsed conformation. In this so-called “forest” model, the bimodal distribution of these “trees” and
“bushes” types of FG domains would create a central transporter structure and two
distinct zones of traffic along the NPC conduit: a peripheral zone, close to the core
scaffold, for small cargos, and a central zone, able to expand to facilitate translocation of large cargos (Adams & Wente, 2013; Yamada et al., 2010 and references
therein).
In all these models, the FG-network does not provide any directionality to the
transport path. In line with this concept, high-resolution single-molecule studies have
revealed that cargos explore the pore channel, until they reach the exit site
(Grunwald & Singer, 2010; Yang & Musser, 2006; see discussion in Adams &
Wente, 2013). In contrast, the asymmetric distribution of some FG-Nups along
the axis of the NPC, along with the observation that specific transport receptors display a higher affinity for FG-Nups positioned at the end of their transport path
through the NPC, had initially led to the hypothesis that an “affinity gradient”
(Ben-Efraim & Gerace, 2001; Pyhtila & Rexach, 2003) may contribute to the polarized transport through the NPC. However, genetic studies in yeast, based on the combined deletion of various FG domains, have indicated that this asymmetric
1.3 The Nuclear Transport Machinery: A Dynamic and Versatile Device
distribution of FG-Nups is dispensable for basal in vivo transport (Strawn, Shen,
Shulga, Goldfarb, & Wente, 2004).
Currently, none of these models may fully account for the complex and multiple
translocation mechanisms through NPCs. Thanks to the multiple in vitro and in vivo
approaches that have already been developed, future studies integrating both the
diversity of FG repeats and their positioning within the NPC framework will likely
help to better understand the fundamental role played by the FG-Nups in the bidirectional transport process.
1.2.8 Noncanonical transport pathways through the NE
In addition to these well-established protein or RNA transport mechanisms, there is
an ever-expanding repertoire of alternative nuclear transport pathways that may be
critical for particular cargos or under specific physiological or developmental
conditions (see Wagstaff & Jans, 2009 and references therein). Transport molecules
distinct from karyopherins include calmodulin (reviewed in Wagstaff & Jans, 2009),
or Hikeshi, which mediates the nuclear import of Hsp70s in a FG-dependent but Ranindependent manner (detailed in Chapter 15). In addition, some proteins harboring
karyopherin-like HEAT repeats, such as b-catenin, cytoskeletal proteins containing
spectrin repeats, or nuclear shuttling/signaling molecules containing ARM repeats
do not require soluble receptors and translocate independently of a carrier (reviewed
in Wagstaff & Jans, 2009; see also Kumeta, Yamaguchi, Yoshimura, & Takeyasu,
2012 and references therein). For such proteins, it is anticipated that the driving
force for their accumulation in the nucleus is their affinity for binding targets in
the nucleoplasm, a mechanism that also contributes to the nuclear accumulation
of small proteins that can otherwise diffuse through the NPCs.
Finally, although most transport through the NE envelope takes place through the
NPC, studies of herpes virus egress have uncovered an unexpected vesicle-mediated
pathway (reviewed in Yarbrough et al., 2013). Subsequently, nuclear export of
mRNP granules harboring specific synaptic transcripts in Drosophila larvae was
shown to occur through a similar pathway (Speese et al., 2012). This suggests that
herpes virus might have subverted a genuine cellular transport mechanism that could
provide an alternative way to travel across the NE, as also recently hypothesized for
INM proteins (Katta et al., 2013).
1.3 THE NUCLEAR TRANSPORT MACHINERY: A DYNAMIC
AND VERSATILE DEVICE
Beyond its role as selective gates between cytoplasm and nucleus, the nuclear transport machinery is also remodeled to adapt to specific stages of cell and organism life,
and further plays a critical role in multiple cellular processes, including cell cycle
regulation, gene expression, and maintenance of genetic integrity. Although any
of these topics would deserve a full chapter, this part aims to highlight the main
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features of these multiple and complex mechanisms, while referring to recently
published reviews in which these aspects have been exquisitely detailed.
1.3.1 NPC biogenesis throughout the cell cycle
The complex NPC architecture is fundamental to maintain the proper functioning of
pores, and consistently, the NPC scaffold is maintained with little to no turnover
throughout the life span of post-mitotic differentiated cells. In fact, the deterioration
of NPCs and the concomitant loss of the nuclear permeability barrier in aging cells
highlight the requirement for cells to maintain the integrity of this assembly
(reviewed in Toyama & Hetzer, 2013). In contrast, NPCs are dynamic structure in
dividing cells that need to assemble new pores to prevent dilution of preexisting
NPCs upon the successive rounds of cell division.
1.3.1.1 NPC disassembly
The most impressive NPC rearrangements are observed in cells characterized by an
open mitosis in which the NE and NPCs disassemble at mitotic onset to allow the formation of the mitotic spindle. Following nuclear envelope breakdown (NEBD), NPCs
are largely dismantled, yet most Nups remain assembled into stable subcomplexes in
the mitotic cytoplasm. By combining GFP-tagged Nups and specific transport reporters, the disassembly kinetics of Nups and transport competence of the NPCs could
be simultaneously monitored by quantitative time-lapse fluorescence microscopy in
single living cells (see Chapter 10 and references therein). In addition, in vitro assays
that recapitulate NPC disassembly at mitotic onset have also been developed, using
in vitro-reconstituted oocyte nuclei, or more recently, semi-permeabilized HeLa cells
(see Chapter 12 and references therein). Together, these approaches have confirmed
the implication of distinct classes of mitotic kinases, including Cdk1, that phosphorylate a broad range of Nups, most frequently on multiple sites (reviewed in FernandezMartinez & Rout, 2009). In particular, hyperphosphorylation of Nup98 was demonstrated to affect the NPC permeability barrier at an early stage of NEBD (see
Chapter 12).
1.3.1.2 Post-mitotic NPC assembly
Starting from anaphase onset, NE and Nups reassemble around the newly formed
nuclei in the daughter cells, a process required to reestablish nuclear compartmentalization. How the simultaneous and rapid reassembly of thousands of NPCs, each
composed of 500–1000 Nups, is spatially and temporally coordinated with the other
cellular rearrangements that take place upon mitotic exit is a fascinating question.
First hints into the sequential stages of post-mitotic NPC reassembly were
obtained by combining distinct antibodies in immunofluorescence studies in mammalian cells (Bodoor et al., 1999). The advent of live cell imaging has provided a
refinement of the kinetics of this highly dynamic process. As with disassembly,
the reassembly kinetics of Nups and the acquisition of transport competence of
the NPCs can be monitored by quantitative time-lapse fluorescence microscopy in
single living cells (see Chapter 10 and references therein). This assay revealed that
Nups are successively recruited to reassemble new import competent NPCs within
10 min of anaphase onset. Live cell imaging of C. elegans embryos (see
Chapter 13) or synticial Drosophila embryos (see Katsani, Karess, Dostatni, &
Doye, 2008; Onischenko et al., 2005 and references therein) provides alternative
models system that allow the simultaneous observation of multiple and successive
disassembly and reassembly reactions. All these assays, when combined with molecular perturbations, such as RNAi depletion, provided insights into the sequential
roles of various Nups in the NPC assembly process. However, in vivo depletion
studies bear several limitations due to the extent of RNAi-induced depletion, loss
of viability caused by altered transport capacities, and other secondary effects that
may occur at other stages of the cell cycle.
In addition, nuclei assembled in vitro using cell-free extracts of Xenopus eggs and
exogenous DNA or chromatin have been used extensively to study NE and NPCs
assembly (see Chapters 2, 8, and 9). This approach, combined with EM, made possible the visualization of potential NPC assembly intermediates (Goldberg, Wiese,
Allen, & Wilson, 1997). Moreover, the Xenopus system provides the possibility
to perform a “biochemical knockout” strategy to study the assembly of NPCs that
occurs during in vitro nuclear formation. Following a highly efficient depletion of
a protein of interest, it is possible to evaluate the recruitment of distinct Nups by immunofluorescence microscopy, to visualize NE integrity and NPC architecture using
EM or SEM, and to assess transport properties of the assembled nuclei, thanks to
specific reporters (Chapters 2, 8, and 9). In these assays (as also in the case of
RNAi-mediated depletion), add-back experiments further enable the characterization of specific functional domains within a given protein, thereby refining the molecular mechanisms contributing to pore biogenesis.
Together, these approaches have helped elucidate the order and interdependence
of the main Nup recruitment steps (see Schooley, Vollmer, & Antonin, 2012 and references therein). Post-mitotic assembly is initiated by the recruitment of the
Nup107–160 complex on chromatin, which is achieved thanks to its interaction with
Elys (also called MEL-28 in C. elegans) that binds directly DNA with its C-terminal
AT-Hook domain. This is followed by the recruitment of the transmembrane Nups
Pom121 (thought to be mediated by its interaction with the Nup107–160 complex),
and Ndc1. The components of the Nup93 complex, that also interact with these two
transmembrane Nups, are then incorporated in a stepwise manner in the assembling
NPCs. Nup93 subsequently recruits the Nup62 complex that, together with another
FG-Nup, Nup98, contributes to the establishment of the NPC selectivity barrier. NPC
assembly is then completed by the formation of the peripheral NPC structures
(cytoplasmic filaments and nuclear basket).
Besides their key role in bidirectional traffic in interphase, the Ran GTPase and
karyopherins also play a critical role in post-mitotic NPC reassembly. Indeed, interaction of several Nups, including Elys, the Nup107–160 complex, Nup53, and
FG-Nups, with importin-b and transportin was shown to inhibit NPC reassembly
(Lau et al., 2009 and references therein). These negative regulatory events are
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counteracted by RanGTP, whose high concentration is maintained around chromosomes during mitosis (Kalab, Weis, & Heald, 2002; see also Chapter 15). RanGTP
thus locally releases these Nups to allow their incorporation into the assembling
NPCs. This spatial information must, however, be coordinated to the temporal regulation of the process, which is likely achieved by the reversal of mitosis-specific
phosphorylation events (Walther et al., 2003; for review, see Schooley et al., 2012).
While these studies have revealed that post-mitotic assembly is initiated by the
recruitment of specific Nups on chromatin, how the ER membranes are reorganized
to enclose the chromatin and form the NE, and how this process is coordinated with
NPC reassembly remain debated (discussed in Schooley et al., 2012). Two distinct
models have been proposed: (i) NPC assembly intermediates (seeds or “prepores”)
may become subsequently enclosed by the outgrowing membranes of the reforming
NE (enclosure model). Alternatively, (ii) the ER network on the chromatin surface
may first form a closed NE into which NPC assembly subsequently proceeds
(insertion model).
Assembly of new NPCs by insertion into the intact nuclear membranes also occurs in all species during interphase, when the number of NPCs doubles in preparation for reentry into next mitosis, and is the unique mode of pore biogenesis in
organisms, such as yeast, that employ closed mitosis for cell division (reviewed
in Doucet & Hetzer, 2010; Jaspersen & Ghosh, 2012; Rothballer & Kutay, 2013).
The post-mitotic NPC insertion model is thus appealing, as it would represent a unifying mechanism for NPC assembly in all stages of the cell cycle and across species.
However, in contrast to the simultaneous post-mitotic assembly of thousands of
NPCs in metazoan cells, interphase NPC assembly occurs as a collection of sporadic
events. Although the NPCs assembled are identical, these two processes may thus
accommodate distinct mechanisms.
1.3.1.3 De novo NPC assembly
First insights into the molecular mechanisms involved in the biogenesis of new NPCs
in the intact NE (a process also termed “de novo” biogenesis) have been provided by
genetic studies in budding yeast (reviewed in Fernandez-Martinez & Rout, 2009). In
particular, members of the Nup84 complex, Pom33, and reticulons (proteins involved
in the maintenance of reticular ER membranes) are all required for proper NPC distribution, suggesting a role in NPC biogenesis or stability. A genetic screen based on
the altered localization of GFP-tagged Nups (nuclear pore assembly mutants, npa,
Ryan & Wente, 2002) revealed the contribution of additional factors in this process.
In vertebrates, approaches to examine interphase NPC assembly have been developed
using in vitro nuclear reconstitution assays based on Xenopus egg extracts (D’Angelo,
Anderson, Richard, & Hetzer, 2006). Visualization of interphase NPC formation in
mammalian cells has long been hampered by the interference of preexisting NPCs
with the observation of nascent NPCs. While total fluorescence intensity of Nups
at the NE in G1 versus G2 cells can provide clues into NPC numbers, high-resolution
cell imaging now enables the visualization of individual NPCs and the detection
of new assembly events at previously pore-free sites. In addition, a photobleaching
approach (FRAP) and an assay based on the cell-fusion technique have also been
developed to tackle this problem (see Chapter 11 and references therein).
Together, these approaches have provided the first clues on the de novo NPC assembly pathway (see reviews by Doucet & Hetzer, 2010; Jaspersen & Ghosh, 2012;
Rothballer & Kutay, 2013 and references therein). A prerequisite for the assembly of
new NPCs during interphase is the formation of aqueous pores in the NE, a step that
requires remodeling of the nuclear membranes. Although this process is far from being understood, interactions between the luminal domains of integral membrane
Nups, such as POM121, and other transmembrane proteins, such as SUN1 in vertebrates or Heh1/2 in yeast, may act in an early step to decrease the distance between
the INM and ONM. In parallel, membrane-binding scaffold Nups, such as vertebrate
Nup53, and membrane-shaping proteins like reticulons (Rtn1/Yop1 in budding
yeast, Rtn4a in vertebrates) may assist pore formation by bending the nuclear membranes toward each other and facilitating their tight approximation. Other membrane
remodeling events, such changes in the lipid composition of the NE, are also likely
involved in the formation of the pore membrane (reviewed in Rothballer & Kutay,
2013). Recruitment of membrane coat-related scaffold Nups, notably the
Nup107–160 complex (yeast Nup84 complex), as well as the previously mentioned
ALPS domain-containing Nups, could then help to stabilize membrane curvature. As
for post-mitotic assembly, this initial step is followed by the recruitment of other
Nups required for the construction of the mature NPC and the establishment of its
barrier and transport functions (reviewed in Rothballer & Kutay, 2013). Of note,
as NPC assembly was shown to proceed from both sides of the NE, preexisting NPCs
are likely required to import Nups, possibly preassembled as subcomplexes, into the
nucleus. Interestingly, both in vivo studies in yeast (Ryan & Wente, 2002) and Xenopus-based cell-free assays (D’Angelo et al., 2006) have revealed the contribution of
Ran and Kaps in de novo NPC biogenesis. However, the specific (possibly multiple)
steps to which they contribute remain uncertain (discussed in Fernandez-Martinez &
Rout, 2009).
1.3.2 Multiple functions of the nuclear transport machinery
during the cell cycle
Besides their roles in nucleocytoplasmic transport and NPC biogenesis, multiple factors of the nuclear transport machinery have also been implicated in diverse mitotic
processes. In particular, the role of Ran and karyopherins in the spatial coordination
of multiple cell cycle events has been extensively reviewed (Clarke & Zhang, 2008;
see also Lau et al., 2009; Chapter 15 and references therein). Among the contributing
mechanisms, the enrichment of RanGTP around the mitotic chromosomes provides
spatial information to locally release spindle assembly factors from Kap-containing
inhibitory complexes.
In addition, the nuclear export factor Crm1 also plays a role in mitosis through its
RCC1-dependent localization to kinetochores, where it was shown to recruit
RanBP2/Nup358 and RanGAP1. The cytoplasmic nucleoporin RanBP2 is an E3
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SUMO ligase that forms a complex with two other proteins, RanGAP1, stably conjugated to SUMO (small ubiquitin-like modifier) and the E2 SUMO-conjugating enzyme Ubc9 (RRSU complex, RanBP2-RanGAP1:SUMO-Ubc9; see Fig. 1.3A). At
kinetochores, this complex may locally regulate the RanGTP gradient and was also
shown to sumoylate both topoisomerase II, a key player in sister chromatid separation, and the chromosome passenger complex (CPC), a mitotic regulator which functions in spindle and kinetochore assembly (reviewed in Bukata, Parker, & D’Angelo,
2013; Wozniak et al., 2010).
This dual location at NPCs in interphase and kinetochores in mitosis is also an
intriguing feature of the mammalian and C. elegans Nup107–160 complex and
Elys/MEL-28 (a property, however, not shared by the Drosophila or yeast complexes). In mammalian cells, efficient depletion of this Y-shaped complex from kinetochores impairs mitotic progression. This mitotic phenotype was correlated with
altered kinetochore recruitment of specific complexes including the abovementioned RRSU and CPC complexes, and the microtubule nucleation complex
(g-tubulin ring complex, g-TuRC). In addition, the Nup107–160 complex is localized to spindle poles and proximal spindle fibers in prometaphase mammalian cells
and was demonstrated to contribute to the in vitro assembly of bipolar spindles in
Xenopus egg extracts (reviewed in Wozniak et al., 2010).
Active mitotic functions have also been assigned for Rae1, a nucleoporin sharing homology with the mitotic checkpoint protein Bub3, that is localized at kinetochores, spindle, and spindle poles in human and Xenopus mitotic cells. Rae1,
through interactions with several partners, seems to regulate spindle assembly
and chromosome segregation through multiple mechanisms (reviewed in Bukata
et al., 2013; Chatel & Fahrenkrog, 2011). Mitotic localizations and functions have
now been reported for an increasing number of Nups including Nup98, Nup188,
and Nup62 (reviewed in Bukata et al., 2013; Chatel & Fahrenkrog, 2011; see also
Hashizume et al., 2013; Itoh et al., 2013). The association of Nups with distinct
parts of the mitotic apparatus (mitotic chromosomes, spindle, centrosomes, and kinetochores) may contribute to the precise choreography of NE disassembly/reassembly with other mitotic events.
Conversely, NPCs behave as an anchor for the localization of key cell cycle regulators. Indeed, besides their localization to kinetochores, the spindle assemble
checkpoint (SAC) proteins Mad1 and Mad2 are also constitutively recruited to
the nuclear pore basket by Tpr (yeast Mlp1/2) (see Fig. 1.3A). In yeast, the shuttling
of Mad1 between unattached kinetochores and nuclear pores upon SAC activation
was demonstrated to inhibit Kap121p-mediated protein import, thus indirectly
changing spindle dynamics (Cairo, Ptak, & Wozniak, 2013). In mammalian cells,
Tpr functions as a scaffold for regulating the stability of Mad1–Mad2 before their
targeting to kinetochores, a mechanism likely underlying Tpr requirement for a robust SAC response (see Schweizer et al., 2013 and references therein). Because Tpr
is also required to recruit the SUMO-isopeptidase SENP1 to NPCs (Fig. 1.3A), these
authors further speculated that the function of Tpr in SAC might involve spatial
control of SUMO-dependent proteostasis at the NPCs. SENP proteins, and possibly
other factors associated with the nuclear basket, may also account for the implication
of Nup153 in the coordination between post-mitotic NPC basket assembly and abscission (discussed in Mackay & Ullman, 2011).
In addition, two distinct NPC-anchored pathways were found to independently
recruit the microtubule motor dynein to the NE in mammalian G2 cells, a process
contributing to the proper localization of centrosomes close to the NE in mitotic prophase. These two G2-specific mechanisms, that depend upon associations with either
the cytoplasmic filament Nup, RanBP2, or the Nup107–160 scaffold constituent,
Nup133, independently recruit dynein through distinct interaction networks
(Bolhy et al., 2011; Splinter et al., 2010). Of particular note, these two modes of
G2-specific dynein recruitment to nuclear pores were recently demonstrated to contribute to another cell cycle-dependent mechanism, namely, the apical migration of
nuclei from radial glial progenitor cells that takes place in G2 (Hu et al., 2013). These
functions in mitosis and/or nuclear migration may contribute to the developmental
defects reported in a functionally null allele of mouse Nup133 (Lupu, Alves,
Anderson, Doye, & Lacy, 2008).
These nonexhaustive examples thus highlight the multiple means of cross talk
between NPC functions and cell cycle progression, and their possible contribution
to developmental processes.
1.3.3 NPCs, nuclear organization, and gene expression
Microscopists originally noticed that chromatin is not randomly distributed at the
periphery of the nucleus and that NPCs are surrounded by heterochromatin-free areas
possibly enriched in transcriptionally active genes (reviewed in Raices & D’Angelo,
2012). This observation raised the possibility that NPCs could act as genome organizers, leading in the 1980s to the so-called “gene gating” hypothesis which stipulated that gene expression could be regulated by physical association with nuclear
pores (Blobel, 1985).
Over the past 10 years, NPCs were confirmed to be important players in the definition of transcriptionally active regions using a panel of experimental approaches,
in particular, in budding yeast (detailed in Chapter 21). On one hand, genome-wide
chromatin immunoprecipitation studies demonstrated the association of several
Nups and Kaps with actively transcribed genes and their importance in the definition
of chromatin boundaries (Casolari et al., 2004; Ishii, Arib, Lin, Van Houwe, &
Laemmli, 2002). On the other hand, GFP tagging of specific chromosomal loci with
the LacO/LacI system indicated that a number of inducible genes relocate to NPCs
upon transcriptional activation (reviewed in Van de Vosse, Wan, Wozniak, &
Aitchison, 2011). NPC anchoring of activated genes requires transient interactions
between multiple players at different stages of the gene expression process. These
include Nups, mRNA export factors, chromatin-associated proteins, specific DNA
sequences, and/or the mRNA itself (reviewed in Dieppois & Stutz, 2010). While
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relocalization of activated loci to NPCs has been shown to contribute to optimal
mRNA production, their retention at NPCs following repression is likely to represent
a form of epigenetic transcriptional memory and to prime them for faster reactivation
(reviewed in Egecioglu & Brickner, 2011). Possible mechanisms of NPC-associated
transcriptional memory include the maintenance of gene loops, as probed by chromosome conformation capture (3C), and/or the integration of specific epigenetic
marks at the anchored locus (reviewed in Egecioglu & Brickner, 2011; Raices &
D’Angelo, 2012). It has to be noted that consistent with their strategic location at
the interface between the NPC and the nuclear interior, nuclear basket-associated
proteins are especially important for these processes.
While the aforementioned experimental data were mainly collected in yeast,
accumulating evidence suggests that NPCs are also involved in the control of gene
expression in metazoans (reviewed in Capelson, Doucet, & Hetzer, 2010; Light &
Brickner, 2013; see also Chapters 13 and 21). A particular feature of the NPC-gene
connection in higher eukaryotes is illustrated by the recruitment of a subset of Nups
to actively transcribed genes in the nucleoplasm. Understanding in detail how the
association of Nups with genes, either at NPCs or within the nucleus, regulates their
expression, will be an important challenge for the future.
1.3.4 NPCs and genetic stability
Beside their functions in chromosome segregation and gene expression, Nups have
crucial roles in the maintenance of genetic integrity by directly influencing the metabolism of DNA damage. The first evidence for a connection between NPCs and the
DNA damage response (DDR) came from genome-wide screens performed in yeast
(reviewed in Bukata et al., 2013). These approaches revealed that Nups mutants are
hypersensitive to clastogen or ionizing radiation, and identified a strong genetic interaction between the NPC and the homologous recombination pathway. In particular, the Y-complex as well as Nups of the nuclear basket appear to be critical for
preventing the accumulation of unrepaired double-strand breaks (DSBs) (reviewed
in Bukata et al., 2013). The combination of imaging approaches with genetics and
ChIP studies in yeast further helped to dissect the molecular function of NPCs in
DSB repair. On one hand, the NPC seems to act as a platform capable of recruiting
several types of damaged DNA, including persistent DSBs, collapsed replication
forks, and eroded telomeres, possibly channeling them into alternative repair pathways (reviewed in Nagai, Davoodi, & Gasser, 2011). On the other hand, the reported
NPC association of enzymes of the SUMO pathway appears to be critical in preventing the accumulation of DNA damage (reviewed in Palancade & Doye, 2008).
The SUMO-deconjugating enzyme Ulp1/SENP2 and the SUMO-dependent ubiquitin ligases Slx5/8, which are both anchored to NPCs likely through interactions with
the Y-complex, are expected to target proteins of the DNA repair machinery for
desumoylation and/or ubiquitination, thereby regulating their activity in the DDR.
Accordingly, NPCs could position damaged chromosomes and their associated factors in the vicinity of these enzymes to finely tune the outcome of the DDR. While
Acknowledgments
dedicated studies as well as proteomic analyses have identified a number of DNA
repair and replication factors whose sumoylation may account for this regulation
(discussed in Nagai et al., 2011; Palancade & Doye, 2008), it is likely that similar
mechanisms could regulate multiple other cellular processes. In this respect, it is
noticeable that Ulp1 was recently shown to target for desumoylation two factors
at the chromatin/NPC interface (Bretes et al., 2014; Texari et al., 2013).
The connection between NPCs and the DDR, albeit studied largely in yeast, appears to be conserved during evolution, as revealed by the genetic instability caused
by the loss-of-function of Y-complex components in several distant species (see
Paulsen et al., 2009 and references therein). However, while the association of
Ulp1/SENP2 with NPCs is conserved in mammals, its role in the DDR remains to be
investigated. Of note, the mammalian-specific NPC localization of another enzyme
of the SUMO pathway, namely, the SUMO ligase RanBP2 (see Werner, Flotho, &
Melchior, 2012 and references therein), could also contribute to regulate SUMO
modification of targets in the vicinity of nuclear pores.
CONCLUDING REMARKS
Over the past 50 years, a combination of approaches in several model organisms has
revealed the main rules of nuclear pore organization and nucleocytoplasmic transport. Despite its repetitive and modular structure, this fascinating assembly appears
more complex and flexible than initially estimated with, for instance, its cell type or
tissue specificities. The reported importance of nuclear pore and nucleocytoplasmic
transport for several cellular processes, as well as its intimate connection with gene
expression, probably explains why several Nups were found to be mutated in cancer
(Chow, Factor, & Ullman, 2012; Kohler & Hurt, 2010) or other diseases (Jamali,
Jamali, Mehrbod, & Mofrad, 2011). Future challenges in the field will include,
among other aims, (i) a refined determination of NPC structure and biogenesis pathways, (ii) an improved understanding of the non-conventional functions of Nups and
nucleocytoplasmic transport components, in and out of the pore, for instance, at gene
loci, and (iii) a deeper comprehension of NPC composition and nucleocytoplasmic
transport pathways and their (mis)regulations occurring during development, differentiation, and disease. For this purpose, some of the traditional methods used in the
field, as well as innovative techniques and tools, are described in this volume. Importantly, certain of these approaches that have revolutionized our view of NPCs
could be usefully applied to improve our understanding of other equally complex
macromolecular assemblies.
Acknowledgments
We thank Brian Burke (Institute of Medical Biology, Singapore) for critical reading of the
manuscript, and colleagues for authorizing the reproduction of previously published images.
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We apologize to colleagues whose work could not be cited directly due to space constraints.
Work in this laboratory is supported by CNRS, University Paris Diderot and “Who am I?” laboratory of excellence (ANR-11-LABX-0071/ANR-11-IDEX-0005-01), the French National
Research Agency (ANR-12-BSV2-0008-01 to V. D.), Fondation ARC pour la Recherche
sur le Cancer (to V. D. and B. P.), and Ligue Nationale contre le Cancer (to B. P.).
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amphipathic analogue forbes

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