Download Macromolecular Transport between the Nucleus and the Cytoplasm

Biochimica et Biophysica Acta 1843 (2014) 2784–2795
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbamcr
Review
Macromolecular transport between the nucleus and the cytoplasm:
Advances in mechanism and emerging links to disease
Elizabeth J. Tran a,b,⁎, Megan C. King c, Anita H. Corbett d,⁎⁎
a
Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907, USA
Purdue University Center for Cancer Research, Purdue University, Hansen Life Sciences Research Building, Room 141, 201 S. University Street, West Lafayette, IN 47907, USA
Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
d
Department of Biochemistry, Emory University School of Medicine, 4117 Rollins Research Center, 1510 Clifton Road, NE, Atlanta, GA 30322, USA
b
c
a r t i c l e
i n f o
Article history:
Received 4 June 2014
Received in revised form 1 August 2014
Accepted 2 August 2014
Available online 9 August 2014
Keywords:
Nucleocytoplasmic transport
Nuclear pore
RNA processing
RNA export
Protein import
Karyopherin/importin/exportin
a b s t r a c t
Transport of macromolecules between the cytoplasm and the nucleus is critical for the function of all eukaryotic
cells. Large macromolecular channels termed nuclear pore complexes that span the nuclear envelope mediate the
bidirectional transport of cargoes between the nucleus and cytoplasm. However, the influence of macromolecular trafficking extends past the nuclear pore complex to transcription and RNA processing within the nucleus and
signaling pathways that reach into the cytoplasm and beyond. At the Mechanisms of Nuclear Transport biennial
meeting held from October 18 to 23, 2013 in Woods Hole, MA, researchers in the field met to report on their recent findings. The work presented highlighted significant advances in understanding nucleocytoplasmic trafficking including how transport receptors and cargoes pass through the nuclear pore complex, the many signaling
pathways that impinge on transport pathways, interplay between the nuclear envelope, nuclear pore complexes,
and transport pathways, and numerous links between transport pathways and human disease. The goal of this
review is to highlight newly emerging themes in nuclear transport and underscore the major questions that
are likely to be the focus of future research in the field.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
The primary cell biological feature of all eukaryotic cells is the presence of a nucleus, a double membrane-bound organelle that encapsulates the genetic material and physically separates the process of
transcription from the translational machinery in the cytoplasm. This
spatial compartmentalization necessitates selective and efficient transport through the nuclear pore complex or NPC, a huge macromolecular
assembly of proteins that provides a selective portal for movement
across the nuclear envelope (NE). Although the major factors required
to recognize and transport macromolecules into and out of the nucleus
through NPCs have been known for well over a decade, substantial gaps
in our understanding of the mechanisms underlying this selective
transit still exist.
A recent biennial meeting of researchers in this field held this past
year in Woods Hole, MA provided an opportunity to report on new
advances in the field of nucleocytoplasmic transport. This meeting
highlighted the cutting-edge, multidisciplinary approaches being
taken to understand not only transport across the NPC but also the
⁎ Correspondence to: E.J. Tran, Department of Biochemistry, Purdue University,
175 S. University Street, West Lafayette, IN 47907, USA.
⁎⁎ Corresponding author.
E-mail addresses: [email protected] (E.J. Tran), [email protected] (A.H. Corbett).
http://dx.doi.org/10.1016/j.bbamcr.2014.08.003
0167-4889/© 2014 Elsevier B.V. All rights reserved.
assembly of this complex structure and the coupling of nuclear transport with other cellular processes. Exciting discussions resulted from
the combined intellectual strengths of geneticists, cell biologists, structural biologists and biophysicists. While some consensus on transport
of cargoes through nuclear pore complexes is emerging, additional
functions and pathways are surfacing for NPC-associated proteins and
for the NPC itself. This complexity indicates that the transport process
is ripe for regulation at various levels, presenting additional opportunities and challenges to scientists as the basis for selective movement
across the NPC continues to be revealed.
2. The nuclear pore complex
The nuclear envelope (NE) of a eukaryotic cell is composed of an
outer and inner nuclear membrane (ONM and INM, respectively) separated by a luminal space. Distributed throughout the NE, the NPCs are
embedded at sites of fusion between the ONM and INM [1,2]. The
NPCs themselves are composed of relatively few protein components
(~30) that are repeated in an 8-fold rotationally symmetric structure,
with an additional two-fold axis of symmetry in the plane of the NE
for many components (Fig. 1) [2,3]. Studies of the mechanism by
which cargoes transit the NPC rely on building a comprehensive list of
the puzzle pieces that comprise the NPC and an image of how these
pieces assemble to form this remarkable macromolecular machine.
E.J. Tran et al. / Biochimica et Biophysica Acta 1843 (2014) 2784–2795
Cytoplasmic Filaments
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Nup120
Nup85
Seh1
Sec13
Nup145c
Nup84
Nup133
Spoke Central Tube
FG Nucleoporins
Nup84
Complex
Nuclear
Envelope
Transmembrane
Ring
Inner Ring
Linker
Nucleoporins
Nuclear Basket
Outer Ring
Core Scaffold
Nucleoporins
Fig. 1. The architecture of the eight-fold, rotationally symmetric nuclear pore complex. A schematic diagram of the primary features of the nuclear complex illustrates the sophisticated
nature of this macromolecular assembly. The NPC is embedded within a NE pore, formed by fusion of the outer and inner nuclear membranes. A transmembrane ring is present on the
outside of the pore, aiding stabilization. Within the core of the NPC are scaffolding proteins that form the inner and outer ring within the channel. At the cytoplasmic and nuclear face
are the cytoplasmic filaments and nuclear basket structures, respectively, which serve as docking sites for nuclear transport events. The FG domains of the FG nucleoporins are shown
protruding into the aqueous channel of the NPC. These domains serve as docking sites for transport receptors, allowing passage through the pore. The Nup84 complex is a substructure
located at the outer periphery of the cytoplasmic face of the NPC, which is required for the proper distribution of individual NPCs around the NE [7].
Provided by Michael Rout, The Rockefeller University, USA.
3. Architectural assembly of the NPC
The NPC is composed of a central core that mediates transit across
the NE as well as peripheral components comprised of cytoplasmic filaments and a nuclear basket that mediate transport receptor–cargo
docking (Fig. 1). The proteins that make up the NPC, collectively termed
nucleoporins or Nups, anchor the NPC to the NE and provide interaction
domains for transport events. While many researchers have helped to
define individual components of the NPC, Michael Rout (The Rockefeller
University, USA) and his collaborators have provided some of the most
comprehensive studies [4–6]. This group first reported the overall composition of the NPC in 2000 [5]. They now continue to refine our understanding of how specific Nups are organized within the larger structure
of the NPC (Fig. 1). At the meeting, Rout reported on his group's detailed
analysis of budding yeast cells harboring mutations in specific subcomplexes within the NPC. Using a combination of electron microscopy,
protein domain mapping data, and yeast genetics, they demonstrated
that mutations in genes encoding components of the Y-shaped complex, which is composed of Nup84, Nup85, Nup120, Nup133,
Nup145C, Sec13 and Seh1 in budding yeast [7,8], have differential effects on mRNA export and NPC distribution. Such functional studies
will continue to define the contributions of distinct sub-complexes
within the NPC, thereby enhancing our understanding of NPC assembly
and function.
Another approach to describe the overall architecture of the NPC is
to define the high-resolution three-dimensional structures of individual
domains of Nups and then map the resulting structures back to lower
resolution models primarily from electron microscopy. Such structural
information allows inference of both function and potential dynamics
of the larger complex [9]. Sozanne Solmaz from the Blobel laboratory
(The Rockefeller University, USA) and Andre Hoelz (California Institute
of Technology, USA) both reported structural studies of Nups that could
provide insight into the overall organization and function of the NPC.
Solmaz described analysis of the mammalian FG Nups, Nup62, Nup54,
and Nup58, using X-ray crystallography. Her studies revealed the
alpha-helical nature of these proteins, which could suggest that these
Nups have the capacity to flex and slide, thus facilitating large diameter
changes within the central transport channel of the NPC [10]. Hoelz described the structure of several Nups from the thermophilic fungi,
Chaetomium thermophilum. His structural studies of the N-terminal
domain of Nup192 reveal two rigid halves of the molecule connected
by a flexible hinge region [11]. Interestingly, Hoelz and others have
shown that the two domains are comprised of HEAT and ARM repeat
motifs [11–13], which share structural similarity with transport receptors of the importin/karyopherin family [14–17]. Complementary biochemical approaches allowed the Hoelz group to define modes of
interaction with both Nup53 and Nic96, two other components of the
NPC [11]. Thus, structural studies are beginning to yield insight into
how individual components of the NPC can be assembled to create
this enormous macromolecular channel.
Productively piecing together the high-resolution crystal structures
of individual Nup domains requires increasingly refined images of the
Nup subcomplexes and intact NPCs, particularly by electron microscopy
[18]. Martin Beck (EMBL, Germany) presented an impressive example
showcasing the value of integrating new methodologies to reconstruct
the architecture of the human NPC. Working in collaboration with the
lab of Joseph Glavy (Stevens Institute of Technology, USA), the Beck
group determined the overall structure of the human Y-shaped complex
(also known as the Nup107 subcomplex and analogous to the Saccharomyces cerevisiae Nup84 complex) by single-particle electron tomography [19]. The team also vastly improved the model of the human NPC
scaffold to 3.2 nm resolution by combining cryo-electron tomography
and sophisticated image analysis that leverages the 8-fold symmetry
of the NPC [20], allowing them to place the Y-shaped complex within
the NPC scaffold using a template-matching approach [19]. This model
was further validated by crosslinking mass spectrometry [19]. Combining these powerful techniques, this study revealed that the stoichiometry of the Y-shaped complex within the NPC may differ in human cells
compared to the Nup84 complex of budding yeast, providing a satisfying explanation to the mysterious difference in NPC mass across eukaryotic systems despite the similarity in individual Nup components [19,6].
Collectively, these studies reveal a remarkably similar overall organization of the NPC across the kingdoms of life [21] as illustrated in images
presented by Martin Goldberg (Durham University, UK) comparing
field emission microscopic images of plant and fungal NPCs (Fig. 2).
As all these multi-faceted approaches continue to improve and converge, the high-resolution structure of the NPC is beginning to come
into focus [18,22]. Researchers in the field continue to push the boundaries of technology to define the complex macromolecular structure
that allows the NPC to serve as both an effective barrier and an efficient
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A
B
*
*
*
*
Fig. 2. Field emission microscopic images of plant and fungal NPCs. . A. SEM image of the
surface of an isolated S. cerevisiae nucleus showing two nuclear pore complexes (green).
Arrowheads indicate prominent cytoplasmic filaments. B. SEM image of the surface of a
tobacco BY-2 cell nucleus. The channel of the NPCs is indicated by asterisks and the cytoplasmic ring is indicated for one NPC with an arrow. These images illustrate the conservation of the symmetric nature of NPCs and the presence of a cytoplasmic ring and/or
filaments across eukaryotes.
Provided by Martin Goldberg, Durham University, UK.
transit channel that can allow rapid and specific transport of numerous
macromolecular cargoes.
4. Transport through the pore
Movement through the NPC involves a series of steps including
docking at the periphery of the NPC, translocation through the
channel, and, finally, release from the NPC into the destination compartment [3]. With the exception of small molecules, metabolites
and proteins b ~ 40 kDa, transport through the NPC is both receptor
and energy-dependent. Nuclear transport receptors often termed
karyopherins or importins/exportins support bi-directional transport
for the vast majority of nucleocytoplasmic transport events by virtue
of their ability to interact with three distinct protein partners: transport
cargo, FG Nups and the small GTPase, Ran [23]. As shown in Fig. 3, Ran
provides directionality to the transport process by regulating the interaction between transport receptors and the selected cargo.
Cargo proteins contain specific transport signals that are recognized
by transport receptors [23,24]. The most well understood and thus
described transport motifs are the classical Nuclear Localization
Signal (cNLS) consisting of a series of basic residues [25] and the classical Nuclear Export Signal (cNES) consisting of spaced leucine and
other hydrophobic residues [26]. Cargoes containing a cNLS motif
are recognized by an importin/karyopherin α adapter protein to
form a complex with the import karyopherin receptor, importin/
karyopherin β. This trimeric import cargo complex assembles in
the cytoplasm in the absence of RanGTP and is disassembled when
RanGTP binds to importin/karyopherin β in the nucleus [27]. However, the cNLS import pathway is a specialized import pathway in that
it utilizes an adaptor, importin α. Indeed, most transport cargoes are
likely recognized directly through binding of a specific transport receptor from the importin/karyopherin β family (Fig. 3). Export
complexes, such as the cNES-dependent pathway that use the
importin/karyopherin β export receptor termed CRM1 [28–30], form
obligate trimeric complexes comprised of the transport receptor, the
cargo, and RanGTP. This export complex is disassembled in the cytoplasm when Ran hydrolyzes GTP to GDP. Congruent with this mechanism, levels of RanGTP are elevated in the nucleus, where export
complexes assemble and import complexes disassemble, due to the nuclear localization of the RanGTP exchange factor (GEF) that promotes
generation of RanGTP from RanGDP and the cytoplasmic localization
of the Ran GTPase Activating Protein (GAP) that stimulates the GTP hydrolysis activity of Ran [31]. Thus, the GTP-bound state of Ran defines
the compartmental requirements for cargo loading and delivery.
The classical import and export pathways represent one route for
cargoes to transit the NPC (Fig. 3). However, there are more than 20
transport receptors in mammals and 14 in budding yeast [32]. Thus,
the absence of a “classical” transport signal in no way precludes
transport receptor-mediated transit of a cargo into or out of the
nucleus. Some of the signals recognized by the other members of
the transport receptor family have begun to be defined through
structural studies of receptor–cargo complexes [23,24]. One transport
signal that has emerged from such an approach is termed a Proline Tyrosine (PY)-NLS, which is found in a number of RNA binding proteins
[33]. Interestingly, the PY-NLS is defined not so much by the primary
amino acid sequence but more by general structural features, making
this motif challenging to identify by simple sequence analysis [34]. An
ongoing goal for the field is to continue to define multiple cargo proteins that interact with specific transport receptors as well as to provide
atomic level insight into mechanisms of recognition. Such approaches
may lead to the development of tools to identify transport pathways/
receptors for specific cargoes, which can only currently be inferred for
the classical nuclear import and export pathways.
5. The NPC permeability barrier
A major open question in the nuclear transport field is the biochemical basis of the selective transport barrier within the NPC. The NPC
supports two fundamental activities: 1) it restricts molecules greater
than ~ 5 nm in diameter from passing freely through the channel
while; 2) simultaneously supporting the signal-dependent transit of
larger cargo molecules through their interaction with transport receptors [3,35]. At the heart of these activities lie the phenylalanine glycine
(FG) repeat-containing Nups or FG Nups (reviewed in [36]) that line
the interior of the NPC (Fig. 4). These natively unfolded FG domains
[37,38] both define the effective sieve size of the NPC through intraand intermolecular interactions and serve as interaction sites for soluble
transport receptor–cargo complexes to facilitate their translocation
across the NPC [39–41]. Several models for the physical nature of the
FG network have been developed in the field, providing rich fodder for
discussion [40,42–44] (Fig. 4).
The selective phase model [45,46], presented by Dirk Görlich (MPI
for Biophysical Chemistry, Göttingen, Germany), relies on two principles. First, barrier-forming FG repeat domains (notably those from
metazoan Nup98 and its yeast paralogues Nup100 and 116) engage in
multivalent cohesive interactions, forming a sieve-like FG hydrogel
whose mesh size sets an upper size limit for passive NPC-passage
(Fig. 4A). Second, FG repeat–repeat interactions disengage locally and
transiently when a transport receptor binds the corresponding FG motifs, allowing the transport receptor to “melt” the gel and consequently
pass through the hydrogel. Harnessing the powerful Xenopus and budding yeast model organisms, Görlich provided evidence that cohesive
FG domains can form an FG hydrogel with NPC-like selectivity, excluding inert macromolecules ≥5 nm, but allowing a N1000 times faster influx of the same macromolecule bound to a transport receptor [42,47].
He also showed that replacing the cohesive Nup98/Nup100/Nup116
FG domains with non-cohesive FG domains is lethal in S. cerevisiae
and disrupts the selectivity barrier of Xenopus NPCs.
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Fig. 3. Many pathways exist for transport into and out of the nucleus. The vast majority of nuclear transport depends on the small GTPase Ran and nuclear transport receptors most often
termed karyopherins but also importins/exportins. During cargo import (top), transport receptors (shown in shades of blue) associate with cargo (shown in gray) in the cytoplasm
through either direct recognition of nuclear localization signals (NLS) or in the case of the classical NLS (cNLS) through the aid of the adaptor protein, importin/karyopherin α (Kap60
in budding yeast). These complexes transit the pore into the nucleus whereby cargo release is triggered by the binding of RanGTP to the receptor–cargo complex. Cargo export (bottom)
occurs through the formation of an obligate trimeric complex consisting of RanGTP, export cargo (shown in gray/black), and transport receptor (shown in shades of blue). This trimeric
complex moves through the pore and is then disassembled in the cytoplasm when Ran hydrolyzes GTP to GDP. The GTPase activity of Ran is specifically localized to the cytoplasm by the
presence of GTPase activating proteins. RanGDP is re-imported into the nucleus and then “re-loaded” with GTP through the activity of the Ran guanine nucleotide exchange factor or
RanGEF, which is associated with chromatin. The compartmentalization of RanGTP/GDP confers directionality on the system. Thus, there are numerous transport pathways to move macromolecular cargoes into and out of the nucleus. The vast majority of the targeting signals required for these transport pathways have not yet been defined.
Another model, termed the Kap-centric model [48,49], was presented at the meeting by Roderick Lim (University of Basel, Switzerland),
who proposed that Kaps (transport receptors) serve as integral, possibly
regulatory constituents of the NPC barrier mechanism. This model is
founded on the idea that each FG Nup can bind multiple Kaps, predicting
that cargo-bearing Kaps might interact more transiently with FG Nups
that are already saturated with Kaps as they escort the cargo through
the central channel (Fig. 4D) [48,49]. Such a model could help explain
the rapid transit time of transport receptor–cargo complexes through
the NPC. Lim further demonstrated how this so-called “dirty velcro”
effect could be applied in a biomimetic manner to regulate the twodimensional diffusion of Kap-functionalized 1 μm-diameter colloidal
beads on glass slides decorated with FG Nup molecular brushes [50]. Extending the question to consider how very large cargo molecules pass
through the NPC is important as fully assembled ribosome subunits
can exit the nucleus following assembly. Siegfried Musser (Texas A&M
University) addressed this question by combining single molecule fluorescence microscopy and mathematical modeling. His work provides
evidence that large cargoes require the binding of multiple transport receptors to transit the NPC [40], a process in which multivalent interactions between FG repeats and transport receptors are essential. While
progress is being made in understanding how the FG Nups can function
simultaneously as an efficient barrier and a medium for rapid transit,
further work is required to ultimately define the precise biophysical
properties that allow such diametrically opposed functions.
Whereas the FG repeats provide the permeability barrier within the
NPC, the directionality of transport has been thought to reside solely in
the docking and release steps of the receptor–cargo complexes with peripheral components of the NPC [51]. In contrast to this predominant
view, Karsten Weis (ETH Zurich, Switzerland) provided evidence for a
Ran-dependent step for receptor interaction with FG repeats. Initial
analyses revealed that the transport receptor importin β can dock at
the NPC but cannot translocate in the absence of Ran. Fluorescence recovery after photobleaching (FRAP) experiments using permeabilized
HeLa cells revealed the presence of ~20 RanGTP-sensitive importin βbinding sites, several of which reside within the FG repeat Nup,
Nup153. Moreover, importin β binding to the FG repeat region of
Nup153 (Nup153FG) is dependent on RanGTP, as evidenced by the
use of the GTPase-deficient Ran-Q69L variant. A Ran-dependent gate
through the NPC would rectify the fact that the NPC FG rich core is not
selective or directional. Whether this model is generally applicable to
other transport receptors and FG Nups or if this Ran-dependent binding
site is exclusive to Nup153 remains to be determined. Complementing
these experiments, Edward Lemke (EMBL, Germany) provided a structural analysis of the Nup153FG-importin β complex through the combined use of single molecule-FRET (sm-FRET) [52] and molecular
dynamics simulations. These analyses suggest that the Nup153FG
region has a collapsed structure in contrast to the extended nature generally attributed to natively unfolded FG domains [38]. Moreover, the
conformation of the structure was insensitive to binding of importin β.
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Fig. 4. Transport selectivity is maintained by the FG barrier within the NPC. The question of how the NPC serves as both an efficient barrier and rapid transit machine remains a point of
debate. Several models were discussed at the meeting. Prevailing transport models advocate that the barrier mechanism is composed of FG domains. In all cases, selective transport is exclusive to karyopherins (transport receptors; dark green) that bind the FG repeats via multivalent interactions. Small molecules (small red watermarked) diffuse freely through the barrier
whereas large non-specific molecules (large red) are withheld due to insufficient interaction with the FG repeats. The models differ with respect to the nature of the FG barrier and its
interactions with transport receptors. A. The hydrogel model is based on cohesive interactions between FG repeat domains that create a sieve-like barrier that is selectively permeable
to transport receptors [39]. B. The brush model is based on the increased extensibility of the FG Nups due to steric repulsion that wins against competing cohesive interactions [143]. C.
The forest model combines aspects of the hydrogel and brush models and is based on the concept that individual FG Nups have distinct properties, being either trees (favoring an extended
conformation) or shrubs (favoring a compact conformation). Cohesive interactions also play a role in this model, which suggests that distinct “zones” through the NPC may be taken by
individual cargo–transport receptor complexes [144]. D. A Kap-centric NPC barrier mechanism model is based on the ability for the FG domains to bind and accommodate large numbers of
Kaps (transport receptors) at physiological concentrations [48,49]. Owing to strong binding avidity, the Kaps that reside within the FG domains (dark green) are slow and form integral
barrier constituents. Nevertheless, this is a prerequisite for weakly-bound Kaps (light green) that dominate fast transport due to limited penetration into the pre-occupied FG domains
(e.g., aka the “dirty velcro” effect).
Adapted from material provided by Roderick Lim and Larisa E. Kapinos.
An interesting question to address will be whether RanGTP binding
might promote a structural change to a natively unfolded state, in line
with other FG repeat domains. Together these studies shed light on
the mechanisms that ensure rapid yet selective transit through NPCs,
a question that has been at the forefront of this field for many years.
6. Not all NPCs are created equal
A long-standing question in the field of nucleocytoplasmic transport
is whether all NPCs within a given organism are equivalent and thus
equally capable of mediating the entire spectrum of nuclear transport
events. Classic experiments performed by Dr. Carl Feldherr in the
1980s demonstrated that gold particles of different sizes could be
transported simultaneously into and out of a single NPC [53]. This experiment has been cited as evidence that all NPCs are competent for
both import and export and sometimes extended to suggest that all
NPCs within a given organism are equivalent. However, more recent
findings bring this point of view into question. At the meeting, several
researchers presented evidence that NPCs can differ from one another
in specific tissues, organisms or developmental stages.
Perhaps the most striking evidence in support of NPC diversity came
from Tokuko Haraguchi (National Institute of Information and Communications Technology, Japan) who reported on her studies in Tetrahymena. Tetrahymena contain two functionally and structurally
distinct nuclei termed macronuclei (MAC) and micronuclei (MIC)
[54]. Haraguchi's group used a biochemical approach to isolate the
two nuclei and identify the Nups present in each form [55]. She reported that the isolated NPCs from the MAC and MIC nuclei have
common core Nups and share similar overall architectures. However,
she also described distinct differences including at least four Nup98 variants with two distinct forms of Nup98 present in each nucleus type [55,
56]. Such differences in composition could underlie some of the functional differences between these nuclei, which are not only distinct in
size but also in transcriptional activity and cell cycle control despite
their presence in the same organism.
Interestingly, Nup98 also emerged as a protein of interest in work
described by Geraint Parry (University of Liverpool, UK). His systematic
efforts to define the function of Arabidopsis Nups using insertional mutagenesis have begun to reveal those Nups that are essential in this
plant model and those that play key roles in specific processes [57].
His genetic studies reveal two distinct Nup98 genes encoding proteins
that appear to participate in different functions. He reported that further
work will be required to define the functions of these dual Nup98
proteins.
Two presentations addressed the related question of whether individual nuclei can have distinct functions within a given tissue. Kate
Loveland (Monash University, Australia) presented studies that examine the expression and function of importin/karyopherin α family members during spermatogenesis [58]. These studies reveal clear functions
for different family members throughout the course of differentiation,
providing evidence that different transport receptors can confer distinct
transport properties. Differences in import receptor expression levels
E.J. Tran et al. / Biochimica et Biophysica Acta 1843 (2014) 2784–2795
also support a model where there are specific functions for importin/
karyopherin α family members. For example, importin α2 levels are
more than 2-fold higher in spermatocytes than in spermatids while no
significant difference was observed for importin α4 [59] suggesting a
model where demands for these receptors differ at specific developmental stages. In another presentation analyzing nuclear protein import
in a specific cell type, Grace Pavlath (Emory University, USA) presented
studies examining import of proteins into the individual nuclei of multinucleated muscle cells. Results from this analysis provide evidence that
the distinct nuclei within multi-nucleated muscle cells can have different import competence from one another despite a common cytoplasm
within the multi-nucleated muscle fiber. As her work employed in vitro
transport assays where all soluble factors are supplied by exogenous,
added cytosol [60], differences in import-competence must be linked
to the nuclei, presumably the NPCs, rather than soluble transport receptors. Taken together, these studies provide evidence that differences in
nuclear transport can occur both through regulation of the transport
receptors and NPCs. The challenge is to define the mechanisms that
underlie this regulation and then understand how such regulation
may influence tissue-specific function(s).
Such work provides evidence that even within a given organism or
cell type, not all NPCs are equivalent. Additional studies in these systems
can provide more detailed insight into how transport mechanisms are
controlled. Once the details of the permeability barrier function of
NPCs are delineated, there will be additional work to define regulatory
pathways that modulate the properties of the NPC in different contexts.
7. Translocation of proteins to the inner nuclear membrane (INM)
A molecular mechanism for integral nuclear membrane protein
movement to the INM represents a major gap in the trafficking
field. There are two major models for INM transport [61]: the
diffusion-retention model [62] and the Kap-dependent model [63].
To define the mechanism for INM transport, Ulrike Kutay (ETH
Zurich, Switzerland) presented an elegant strategy for measuring
the rate of transport of an integral membrane protein reporter.
This approach involved the knowledge that INM transport is sizedependent, with proteins bearing extra-luminal domains greater than
60 kDa unable to translocate through NPCs to the INM. This feature was
exploited to uncouple membrane integration into the ER from transport
to the INM. Reporter proteins were then rendered transport-competent
by size reduction using protease cleavage in semi-permeabilized cells,
allowing measurement of INM transport kinetics. Transport of two different reporter proteins derived from the INM proteins, Lamin B receptor
(LBR) and SUN2 [64,65], could be reconstituted by the addition of a
HeLa cell lysate and an energy-regenerating system. However, the INM
reporter proteins translocate to the INM in the absence of karyopherins,
suggesting that energy is required for a poorly-understood step in the
process, perhaps to support mobility of cargo from their site of synthesis
to the outer aspect of the NPC. RNAi of specific Nups revealed “leaking” of
the large LBR reporter into the INM, consistent with transport of membrane proteins being hindered by the central NPC scaffold. The Kutay
group then utilized mathematical modeling to integrate the kinetic
data. Their preliminary model revealed that targeting of LBR can, in principle, be described through a diffusion-retention mechanism. Further
work is underway to bring together the results of this work into a
comprehensive model for the targeting and localization of INM proteins.
In support of a Kap-dependent model for INM transport, Gino
Cingolani (Thomas Jefferson University, USA) provided evidence for a
direct interaction between the NLS of the yeast INM protein, Heh2,
and karyopherin Kap60 (importin α in vertebrates). Interestingly, binding studies revealed a complex series of mutually exclusive interactions
between the Importin β-binding domain of Kap60, the NLS of Heh2, and
the nucleoporin, Nup2. Moreover, Nup2 is required for the proper localization of both full-length Heh2 [63] and a Heh2-NLS-containing membrane protein reporter to the INM. Future studies will reveal if there are
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multiple mechanisms for the translocation of integral membrane proteins to the INM or if these alternative pathways represent divergent
trafficking mechanisms between humans and fungi.
8. The NE, lamins and LINC complex
In multicellular eukaryotes, the INM is associated with a class of intermediate filaments called lamins. The proteins that make up the nuclear lamina, lamin A, B and C, were described almost 35 years ago
[66]. Despite this fact, the precise function(s) of the nuclear lamins are
not fully understood. Key clues into their molecular roles have come
from the study of human lamin diseases called laminopathies, which
point to roles in cellular signaling pathways as well as maintenance of
nuclear architecture and NPC positioning within the NE [67]. Larry
Gerace (The Scripps Institute, USA) presented evidence that the
lamin-interacting protein Lem2 may be a key integrator for cell signaling pathways during myogenesis. Lem2 is one of four LEM domain proteins in humans (LAP2, emerin and Man1 are the other three). The LEM
proteins are thought to carry out multiple functions, including playing
roles in cell signaling [68]. Gerace's group demonstrated that knockdown of Lem2 in muscle cells results in a MEK1-dependent myogenic
defect, indicating a deficiency in mitogen-activated protein kinase
signaling (MAPK signaling). Interestingly, Yosef Gruenbaum (Hebrew
University, Israel) connected the lamin network to mTOR signaling
and regulation of fat content in Caenorhabditis elegans. He showed that
this interplay is mediated through ATX-2, whose expression level is inversely proportional to body size [69]. Furthermore, the function of
ATX-2 requires lamin, S6K and an intact mTOR signaling pathway. Because the mTOR pathway plays a major role in translational control
[70], these data suggest that lamins may transmit signals not only to
the gene expression network in the nucleus but also to the translational
pool within the cytoplasm.
A complex of transmembrane proteins termed the LINC complex
(linker of nucleoskeleton and cytoskeleton) spans the NE and connects
the cytoplasmic cytoskeletal filaments with the lamin network [71]. Iris
Meier (The Ohio State University, Ohio) presented an elegant complementary genetic and cell biological study connecting plant-specific
LINC proteins to male fertility in Arabidopsis. The LINC complex is composed of SUN and KASH domain proteins, which are transmembrane
proteins that interact with each other in the NE lumen and extend
into the nucleus or cytoplasm, respectively [72,73]. Arabidopsis encodes
two plant-specific, KASH-like proteins called WIP1 and WIP2 (WPPinteracting proteins) [74]. Consistent with a role in nuclear membrane
architecture, Meier reported that loss of the WIP proteins causes severe
nuclear morphology defects. Furthermore, her group identified two additional NE proteins, termed WIT1 and WIT2 (WPP-interacting tail proteins) and demonstrated that the WIT proteins are required for pollen
tube nuclear movement and sperm cell delivery [75]. Because simultaneous mutation of the two WIT and two WIP genes results in reduced
seed formation, this discovery may have broad agricultural applications.
Furthermore, a conserved role for LINC complexes in meiosis across
eukaryotes [76], suggests that insights from this model system will
contribute to our knowledge of fertility defects in humans.
9. Messenger RNA processing, assembly and export
Transport receptor-mediated mechanisms move most soluble proteins, small RNAs and ribosomes through NPCs. In contrast, messenger
RNA (mRNA) export is not dependent on any exportin/karyopherin or
Ran [51]. Rather, mRNA export requires recruitment of the mRNA export receptor Mex67 in fungi (Tap or NXF1 in multicellular eukaryotes)
and a heterodimerization partner, Mtr2 (p15 or NXT1), whose binding
to mRNA is dependent on proper pre-mRNA processing in the nucleus
(Fig. 5). Recruitment of Mex67/Mtr2 in multicellular eukaryotes is highly linked to splicing [77] and 5′ capping [78] or specific recruitment sequences [79,80], whereas, in budding yeast, recruitment is dependent
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Fig. 5. Messenger RNA (mRNA) assembly is required for transport through the NPC.
Export-competent mRNAs are assembled in the nucleus during transcription and mRNA
processing steps. This includes binding of transport adaptor proteins such as Yra1, Npl3
and Nab2 to mRNA. These adaptor proteins (shown in gray) recruit the mRNA export
receptor heterodimer, Mex67/Mtr2 (TAP/p15 in mammals), and facilitate docking of the
mRNA–protein complex (mRNP) with the proteins at the nuclear face of the NPC
(i.e., Mlp1/2) [145]. Direct binding of Mex67/Mtr2 to FG repeats within the NPC facilitates movement of the mRNP to the cytoplasm where the activity of the RNA helicase
Dbp5 promotes mRNP remodeling and release into the cytoplasm. Proteins that remain associated with the mRNP in the cytoplasm can then influence mRNA stability,
cellular localization and/or translational efficiency of the message.
Provided by Benoit Palancade, Institut Jacques Monod, France.
on multiple processing steps [81] that are thought to be linked to
accurate termination and 3′ end formation [82], although defining
mechanisms that underlie this coordination remains an area of active
investigation [83]. Because Mex67/Mtr2 lacks the ability to recognize
RNA directly, recruitment is accomplished through interaction with
other RNA-binding proteins such as Yra1 (Aly in humans) [84,85],
Npl3 [86,87], and Nab2 [88,89]. Many questions about how adaptor recruitment and assembly is coordinated with assembly of an exportcompetent mRNP remain. While some studies show recruitment of different factors at specific stages of maturation, e.g., splicing, termination,
polyadenylation (Fig. 5), other work shows that Mex67 is recruited cotranscriptionally [90]. The coupling of mRNA maturation with nuclear
transport provides a layer of quality control to the gene expression process by preventing translation of potentially deleterious gene products
[91,92]. Following recruitment of Mex67/Mtr2 to a given mRNA, the
complex docks at the NPC and then migrates through the channel via
interaction of Mex67/Mtr2 with FG Nups [93]. The mRNA export receptor–cargo complex is disassembled at the cytoplasmic face of the NPC
through the action of the ATPase and RNA helicase, Dbp5, which
triggers release of Mex67/Mtr2 from the mRNA in a manner analogous
to the role of Ran in transport receptor-dependent export [87,89].
Whereas the identity of the factors that are required for efficient
mRNA export is established, the mechanisms for choreographing the
numerous sequential steps in the formation of an export-competent
mRNP are not well understood. These steps include capping, premRNA splicing, termination and polyadenylation, all of which have
been linked to mRNP assembly and efficient export through the NPC
(Fig. 5). Elizabeth Tran (Purdue University, USA) presented evidence
that the RNA helicase Dbp2 in budding yeast (p68/DDX5 in humans)
controls RNA structure during transcription. Dbp2 is an active helicase
in vitro that associates directly with transcribed chromatin [94,95].
Moreover, Dbp2 is required for the efficient assembly of Yra1 and
Mex67 onto mRNAs as well as for efficient transcription termination.
A model was presented whereby Dbp2 controls RNA folding during
transcription to prevent the formation of RNA structures refractory to
mRNP assembly, suggesting that RNA structure may add an additional
layer of complexity to the mRNA export process.
To examine the regulation of key steps in the assembly of exportcompetent mRNPs and interactions with the NPC during docking and
export, Catherine Dargemont (Hospital Saint Louis, France) and Benoit
Palancade (Institut Jacques Monod, France) explored the role of posttranslational modifications [96–98]. Dargemont reported on her studies
to examine post-translational modifications of Nups. Her work revealed
interplay between ubiquitin and SUMO modification of Nup60 that is
linked to cell cycle progression. Using a newly developed spinach
aptamer-based mRNA visualization method in living yeast [99],
Dargemont showed that specific post-translational modifications of
Nup60 are involved in nuclear export of polarized mRNAs. Understanding how these modifications modulate the function of the NPC and defining the fraction of Nups modified at any given time remains a goal
of her research. In contrast to examining modification of Nups,
Palancade analyzed the requirement for SUMO modification of RNA
binding proteins within mRNP export complexes. His group exploited
budding yeast mutants of the Ulp1 SUMO protease [100] coupled with
a proteomic approach to identify changes in mRNP complexes when
SUMO modification is impaired. While several complexes analyzed
were unaffected by this perturbation, Palancade reported specific
changes in the association of the THO complex with mRNPs. Further
studies identified a specific class of stress–response mRNAs that showed
altered levels in the absence of SUMO modification [101]. These studies
lay the groundwork for understanding how SUMO modification as well
as other post-translational modifications modulates biogenesis and export of specific classes of mRNAs. Such experiments also highlight the
question of whether specific RNA binding proteins mediate export of
classes of mRNAs or whether a core cadre of mRNA export factors is required for all mRNA transcripts. These are future challenges for the field.
Once mature mRNPs are assembled within the nucleus, there is great
interest in understanding the kinetics of the export of these complexes.
Single transcript analyses of mRNA export have revealed that the
docking and release steps are rate limiting for mRNA export [102]. Furthermore, mRNAs scan along the NE prior to export [103]. However,
these studies have largely been performed in mammalian tissue culture
systems. David Grünwald (University of Massachusetts Medical School,
USA) reported on attempts from his lab, Karsten Weis' lab and Ben
Montpetit's lab (University of Alberta, Canada) to combine the power
of S. cerevisiae genetics with single transcript studies. Their team is focused on determining the kinetics of mRNA export in the budding
yeast system and the impact that mutations in various export factors
have on NE scanning, docking, transport, and release. To accomplish
this goal, these laboratories have worked to develop a microscopy
method to analyze the movement of individual transcripts in living
yeast cells (Fig. 6). Preliminary data suggest that mRNAs are rapidly
exported following their interaction with the NE and these mRNAs do
not exhibit observable NE scanning in wild type yeast cells. However,
cells harboring mutations in the genes encoding either the RNA helicase,
Dbp5 [104], or the cytoplasmic nucleoporin, Nup159 [105], display increased NE scanning events. This finding suggests that scanning may
be an indicator of slower or inefficient mRNA export in these mutants.
Furthermore, Grünwald noted that transcripts can be re-imported into
the nucleus following initial transport into the channel in dbp5 and
nup159 mutant cells, supporting the idea that mRNAs can move bidirectionally within the NPC [102]. The model that emerges from this
study is that failure of Dbp5 to remodel the exported mRNP on the cytoplasmic side of the NPC and remove export factors, such as Mex67/Mtr2,
allows for re-import. His group is now expanding their analysis to
E.J. Tran et al. / Biochimica et Biophysica Acta 1843 (2014) 2784–2795
2791
Fig. 6. Improved methods for imaging single mRNA molecules in S. cerevisiae. A. Budding yeast cells imaged in buffer show high contrast in Phase and DIC. B. Refractive Index (R.I.) differences between immersion and buffer are high for use of high N.A. objectives leading to a loss of emission light. The R.I. of yeast cells is unknown. C. Consequently, at viable excitation light
power mRNA fluorescent signal from mRNA in the budding yeast cell is very dim. D & E. Modification of imaging conditions leads to reduced contrast in Phase (DIC contrast does not yield
image) resulting in (F) improved mRNA signal for low light imaging.
Provided by David Grünwald, University of Massachusetts Medical School, USA.
include other mRNA export mutants with the hopes of building a spatiotemporal model of when and where these various mRNA export factors
function.
Cytoplasmic events such as translational control and mRNA degradation can also be coupled to mRNA export. Susana RodriguezNavarro (Principe Felipe Research Institute, Spain) provided characterization of a largely unstudied Mex67/Mtr2-adaptor protein in
budding yeast called Mip6. Mip6 was originally identified as a Mex67interacting protein (MIP) in a yeast two-hybrid screen [106]. However,
the role of Mip6 in mRNA export was not defined. Rodriguez-Navarro
showed that Mip6 shuttles between the nucleus and the cytoplasm in
a Mex67-dependent manner, suggesting a possible role in mRNA export. However, the deletion of MIP6 did not cause the accumulation of
poly(A) RNA within the nucleus, inconsistent with a general mRNA
export role. Instead, Rodriguez-Navarro found that the Mip6 protein accumulated in heat-induced stress granules in response to heat shock.
These observations suggest that Mip6 may couple mRNA export with
translational control.
These studies highlight some of the complexities of defining the molecular nature of export-competent mRNPs, the selectivity for export of
mature mRNPs through the NPC and the remodeling necessary to prepare the exported mRNAs for a productive life in the cytoplasm. Layered
on top of these basic questions are several issues including whether all
mRNAs are packaged and exported in the same manner and how
these pathways are controlled. Cutting-edge approaches will continue
to address these questions as there is still much to learn.
10. Transcriptional control at the NPC
Both the NPC and the NE associate with transcriptionally active and
repressed genes, suggesting that factors at the nuclear periphery contribute to control of gene expression [107,108]. This control could be accomplished by the regulation of transcription factors and/or chromatin
structure to alter the accessibility for RNA Polymerase II (RNAP II).
Studies in a number of different model systems have documented the
re-localization of inducible genes from the nucleoplasm to the nuclear
periphery upon transcriptional activation, suggesting a link between
transcriptional induction and NPC association [109]. Both Francoise
Stutz (University of Geneva, Switzerland) and Richard Wozniak
(University of Alberta, Canada) provided evidence that this process involves a cycle of SUMOylation. Using the galactose-inducible GAL1
gene in budding yeast, the Stutz laboratory found that the NPCassociated SUMO protease Ulp1 [100] is required for normal induction
of the GAL1 gene. The GAL1 transcriptional co-repressor Ssn6 is a target
of Ulp1 deSUMOylation, suggesting that recruitment of the GAL1
gene to the NPC contributes to optimal activation kinetics by favoring deSUMOylation of chromatin-associated factors [110]. Richard
Wozniak then showed that both SUMOylation and deSUMOylation
(also mediated by Ulp1) events are necessary for NPC targeting of
the inducible gene INO1 in budding yeast. For example, cells that express a Ulp1 mutant that does not associate with NPCs exhibited
both drastic decreases in INO1 recruitment to the NPC and reduced
INO1 transcription. Taken together, these studies suggest that
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changes in the SUMOylation state of chromatin-associated proteins
regulate NPC targeting and the transcriptional state of inducible
genes.
A unique mechanism for transcriptional control at the NPC is the
modulation of gene expression through epigenetic memory, an area of
research largely pioneered by Jason Brickner (Northwestern University,
USA). The concept of transcriptional memory first originated from the
observation that genes are more rapidly re-activated in subsequent
rounds of induction than the first activation event [111,112]. This activity is associated with the movement of inducible genes to the NPC, suggesting that specific elements within the chromatin may be involved in
memory. The Brickner lab has been exploring the DNA sequences responsible for the inducible association of target genes with the NPC.
These sequences, called “zip codes”, are found in the INO1, TSA2, and
HSP104 genes of S. cerevisiae [113]. Strikingly, Brickner presented evidence that epigenetic memory also occurs in human cells at the Interferon γ gene [114]. Using HeLa cells as a model system, he showed that the
Interferon γ gene is more rapidly reactivated as compared to the initial
activation event consistent with prior work in budding yeast [115].
Moreover, this rapid reactivation is associated with retention of RNAP
II at the gene even after transcriptional shut off. RNAP II retention
requires Nup98 as well as the H3K4me2 histone methylation mark
catalyzed by MLL1 (Set1 in S. cerevisiae). Interestingly, INO1 epigenetic memory in yeast also requires H3K4me2, the Nup98 ortholog,
Nup100, as well as the histone deacetylase Set3. Because the Set3containing complex, Set3C, recognizes the H3K2me2 modification
[116], Brickner proposed that histone dimethylation and subsequent deacetylation are essential for the transcriptional memory
process. These findings suggest that heritable epigenetic states are
controlled through physical association of the NPC with chromatin
[117], representing an emerging area of research ripe for future
discoveries.
11. Emerging connections between nucleocytoplasmic transport
and human disease
For some time, laminopathies have represented one of the most
established links between nuclear transport/structure and disease
[118,119]. At this meeting, however, a number of new connections
emerged including those with potentially exciting clinical implications.
Dr. Yosef Landesman from Karyopharm Therapeutics Inc. presented
recent research on Selective Inhibitors of Nuclear Export (SINE), compounds that target protein export from the nucleus, which have
shown promising results for the treatment of a variety of human medical conditions. Potential targets include both hematological and solid
malignancies [120]. Specifically, he presented evidence on Selinexor, a
SINE compound, which shows promising results for the treatment of
several types of cancers [121,122]. These promising results provide
evidence that nuclear transport pathways can be druggable targets for
therapeutic intervention and set the stage for further exploration of
nuclear transport pathways as potential therapeutic targets.
A number of the emerging connections link mRNA processing/
export to human disease [123,124]. Susan Wente (Vanderbilt University, USA) spoke of her recent structure–function studies of the evolutionarily conserved Gle1 protein [125,126]. Gle1 plays a key role in RNA
export from the nucleus through modulating the DEAD box RNA
helicase, Dbp5 [104,127,128], during mRNP remodeling upon nuclear
export [87,89] at the cytoplasmic face of the NPC (Fig. 5; [129,130]). In
recent years, mutations in GLE1 have been linked to several devastating
autosomal recessive diseases with tissue-specific consequences [131].
These diseases include lethal congenital contracture syndrome 1
(LCCS-1) and lethal arthrogryposis with anterior horn cell disease
(LAAHD), which ultimately result in fetal death. Wente reported on
experiments that used structure–function approaches to assess the
functional consequences of specific mutations identified in patients.
These studies reveal how a mutation in GLE1 termed FinMajor,
which is named due to the high prevalence in the Finnish population, causes production of a Gle1 protein with an altered capacity
for nucleocytoplasmic transport. She also reported that human
Gle1 can form higher order complexes and utilized a CryoEM approach
to assess how alterations within the Gle1 protein impact this selfassociation [125]. Such studies that combine multiple approaches to assess the functional consequences of patient mutations provide insight
not only into the defects that underlie the human disease but also into
mechanisms critical for nuclear transport.
The example of mutations in GLE1 that cause tissue-specific disease
is just one example of genes that encode ubiquitously expressed RNA
binding proteins which are mutated in tissue-specific disease [132]. Another example is spinal muscular atrophy, a motor neuron disease
caused by mutation in the human SMN gene [133], which is required
for spliceosome assembly and hence implicated in global splicing
events. Anita Corbett (Emory University, USA) described work relevant
to two additional examples where the alteration of ubiquitously
expressed RNA binding proteins leads to tissue-specific disease. Her
presentation touched on the ubiquitously expressed, nuclear,
polyadenosine RNA binding protein, PABPN1, which is mutated in
oculopharyngeal muscular dystrophy [134], and then described more
detailed efforts to define the role of another polyadenosine RNA binding
protein, Nab2/ZC3H14 [135,136]. Whereas the S. cerevisiae Nab2 protein
is essential for viability [137], mutation of human ZC3H14 causes an autosomal recessive form of intellectual disability [136,138]. A Drosophila
loss-of-function model has been of significant value in demonstrating
that Nab2/ZC3H14 function is most critical in neurons, consistent with
the brain-specific phenotype in patients [138,139]. This model also reveals aberrant brain morphology, suggestive of axon guidance defects.
As with other cases where mutations in such ubiquitously expressed
RNA processing/export factors lead to tissue-specific consequences,
the challenge is both to define what could be a multitude of functions
and then subsequently understand the critical requirement for those
functions within the relevant tissue.
Other links between human disease and RNA export processing extended both into the nucleus and to the cytoplasmic translation machinery. Kathy Borden (University of Montreal, Canada) reported on
how deregulation of the eIF4E protein, which is traditionally thought
of as a key translation initiation factor [140], can contribute to defects
in numerous steps of gene expression. Mechanistic studies of eIF4E
are of critical importance as eIF4E is overexpressed in 30% of human
cancers [141]. Borden showed evidence that eIF4E is a shuttling protein
that is exported from the nucleus in an mRNA export-dependent manner. Her work suggests that nuclear functions of eIF4E could contribute
to the oncogenic potential of eIF4E. She also reported on their identification of the previously unknown import receptor for eIF4E. An important
extension of this work is an effort to define the mechanism by which ribavirin triphosphate, the only direct inhibitor of eIF4E to reach Phase II
clinical trials for acute myeloid leukemia, impairs the function of eIF4E
[142].
With the expanded ability to define the specific mutations that underlie human diseases, many of the key players involved in the critically
important processes broadly termed nuclear transport are likely to be
implicated. Such mutations may have very specific consequences, as
complete loss of function of many of these vital factors would likely
not be compatible with life. The lamins, however, provide an example
of how distinct changes within a family of proteins can cause diverse
disease phenotypes.
12. Future challenges
As technology advances, our ability to peer into the NPC has
grown exponentially, shifting the field from viewing the NPC as a
static structure embedded within the nuclear envelope, to a dynamic
macromolecular complex for exquisite control of nuclear transport,
cell signaling and gene expression. These advances add new and
E.J. Tran et al. / Biochimica et Biophysica Acta 1843 (2014) 2784–2795
exciting challenges to the transport field while simultaneously
pinpointing the gaps in our knowledge regarding the fundamental
process of transport into and out of the nucleus. Some of the basic
mechanistic questions that remain to be addressed include: can multiple cargoes can be simultaneously transported through a single
NPC at the same time in a bidirectional manner or, alternatively,
are individual NPCs dedicated to specific transport events? If NPCs
are transport event-specific, this property could provide an explanation for why NPC composition varies across organisms and tissues.
Understanding the biophysical parameters of the NPC permeability
barrier is also of key importance. Whereas FG domains are clearly
critical for transport, these regions are non-selective and inefficient.
Moreover, as eloquently stated by Dirk Görlich, it is paradoxical that
energy-dependent, receptor-mediated transport of cargoes is faster
than diffusion through the NPC. More analysis is necessary to define
the rules for transport selectivity and determine the generality to
specific transport events.
The NPC is clearly a core integrator for multiple, biochemically distinct nuclear processes. Thus, a future goal is to determine how different
modes of regulation influence one another. For example, how or whether global regulation of transport receptors impacts mRNP assembly or
the many subsequent steps in gene expression is not yet known.
Given the intimate connectivity between these processes and the NPC,
there is likely to be a high degree of synergy at both the cellular
and molecular level. Uncovering the molecular connections that
link early and late steps in gene expression with mechanisms for
transport through the NPC and regulation of that transport will be
necessary to determine the underlying basis for human diseases
linked to defective NPC components, transport receptors, and/or NE
integrity. The future of the nuclear transport field is bright with the
prospects of novel discoveries, unique interdisciplinary collaborations, and applications to biomedical research.
Acknowledgements
We are most grateful to Thomas Schwartz (MIT, USA) and Karsten
Weis (ETH Zurich, Switzerland) for organizing an outstanding meeting.
We also thank all of our colleagues in the field for sharing unpublished
information. We apologize to those colleagues whose work is not specifically mentioned due to the space limitations.
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