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Comprehensive summaries of Uppsala Dissertations from the Faculty of
Science and Technology 786
Nuclear pore membrane glycoprotein 210 as a
new marker for epithelial cells
BY
MAGNUS OLSSON
Dissertation for the degree of Doctor of Philosophy in Molecular Cell Biology presented at
Uppsala University 2003.
ABSTRACT
Olsson, M. (2003). Nuclear pore membrane glycoprotein 210 as a new marker for epithelial
cells. Comprehensive summaries of Uppsala Dissertations from the Faculty of Science and
Technology 786. 55 pp. Uppsala. ISBN 91-554-5486-0.
Epithelial cell polarisation is a prerequisite for the branching morphogenesis in several
organs. Differential screening techniques were used to identify genes, which are upregulated
during induction of epithelium in early kidney development. This investigation revealed two
separate genes, Nuclear localising protein 1 (Nulp1), a previously undescribed gene with
sequence characteristics of the basic helix-loop-helix transcription factor family, and
glycoprotein 210 (gp210, POM210), an integral membrane protein constituent of the nuclear
pore complex (NPC). Of these, gp210 was found to be upreglated during conversion of
mesenchyme to epithelium.
The nuclear envelope, which demarcates the nuclear region in the eukaryotic cell,
consists of an inner and an outer membrane that are fused at the locations for NPCs. These
large macromolecular assemblages are tube like structures connecting the cytoplasmic and
nuclear compartments of the cell. NPCs serve as the only conduits for exchange of molecular
information between these cellular rooms. Electron microscopy techniques have revealed
detailed information about the NPC architecture. A number of its proteins (nucleoporins)
have been characterised and embodied as components of the NPC structure. Active, energy
dependent nucleocytoplasmic transport of RNAs and proteins is mediated by a group of
soluble receptor proteins, collectively termed karyopherins.
Gp210 has been suggested to be important for nuclear pore formation. Nevertheless,
our analyses showed a limited expression pattern of gp210, with its mRNA and protein
largely confined to epithelial cells in the mouse embryo. Furthermore, in several cell lines,
gp210 was undetectable. The expression pattern of gp210 was not synchronised with some
other nucleoporins, indicating NPC heterogeneity. Characterisation of the structure of the
human gp210 gene, including its promoter region, gave insight about possible cell-type
specific gene regulatory mechanisms.
Regulation of molecular traffic between the nucleus and the cytoplasm leads to
transcriptional control. Cell specific configuration of the NPC structure, due to diffential
expression of gp210, could be involved in this control. Gp210 could be of importance for the
development of epithelial cell polarisation.
Key words: Nuclear pore complex, gp210, Nulp1, epithelial cells, kidney development.
Magnus Olsson, Department of Cell and Molecular Biology, Biomedical Center, Uppsala
University, Box 596, SE-751 24 Uppsala Sweden
” Magnus Olsson 2002
ISSN 1104-232X
ISBN 91-554-5486-0
Printed in Sweden by Fyris-Tryck AB, Uppsala 2002
2
3
The thesis is based on the following articles, referred to in the text by their Roman numerals
I-IV:
I.
Olsson, M., Ekblom, M., Fecker, L., Kurkinen, M., and Ekblom, P. (1999). cDNA
cloning and embryonic expression of mouse nuclear pore membrane glycoprotein 210
mRNA. Kidney International 3, 827-838.
II.
Olsson, M., Schéele, S., and Ekblom, P. (2002). Limited expression of the nuclear
pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific
nuclear pores in metazoans. In manuscript.
III.
Olsson, M., Mason, J. M., English, M. A., Licht, J. D., and Ekblom, P. (2002).
Genomic structure and promoter sequence analysis of nucleoporin gp210, a marker
for epithelial cells. In manuscript.
IV.
Olsson, M., Durbeej, M., Ekblom, P., and Hjalt, T. (2002). Nulp1, a novel basic
helix-loop-helix protein expressed broadly during early embryonic organogenesis and
prominently in developing dorsal root ganglia. Cell & Tissue Research 308, 361-370.
Reprints were made with permission from the publishers.
4
TABLE OF CONTENTS
ABBREVIATIONS
…………………………………………………………6
INTRODUCTION
…………………………………………………………8
EPITHELIAL CELLS
………………………………………………....9
KIDNEY ORGANOGENESIS
Basic features of kidney organogenesis
Regulatory molecules in early kidney development
TECHNIQUES
Differential hybridization
Differential display
…………………9
………………..10
………………………………………..12
………………………………………..12
SPECIFIC AIMS OF THIS THESIS
………………………………………..14
THE NUCLEAR PORE COMPLEX
The nuclear envelope
Lamins and the nuclear lamina
Architecture of the nuclear pore complex
Nuclear pore complexes in the Annulate lamellae
Nuclear pore proteins
Function of nucleoporins
Integral membrane nuclear pore proteins
The nuclear pore complex during mitosis
………………..14
………………..15
………………..15
………………..18
………………..18
………………..22
………………..23
………………..25
BIDIRECTIONAL TRANSPORT OF MOLECULES BETWEEN THE
NUCLEUS AND THE CYTOPLASM
Passive diffusion
………………………………………………..26
Active import
………………………………………………..26
Active export
………………………………………………..28
RESULTS AND DISCUSSION
………………………………………………..31
FUTURE PERSPECTIVES
………………………………………………..35
POPULÄRETENSKAPLIG SAMMANFATTNING PÅ SVENSKA
………..35
ACKNOWLEDGMENTS
………………………………………………..37
LITERATURE CITED
………………………………………………..38
5
ABBREVIATIONS
bp
Base pair
cDNA
Complementary deoxyribonucleic acid
ER
Endoplasmic reticulum
FG Nup
Nucleoporin containing FG, GLFG or FXFG amino acid repeats
kb
Kilobase
kDa
Kilodalton
mAb
Monoclonal antibody
mRNA
Messenger ribonucleic acid
N-linked
Asparagine-linked
NE
Nuclear envelope
NES
Nuclear export signal
NLS
Nuclear localizing signal
NPC
Nucleo pore complex
Nup
Nucleoporin
PCR
Polymerase chain reaction
6
7
INTRODUCTION
Large-scale DNA sequencing efforts have provided us with the assembled and annotated
genome sequences from a diverse set of organisms. Fairly precise estimations of the number
of genes contained in several of these genomes have been done. The human genome contains
approximately 30.000 different genes (Venter et al., 2001), of which only a fraction are
simultaneously expressed in any individual cell (Adams et al., 1995). The major control of
gene expression occurs at the transcriptional level, the initial step in gene expression.
Transcriptional activation proceeds in coordinated stages, from the packaging of a gene into
chromatin and its localization within the nucleus to the recruitment of transcription factors
whose inhibitory or activating influences is a result of specific protein-protein or proteinDNA interactions. The ability of each cell to program its genome and determine which genes
are to be expressed is central to tissue differentiation, organogenesis, organismal
development and disease. Thus, the ability to compare expression of genes in different cell
types provides the underlying information we need to analyse these events.
A number of techniques have evolved aiming to isolate genes expressed at a given
time and under specific stimuli. In this thesis, two of these methods where applied in order to
specifically identify genes involved in the induction of epithelial cell polarisation in early
kidney development, a generally accepted model system for organogenesis.
8
EPITHELIAL CELLS
The ability of cells to generate biochemically and functionally distinct plasma membrane
domains is a prerequisite for the development of multicellular organisms. The best-studied
examples of polarized cells are columnar epithelial cells, such as the cells lining the lumen of
the intestine or of nephrons. The plasma membrane of epithelial cells is typically divided into
two domains: an apical cell surface facing the organ lumen and a basolateral cell surface that
is in contact with neighboring cells and the underlying extracellular matrix. The two domains
are separated by tight junctions, which form a morphological border and act as an
intramembrane diffusion barrier.
KIDNEY ORGANOGENESIS
Basic features of kidney organogenesis
Most vertebrate organs, once formed, continue to perform the function for which they were
generated until the death of the organism. The kidney is a notable exception to this rule.
Vertebrates utilise a progression of more complex kidneys as they grow and develop. In
mammals, three successive sets of excretory organs develop during embryogenesis: the
pronephros, the mesonephros and the metanephros. The first two are only transiently present
during early fetal life in mammals, and the mature kidney develops from the metanephros.
The development of the human metanephric kidney starts in the fifth gestational
week, when an inductive signal originating from the metanephric mesenchymal blastema
induces the uretic bud to grow out from the mesonephric (Wolffian) duct. This stage is
reached at embryonic day 10.5 in mouse. The mesenchyme induces the ureteric bud to
undergo branching morphogenesis, which eventually will lead to the formation of the ureter
and the collecting duct system. In turn, the ureteric bud induces a part of the surrounding
mesenchyme cells to condensate and to convert into new epithelium (Gruenwald, 1943;
Grobstein, 1953; Erickson, 1968). All different cell types included in the adult kidney apart
from the endothelial cells of the vascularization system originate from this initial reciprocal
cell-cell interaction. The mesenchymal condensates transform into a defined comma-shaped
body and further into the S-shaped tubule (Huber, 1905). This cell configuration is the
rudiment for proximal and distal tubules, and for the different epithelial cells of the
glomerulus, the basic functional unit of all kidneys. Uninduced mesenchyme cells will
become the stromal cell compartments. The first branching of the growing ureteric bud is
symmetric and dichotomous. In nephron formation, each tip (ampullae) of the bifurcate
merge with the distal tubule and the glomerular podocytes become folded into the
glomerulus. The outcome of repeated branching of the ureter with new condensates induced
9
at every branch point is a treelike architecture with a total number of one million nephrons in
the mature human kidney.
Microsurgical studies on embryonic human material, performed by Osathanondh and
Potter gave a detailed description of the branching pattern (Osathanondh and Potter, 1963).
According to this work, the branching process is divided into four periods. In the first step
two tubules are formed, followed by elongation and formation of new branches. Arcades are
formed during the second step. These nephric chain structures results from a repeated joining
of new nephrons to the same ampullae, giving four to seven nephric units where only the
youngest one are attached to the collecting duct system itself. In the third phase, no branching
occurs but the collecting duct system extends themselves past the attachment points of the
arcades. At this stage terminal nephrons are induced. These are, unlike the arcadic nephrons,
directly connected to the collecting duct. In the fourth step the terminal ureter disappears, and
the formation of nephrons stop.
Regulatory molecules in early kidney development
Functional aspects of regulatory molecules have emerged from gene targeting experiments in
mice, and from organ culture techniques. A few examples may be sufficient to illustrate this
point.
Pax2, a transcriptional regulator of the paired-box family is one of the earliest markers
of kidney development and also widely expressed during the development of both ductal and
mesenchymal components of the urogenital system. When Pax2 is homozygously mutated,
the formation of the Wolffian duct is incomplete, which leads to renal agenesis (Torres et al.,
1995). Wilms’ tumour gene 1 (Wt1) is normally expressed in the uninduced mesenchyme,
and in the case of null mutants, this cell population undergoes apoptosis at embryonic stage
11, suggesting a role for this gene in the survival of the mesenchyme surrounding the
Wolffian duct (Kreidberg et al., 1993). Induction of the outgrowth of the ureteric bud from
the mesonephric duct depends on secretion of glial derived neurotrophic factor (GDNF),
expressed by the metanephric mesenchyme, and the activation of its receptor c-ret, presented
exclusively at the tip of the epithelial bulging (Pachnis et al., 1993). GDNF knockout mice
(Pichel et al., 1996), as well as mice lacking c-ret (Schuchardt et al., 1994) have no kidneys
due to failure of the ureteric bud to grow out. Eya1, a transcription factor belonging to the
homeo box gene family has been shown to be the upstream activator of GDNF (Xu et al.,
1999). Conversely, counter induction by the ureteric bud itself induces condensation of the
metanephric mesenchyme, which sequentially results in tubulogenesis and formation of
nephrons. One or several signalling molecules specifically expressed in a spatially and
temporarily correct fashion by the ureteric bud probably initiate this event. A member of the
interleukin-6 (IL-6) family, leukaemia-inhibiting factor (LIF) have been shown to act as an
inducer of mesenchme to epithelial conversion in organ cultures. Other IL-6 type cytokines
10
shared this ability, and the deletion of their common receptor reduced nephron development
(Barasch et al., 1999). Additional factors, such as Bone morphogenetic protein 7 (Bmp7) and
members of the Wnt gene family have been suggested to act in a synergy with LIF and to be
required for nephrogenic induction (Herzlinger et al., 1994; Vukicevic et al., 1996). Each of
these ligands is recognised by a receptor in the blastema and it is believed that transmembrane heparan sulphate proteoglycans serve as presenting agents directing these ligands
to their proper location. The activation of specific signal transduction pathways will in turn
generate gene products needed for nephrogenesis and the reciprocal induction of branching.
Bmp7 and different transcription factors, including Pax2, Hoxa11/Hoxd11, Lim1, Pod1 and
Wt1 are likely candidates for co-ordinating this activating cascade (reviewed in Burrow,
2000; Dressler, 1999; Schedl and Hastie, 2000). For example, Hoxa11 and Hoxd11 are
members of a paralogous subgroup of genes, which also contains Hoxc11. The kidneys in
Hoxa11/Hoxd11 double mutants have been described. In those embryos ureteric bud
initiation occur normally but branching is perturbed. The expression of several genes,
including Wnt11, Pax2, Wt1 and Bf2 was examined and found to be altered compared to
controls, particularly with respect to expression along the dorsoventral axis (Patterson et al.,
2001).
During kidney development, each formed epithelial cell layer becomes coated with a
basement membrane, a thin sheet of specialised extracellular matrix composed primarily of
laminins, collagen IV, nidogens and sulphated proteoglycans. A constant extracellular matrix
remodelling occurs through a concerted action of different metalloproteinases and by
differential expression of matrix components. Mice mutated in the nidogen-binding domain
of the laminin-g1 chain were recently shown to suffer from renal agenesis due to ruptures in
the Wolffian duct basement membrane (Willem et al., 2002). These results are in line with
previous in vitro investigations (Ekblom et al., 1994), and clearly demonstrate that
interactions between basement membrane components are important for early kidney
morphogenesis.
A unique feature of kidney development is the conversion of mesenchymal cells into
epithelium. Changes in gene expression can be precisely timed by taking advantage of the
transfilter induction method (Grobstein, 1956; Saxen et al., 1968). This organ culture system
mimics the in vivo situation of induction of the undifferentiated metanephric mesenchymes,
which is completed in 18-24 h. At this stage, the cells are not yet polarized, but during the
next few days polarized epithelial cells attached to a basement membane form. Wellcharacterised cell surface receptors for laminins include different integrin chain heterodimers
and dystroglycan. Antibody perturbation of the binding sites for laminin-1 in dystroglycan as
well as in integrin a6b1 in organ culture studies blocked kidney tubologenesis (Durbeej et
al., 1995; Falk et al., 1996), suggesting that proper interactions between the basal lamina and
its receptors are crucial for epithelial morphogenesis and kidney formation. These studies
11
also demonstrate that in vitro organ culture systems can be used to identify components
involved in epithelial cell polarisation. Obviously, this process must be controlled at many
levels, and it remains an important task to identify components that are specific for the
polarised epithelial phenotype. A major aim of the present study was to identify molecules
that appear early during the converson of mesenchyme to epithelium. A novel marker for the
epithelium was identified using the in vitro organ culture system.
TECHNIQUES
Differential hybridisation
Finding genes that are transcriptionally regulated, in a spatial or temporal fashion is one way
to clarify developmental pathways as well as an approach to localise disrupted genetic
programmes leading to different malignancies such as cancer. Classical methods for isolating
regulated genes include differential screening of cDNA libraries or subtractive enrichment
methodologies, and their modifications. They both proceed from two cellular pools of RNA
that differ in a biological aspect of interest. Transcripts common to both pools are eliminated
and genes exclusively expressed in either of them are eventually cloned and further
characterised. In differential hybridisation, duplicate lifts from a plated library are probed
with labelled cDNA from the two conditions being compared. A strong signal present in one
cDNA pool but not in the other is indicative of a candidate differentially expressed message
(Dworkin and Dawid, 1980). Subtractive hybridisation, on the other hand, is carried out with
an excess amount of biotinylated cDNA from one sample (driver) that is hybridised with
cDNA from another (tracer). Biotinylated driver/tracer hybrids are subsequently removed by
streptavidin precipitation of biotin (Sive and St John, 1988). Since both these methods suffer
from high complexity of the probe material, usually only high abundance cDNAs are detected
and consequently eliminating low abundance genes, which may be of interest. Another major
disadvantage is the fact that differences in gene expression only can be studied between two
transcriptomes at a time.
Differential display
Two similar methods for fingerprinting of eukaryotic RNA have been developed; mRNA
differential display (Liang and Pardee, 1992) and RNA arbitrarily primed PCR (RAP-PCR)
(Welsh et al., 1992). They allow the detection of differentially expressed messages based
upon arbitrarily priming and amplification of cDNAs representing two or more physiological
states. The two protocols are distinguished in the way cDNA initially is generated. In the
differential display method an oligo-dT primer, containing one or two specific additional
nucleotides at the 3´end, is used whereas an arbitrarily primer is used in RAP-PCR.
Following the reverse transcriptase step, a low stringency PCR amplification will result in 5012
100 gene products with a size of 100-500 bp that are resolved on a polyacrylamide gel and
visualised by autoradiography. Analogous cDNA pools obtained from the compared RNA
samples are resolved side by side. PCR products with sample-to-sample variations indicate
regulation of the corresponding gene. Advantages of RNA fingerprinting methods compared
to differential hybridisation and subtractive library constructions are primarily that they
require very small amounts of total RNA or polyA+ RNA as starting material. Through
visualisation, side-by-side, it is also possible to perform a simultaneous comparison of several
RNA pools in which both up regulation and down regulation of gene expression can be
detected. However, a large number of primer pair combinations are required to fully cover the
wide representation of mRNA species presented in any cell or cell population.
In recent years, new high-capacity devices for high-throughput determination of gene
regulation have been developed to satisfy the increasing demands for genetic tests. Much
attention has focused on the use of the cDNA micro arrays technique (Lockhart et al., 1996;
Schena et al., 1995). In this method, oligonucleotides are bound to a microchip, which is then
hybridised with labelled mRNA from the sample or samples to be analysed. Technically,
there is no limitation in how many genes that can be screened simultaneously and in contrast
to other methods described, an expression profile is given for every single gene spotted on the
array. One limitation is that only predetermined genes can be processed, although this
disadvantage in a near future will be overruled by the completion of the genome projects.
13
SPECIFIC AIMS OF THIS THESIS
The molecular map of kidney organogenesis is at present fragmentary and it is not yet
possible to identify a complete genetic pathway required for any one of the many steps in
nephrogenesis. The specific aims for this thesis have been to identify missing links in this
process. In particular, the aim was to identify components upregulated early during
conversion of the metanephric mesenchyme to epithelium.
Although this work generated publications of two separate genes, my efforts have
primarily been centred on one of them, gp210, a nuclear pore membrane protein that
unexpectedly was found to be differentially expressed during induction of kidney epithelial
cell polarisation. Background and discussion in this thesis will therefore mainly be focused
on the nuclear pore complex.
THE NUCLEAR PORE COMPLEX
The nuclear envelope
The eukaryotic cell nucleus is highly organised into discrete functional regions separating
different activities such as transcription, RNA processing, DNA synthesis and ribosome
assembly. In turn, these events are physically separated, and thereby regulated independently
from the translation in the cytosol, by two concentric lipid membranes forming the nuclear
envelope (NE). Partitioning of the nucleus in eukaryotic cells give a more sophisticated
control over gene expression than is possible in prokaryotes.
The nuclear envelope is composed of three biochemically distinct domains. The outer
nuclear membrane is continuous with the membrane of the endoplasmic reticulum (ER) and
consequently shares a set of proteins and functional properties with this domain. In contrast,
the inner nuclear membrane has unique characteristics, mainly because of a close association
with the chromatin and the nuclear lamina, a supportive polymeric network located subjacent
to the NE. The inner and the outer membranes make contact at several points forming holes
occupied by nuclear pore complexes (NPCs), thus defining the third membrane domain. The
NPCs provide channels for nucleocytoplasmic transport and serve dual roles as both
mediators and regulators of transport. Beside these classical features of the nuclear envelope
it also exhibits considerable plasticity. Confocal imaging has demonstrated that the entire
envelope is able to form deep invaginations into the nuclear interior (Fricker et al., 1997).
These straight or branching membrane tubules bring the envelope to the centre of the nucleus
and may facilitate the molecular export to the cytoplasm by reducing the distance to the
nearest NPC.
14
Lamins and the nuclear lamina
The nuclear lamina is a flattened fibrous matrix assembly of nuclear specific intermediate
filaments that provide stability to the nuclear envelope. Associated with the lamina meshwork
are several integral membrane proteins of the inner nuclear membrane, including the laminaassociated proteins LAP1 and LAP2, emerin, nurim, otefin, MAN1, nesprin and the lamin B
receptor (LBR) (reviewed in Goldman et al., 2002). The targeting of integral proteins to the
inner membrane is thought to involve lateral diffusion along the ER and the pore membrane
domain, followed by retention at the inner membrane mediated by binding to lamins or
chromosomes (Ostlund et al., 1999). In humans, three lamin genes have been identified
(LMNA, LMNB1 and LMNB2), encoding in total seven alternatively spliced isoforms. The
isoforms are divided into type-A or type-B lamins, depending on their isoelectric point,
behaviour during mitosis and expression profile. Nearly all mammalian cells express at least
one type-B lamin whereas the major A-type lamin variants, lamin A and C, are
developmentally regulated and preferentially expressed in differentiated non-proliferating
tissues ( Stewart and Burke, 1987; Rober et al., 1990; Rober et al., 1989). The correlation of
the induction of lamins A/C has led to the suggestion that these lamins are involved in the
maintenance of the differentiated phenotype.
All lamin isoforms possess a tripartite secondary structure with a centrally located
alpha-helical rod domain flanked by a non-helical NH2-terminal and globular COOHterminal tail domain. The rod domain mediates the lateral association between lamins
required for polymerisation (Stuurman et al., 1996). Attachment of the nuclear lamina to the
inner nuclear membrane is mediated by interactions with several integral membrane proteins.
These include emerin that primarily interacts with type A lamins (Clements et al., 2000;
Fairley et al., 1999), LAP1b that interacts with both lamin A and B (Foisner and Gerace,
1993) and LBR, which bind independently to lamin B (Ye and Worman, 1994). Lamins have
been suggested to be involved in various nuclear functions, including the maintenance of
chromatin organisation and in DNA replication (reviewed in Goldman et al., 2002). Naturally
occurring lamin A/C mutations have been implicated in the pathogenesis of muscular
dystrophy, certain forms of lipodystrophy (Shackleton et al., 2000; Bonne et al., 1999) and in
nuclear envelope defects (Raharjo et al., 2001; Vigouroux et al., 2001), suggesting that lamin
A/C has additional functions that remain to be elucidated morefully.
Architecture of the nuclear pore complex
The nuclear pore complexes (NPCs) are large, supramolecular assemblages of proteins
forming the only conduit for the exchange of information between the nucleus and cytoplasm.
NPCs from different organisms share a common fundamental architecture. However,
significant alterations in NPC architecture have arisen during evolution that may be
15
correlated with differences in nuclear transport regulation or mitotic behaviour (Yang et al.,
1998). The vertebrate NPC (vNPC) has an estimated molecular weight of 125 mDa, which
makes it approximately twice as large as the NPCs found in yeast (yNPC) (Reichelt at al.,
1990; Rout and Blobel, 1993).
Our current knowledge about the vNPC structure is to a large extent based on studies
using large amphibian oocytes. Ultrastructural analyses of the NPC using transmission
electron, scanning electron and atomic force microscopy have revealed a three-dimensional
model visualising the Xenopus NPC basic framework as a cylindrical assembly with
octagonal symmetry comprised of several distinct structural elements (Fig. 1). Signal
mediated transport is believed to occur through a cylindrical element, referred to as the
central transporter, which is located along the central axis of the pore complex (Rutherford et
al., 1997). Eight spokes extend from a middle, inner ring that embraces the centrally located
transporter. These spokes penetrate the nuclear envelope and project into the membrane
lumen where they are bridged by a distal ring formed by eight loops named radial arms (Akey
and Radermacher, 1993). The spoke ring complex is sandwiched between the cytoplasmic
and the nucleoplasmic coaxial rings (Goldberg and Allen, 1993), where the cytoplasmic
coaxial ring is located vertically above the plane of the outer nuclear membrane and consists
of a thin smooth ring onto which eight subunits are attached, each supporting one short fibre
that extend approximately 35-50 nm into the cytoplasm (Jarnik and Aebi, 1991). A different
set of eight fibres extends approximately 40-50 nm from the nucleoplasmic coaxial ring into
the nucleus, and is joined at their distal ends to form a basket structure (Ris, 1997; Ris and
Malecki, 1993). Tethered to the nuclear basket is Tpr, a coiled coil protein able to form
filament bundles, which have been traced into the nuclear interior for up to 350 nm (Cordes
et al., 1997b). Tpr has been implicated in nuclear export (Frosst et al., 2002; Bangs et al.,
1998) and is also hypothesised to constitute an interchromosomal channel-based nuclear
matrix (Zimowska et al., 1997). Additional features of the vertebrate NPC are the “star ring”
underlying the cytoplasmic ring and two sets of internal filaments joining the cytoplasmic
and nucleoplasmic coaxial rings to the central transporter (Goldberg and Allen, 1996;
Goldberg et al., 1997).
Studies of the passage of gold particles through the central transporter by transmission
electron microscopy indicate a central channel that has the ability to vary its opening
diameter, from potentially closed to 26 nm (Feldherr and Akin, 1997). The internal filaments
have been proposed to participate in the gating process by open or close the channel at the
entrance to the transporter (Kiseleva et al., 1998). However, it should be mentioned that there
are controversial opinions regarding the central transporter as a distinct element of the NPC.
Alternative views claim that this structure is merely a cargo caught in transit during specimen
preparation or collapsed cytoplasmic fibres.
16
Although it is similar to the vNPC, the yNPC is much simpler in structure. It lacks the
cytoplasmic and nuclear rings and the lumenal radial arms of the spoke complex. Peripheral
filamentous domains are present but in a comparison to vNPC, they are shorter. Overall, the
yNPC is smaller (96 nm in diameter by 35 nm high) than the vNPC (145 nm diameter by 80
nm high) and occupies one fifth of the volume (Yang et al., 1998; Fahrenkrog et al., 1998).
Figure 1. Schematic model of the vertebrate nuclear pore complex. The figure indicates
major NPC constituents. Individual parts are not scaled. Note that only two out of eight
central spokes are outlined and that the radial arms connecting the spokes in the perinuclear
space are missing in the picture.
17
Nuclear pore complexes in the Annulate lamellae
Nuclear pore complexes are not merely present in the nuclear envelope. Annulate lamellaes
(ALs) are cytoplasmic stacks of double membrane containing large numbers of tightly packed
pore complexes. Each double membrane possesses hexagonally packed pores and is aligned
vertically by en face contact between pores. ALs are heavily represented in rapidly growing
or differentiating cells, such as tumour cells, male and female gametes and virally infected
cells (reviewed in Kessel, 1992). At present, there is no defined function for ALs but it has
been suggested that they represent a storage form of pore complexes used in the reassembly
of daughter nuclei during cell division.
Evaluation of comparison between nuclear and cytoplasmic pores by electron
microscopy concludes that they are morphologically indistinguishable and AL pores have
been shown to interact with the soluble mediators of nuclear protein import and their
karyophilic protein substrates (Cordes et al., 1997a). At the molecular level, however, it has
been reported that AL pores in rat cells (line RV) lack POM210 and POM121 (Ewald et al.,
1996), two integral membrane components of the NPC, although this finding has been
questioned, at least for POM121 (Imreh and Hallberg, 2000). Overexpression of rat POM121
in COS-1 cells (African green monkey kidney cells) induced AL formation, indicating that
this protein indeed can be a component of cytoplasmic NPCs, also suggesting that it may
have a function in NPC biogenesis (Soderqvist et al., 1996).
Nuclear pore proteins
Stable protein components of the nuclear pore complex are referred to as nucleoporins or
nups. The octagonal symmetry of the NPC results in that each nucleoporin occurs mainly in 8
or multiples of 8 copies per pore. Recently, a comprehensive characterisation of all yNPC
constituents, including localisation studies performed by immunoelectron microscopy, was
done. This investigation revealed approximately 30 distinct nucleoporins, each with a mainly
symmetrical distribution within the vertical plane of the NPC structure (Rout et al., 2000). A
similar analysis of the mammalian NPC has been presented, based on mass spectrometry
identification of all proteins included in a highly purified NPC fraction from rat liver nuclei
(Cronshaw et al., 2002). According to this study, the mammalian NPC seems to be composed
of essentially the same number of nucleoporins as its yeast counterpart. This is less than
expected considering the striking disparity in NPC size and complexity between the two
species. Previously, others have estimated a larger number of nucleoporins (Fontoura and al.,
1999; Miller and Forbes, 2000), although the discrepancy may be explained by the presence
of pore associated proteins in the preparations, for example transport receptor proteins and
molecules involved in RanGTP hydrolysis. To date, more than twenty vertebrate nups have
been molecularly described and localised to discrete NPC regions (listed in Table 1). Among
18
these is a group of polypeptides that withhold multiple copies of glycine-leucinephenylalanine-glycine (GLFG), FXFG or FG repeats interrupted by characteristic spacer
sequences, preferentially in their COOH-terminal region (Table 1). Many nucleoporins with
repeats dispersed through a portion of their sequence have been found to bind directly to
nuclear transport receptors (Fornerod et al., 1997; Hu et al., 1996; Radu et al., 1995; Shah and
Forbes, 1998; Shah et al., 1998), and appear to represent binding sites for transport
complexes during their translocation through the NPC. Interactions between nucleoporins and
nucleoporin subcomplexes are thought to be mediated through a-helical coiled coil domains
(Fornerod et al., 1996). Predicted coiled coil regions have been found in a number of
nucleoporins, including Nup88, CAN/Nup214 and the Nup62 complex (Table 1).
Common posttranslational modifications of nucleoporins include different kinds of
phosphorylations and glycosylations by attachment of O-linked N-acetylglucosamine to
serine and threonine residues (O-GlcNAc). Whereas nups are phosphorylated in a dynamic,
cell-cycle dependent manner, levels of O-GlcNAc remains constant (Miller et al., 1999;
Macaulay et al., 1995; Favreau et al., 1996). The role for glycosylation of NPC subunits
remains elusive. M-phase specific phosphorylation, on the other hand, is shared with lamin
components. It has been shown that specific phosphorylation of lamins during cell division
destabilise lamin polymers, leading to filament dissolution and that the elimination of these
phosphorylation sites prevents polymer disassembly during division (reviewed in (Moir et al.,
1995)). Thus, it has been suggested that the function of phosphorylation of nups may parallel
that shown in lamins leading to a reversible disassociation of the NPC during mitosis.
To elucidate the hierarchy of interactions between metazoan nucleoporins has been a
main goal in order to clarify their function. This work has partly been accomplished by a
comparison with the yeast NPC and partly by either biochemical methods such as the yeast
two-hybrid system or by the separation of sub complexes formed during NPC breakdown
during mitosis. During this work, individual nucleoporins as well as interacting nucleporins
forming sub complexes have further been localised to the central transporter, the cytoplasmic
filaments or to the nucleoplasmic basket. Interestingly, vertebrate nucleoporins seems to have
a more asymmetrical distribution within the vertical plane of the NPC, compared to yeast
nucleoporins. To date, the nucleoporins that contribute to the nuclear basket composition
include Nup153 (Pante et al., 1994; Sukegawa and Blobel, 1993; Walther et al., 2001),
Nup98 (Fontoura et al., 1999; Radu et al., 1995), Nup93 (Grandi et al., 1997), Nup50 (Guan
et al., 2000) and a new Nup160 sub complex containing Nup160-Nup133-Nup96-Nup107
(Vasu et al., 2001). Nup133 and Nup107 have also been localised to the cytoplasmic face of
the NPC. Throughout mitosis, a fraction of Nup133 and Nup107 localizes to the
kinetochores, thus revealing an unexpected connection between structural NPCs constituents
and kinetochores (Belgareh et al., 2000). Potential members of the central scaffold of the
pore are the Nup93-Nup205-Nup188 complex (Miller et al., 2000) and Nup155 (Radu et al.,
19
1993), where the latter is found on both the nucleoplasmic and the cytoplasmic side of the
pore. Also associated to the central domain of the NPC, at the periphery of the central gated
channel is a sub complex consisting of Nup62-Nup58-Nup54-Nup45 (Guan et al., 1995; Hu
et al., 1996; Finlay et al., 1991). Three components have been found to be restricted to the
cytoplasmic face of the NPC, at or near the cytoplasmic fibrils: Nup88/Nup84 (Fornerod et
al., 1997), CAN/Nup214 (Kraemer et al., 1994) and RANBP2/Nup358 (Delphin et al., 1997;
Wilken et al., 1995), of which the former two form a sub complex (Bastos et al., 1997).
20
Name
Characteristic
motifs
Localization
in NPC
Function in
Transport
References
RanBP2/Nup358
Zn fingers
Ran binding domain
FXFG
Cytoplasmic
filaments
May have an
essential role in
nuclear import.
Wu et al., 1995
Yokoyama et al., 1995
Walther et al., 2002
Nup88 (Nup84)
Coiled coil
Protein import
CAN/Nup214
FG
Coiled coil
At or near the
cytoplasmic
filaments.
Fornerod et al., 1997
Uv et al., 2000
van Deursen et al., 1996
Nup180
Nup45 (p45)
Nup54 (p54)
Nup58 (p58)
Nup62 (p62
Nup155
Nup96
Nup107
Nup133
Nup160
Nup93
Nup188
Nup205 (p205)
Nup50 (Npap60)
Nup98
Nup153
NLP-1
Tpr
POM121
gp210 (POM210)
FG
Coiled coil
FXFG, Coiled coil
Leucine zipper
Coiled coil
Leucine zipper
Ran binding domain
FG, FXFG, GLFG
RNA binding domain
Zn-finger
FG-repeats
M9-like domain
FG, PAFG
Coiled coil
Leucine zipper
Coiled coil
NLS
Transmembrane
FXFG
Transmembrane
Cytoplasmic
coaxial ring
Centraly, near the
gated channel, at
both nuclear and
cytoplasmic face.
Both cytoplasmic
and nuclear face
Maps to the
Nuclear face of
NPC.
Nuclear basket
and/or close to the
nuclear entry of
the gated channel.
Nuclear basket
Protein import
and mRNA export
Wilken et al., 1993
Role in nuclear
Import.
Finlay et al., 1991
Hu et al., 1996
Hu and Gerace, 1998
Radu et al., 1993
mRNA export
Fontoura et al., 1999
Radu et al., 1994
Vasu et al., 2001
NPC biogenesis
Grandi et al., 1997
Miller et al., 2000
Protein export.
Stimulates NLS
mediated import.
Export of multiple
types of RNA.
Associated with
export of most
RNAs except
tRNA.
Importin a/b
mediated import.
Interacts with
CRM-1
Guan et al., 2000
Lindsay et al., 2002
Nuclear basket
and nuclear interior.
Pore membrane
Export of mRNA
Cordes et al., 1997
Shibata et al., 2002
Soderqvist et al., 1997
Pore membrane
Implicated in
NPC biogenesis
and pore dilation.
Wozniak and Blobel,
1992; Drummond and
Wilson, 2002
Nuclear basket
Nuclear basket
Radu et al., 1995
Powers et al., 1997
Shah and Forbes, 1998
Ullman et al., 1999
Walther et al., 2001
Farjot et al., 1999
Table 1. Description of isolated and characterised mammalian nucleoporins. Nucleoporins
are listed in their vertical order of appearance except pore membrane proteins and NLP-1.
Major subcomplexes are boxed. Coiled coil, a-helical coiled coil domain; NLS, nuclear
localizing sequence.
21
Function of nucleoporins
The synthetic lethal screen has been one of the most successful genetic approaches for
dissecting function for individual nucleoporin members in yeast. By this method, overlapping
functions can be envisioned either as redundant function or as function that depend on each
other (reviewed in Fabre and Hurt, 1997). A similar approach is not available for vertebrate
cells. Instead, information regarding nucleoporins and their function in nucleocytoplasmic
transport, NPC assembly and in aspects of intranuclear organisation are primarily derived
from alternative methods, such as antibody perturbations and nuclear assembly reactions in
cell free systems. Demembranated sperm chromatin induces nuclear assembly when it is
added to Xenopus egg extract. These nuclei are functionally normal with respect to protein
import, DNA replication, and disassemble in response to mitotic signals (Lohka and Maller,
1985). Nuclei assembled in vitro in the presence of Xenopus egg cytosol, biochemically
depleted from the Nup62 complex, has been shown to be defective in nuclear transport with a
linear correlation between the amount of complex present in cytosol and the amount of
nuclear transport of which those nuclei are capable (Finlay et al., 1991). The same approach
has also successfully been used for Nup153, the Nup93 complex and for Nup358. Consistent
with its localisation at the base of the nuclear basket, Nup153 deficient NPCs where found to
lack basket components Nup93, Nup98 and Tpr. These nuclei where further shown to have a
reduced capacity for basic NLS mediated cargo import and became mobile within the NE
(Walther et al., 2001). The latter dysfunction may be explained by the recent finding which
indicates a direct interaction between Nup153 and lamin LIII, the major lamin form present
in Xenopus oocytes (Smythe et al., 2000). Nuclear import for NLS-containing karyophilic
substrate appears fairly normal in Nup93-complex-depleted nuclei but show low NPC
frequency and reduced size of the nucleus. Thus, Nup93 or its interacting nucleoporin
partners seems to be important for NPC biogenesis (Grandi et al., 1997). Interestingly and
somewhat unexpectedly, the cytoplasmic filaments, which have been proposed to act as a
docking site for both import and export complexes, seem not to be needed for nuclear import.
NPCs devoid of RanBP2/Nup358 or CAN/Nup214 or both show no deficiency in NLS or M9
import signal mediated nuclear accumulation (Walther et al., 2002). Nuclear microinjection
of anti-Nup98 specific antiserum revealed this nucleporin as an essential element of the RNA
export pathway. Cytoplasmic entry for several classes of RNAs, including snRNAs, 5S RNA,
large ribosomal RNAs, and mRNA was prevented. In contrast, the export of tRNA was
unaffected (Powers et al., 1997).
A limited number of nucleoporins have been analysed by genetic methods in
metazoans. CAN/Nup214 -/- mouse embryonic stem (ES) cells are not viable but maternally
derived CAN account for normal development of homozygous offspring until between 4.0
and 4.5 days postcoitum when they die due to a blastocoel collapse. In 3.5-day-old mutant
22
embryos, cultured in vitro, progressive depletion of CAN leads to cell cycle arrest in the G2
phase, nuclear accumulation of polyadenylated RNA and in sharp contrast to the results
presented by Walther and his colleagues (Walther et al., 2002), impaired NLS-mediated
protein uptake (van Deursen et al., 1996). Embryonic lethality is also seen in Nup50 deleted
mice, although at a later stage compared to CAN-/- mice. Phenotypically, the homozygous
Nup50 deletion is characterised by severe defects in the neural tube development and
abnormalities in p27kip1 expression (Smitherman et al., 2000). p27kip1 is a Nup50 interacting
cyklin-dependent kinase inhibitor involved in regulation of the cell cycle (reviewed in (Sherr
and Roberts, 1999)). On the other hand, microinjection studies implicate Nup50 in the
nuclear export of proteins containing leucine-rich export sequences and was, in consistent
with this finding shown to directly bind Crm1 (Guan et al., 2000). Apart from being a
structural component of the NPC, Nup50 may also serve as a soluble co factor in importin-b
mediated nuclear import (Lindsay et al., 2002).
A current model for translocation of transport complexes through the NPC involves
interaction between the transport receptor and FXFG repeat containing nucleporins (FG
Nups). Sequential repeat domains are proposed to act as stepping-stones for karyopherinmediated transport reactions (Radu et al., 1995). The yeast NPC contains thirteen FG Nups
(Rout et al., 2000). When Allen and co-workers sampled eight of these individually using
immobilised FG domains as bait for proteins in whole yeast extract it was revealed that they
all bound transport receptors, in a more or less selective manner (Allen et al., 2001). A
selective binding efficiency between a FG Nup and a transport complex may indicate the
existence of separate translocation routes. The observation that Nup98 disruption impairs M9
signal mediated import but leave the import of ribosomal protein L23a and spliceosome
protein U1A intact is in agreement with this theory (Wu et al., 2001).
Integral membrane nuclear pore proteins
Metazoan NPCs are during interphase tightly connected to the bilayered nuclear envelope and
moves sparsely, in an elastic manner, in the plane of the NE. Synchronised movements
between NPCs and the lamina meshwork clearly indicate that they belong to a common
network (Daigle et al., 2001). In contrast, in vivo data on the dynamics of the NPC from
budding yeast suggests a high mobility of NPCs in the NE (Bucci and Wente, 1997), maybe
as a consequence of the absence of true lamin genes in the yeast genome.
Anchoring of the NPC to the NE has been suggested to be mediated by integral
membrane nuclear pore proteins (POMs). To date, only two pore membrane proteins have
been described in metazoans, namely POM121 (Hallberg et al., 1993) and gp210 (POM210)
(Wozniak et al., 1989; Gerace et al., 1982). Both proteins possess a single transmembrane
domain with their C-terminal end protruding into the cytoplasm. However, while POM121 is
mainly cytoplamic, most of the mass (95%) of gp210 occurs in the perinuclear lumen
23
(Soderqvist and Hallberg, 1994; Greber et al., 1990). Although the NPC target signal have
been determined for gp210 and POM121 (Wozniac and Blobel, 1992; Soderqvist et al.,
1997), their respective binding partner(s) within the NPC remains elusive. The early post
mitotic appearance of POM121 in the nuclear envelope relative to other nucleporins supports
its role as an anchoring molecule (Bodoor et al., 1999). Repeat elements of the XFXFG type
is indeed present in the primary sequence of POM121 but whether these represent binding
domains for other nucleoporins or for molecules of the soluble transport machinery is
unclear. Gp210, on the other hand, lack pentapeptide repeats but contain a hydrophobic
domain distinct from its transmembrane region. By referring to segments present in the
luminal domain of certain viral fusion proteins, Wozniak and Blobel (1992) suggested that
this hydrophobic domain might interact with lipid bilayers and act as a fusogenic peptide
during formation of the pore. However, comparison of gp210 primary sequences from several
species revealed no significant evolutionary conservation of this putative second hydrophobic
domain (Cohen et al., 2001). Evidence for an early role of POM210 in NPC assembly and
pore dilation comes from studies where recombinant cytoplasmic tails and tail specific
antibodies was used in vitro to inhibit the functionality of this domain. It was shown that
inhibited nuclei were growth arrested and that only mini pores was formed, these pores
lacked several nucleporins, including Nup214, Nup153 and Nup98, but not Nup62
(Drummond and Wilson, 2002). In vivo, antibodies directed to the lumenal part of POM210
interfered with passive diffusion as well as signal-mediated import, data arguing for a direct
or indirect link between POM210 and nucleo pore constituents involved in these events
(Greber and Gerace, 1992). Furthermore, POM210 has been shown to form homodimers and
even higher orders of oligomerisation, at least in vitro (Favreau et al., 2001).
A new family consisting of three putative pore membrane proteins in human was
recently isolated and characterised (Coy et al., 2002). The encoded proteins of this family
(POMFIL1-3) are distantly related to POM121 and to genes involved in axon guidance. In
the comprehensive classification of yeast NPC constituents performed by Rout and his
colleagues (Rout et al., 2000), three true pore membrane proteins where found, POM152p
(Tcheperegine et al., 1999; Wozniak et al., 1994), Ndc1p (Chial et al., 1998) and a previously
uncharacterised protein named POM34p. Neither of them shares any significant sequence
homology to any POMs found in vertebrates. Strikingly, examinations of yeast strains
mutated in POM152p or Ndc1p show that these genes are not essential for cell survival.
Deletion mutants of POM152 are lethal in combination with mutations in Nup170p
(Aitchison et al., 1995), which are proposed to help establish the functional diameter of the
NPC central channel (Shulga et al., 2000).
24
The nuclear pore complex during mitosis
De novo assembly of NPCs takes place partly during interphase, in order to accommodate
nuclear growth, and partly postmitoticallay, in order to reassemble nucleoporins that have
been dispersed in the cytpolasm during the mitotic nuclear envelope breakdown, a process
needed to allow the condensed chromosomes access to the mitotic apparatus. In higher
eukaryotes, the nuclear membranes disassemble during prometaphase and reassemble around
daughter chromosomes during late anaphase and telophase (reviewed in Gant and Wilson,
1997). Soluble components of the NPC are thought to break down into either single units, or
into mitotic sub complexes during this process. Whether nuclear membrane proteins are
released into domain specific vesicles during cell division (Chaudhary and Courvalin, 1993;
Buendia and Courvalin, 1997), or released throughout all ER membranes is still controversial
(Yang et al., 1997).
Dispersed nucleporins are in the end of mitosis reutilised to form new pore
complexes. This seems to occur in a sequential fashion and high resolution scanning EM has
revealed morphology distinct NPC assembly intermediates (Goldberg et al., 1997)
(Drummond and Wilson, 2002). In an attempt to resolve the temporal reassociation of
different nucleoporins into newly formed NPCs, it was shown that Nup153 and POM121
where reintroduced at late anaphase, thereby preceding that of the Nup62 complex and
CAN/Nup214 together with Nup84 in early to middle telophase. Interestingly, POM210,
proposed to be important for NPC biogenesis, do not reappear until late telophase/early G1
phase, in parallel with tpr (Chaudhary and Courvalin, 1993; Bodoor et al., 1999).
25
BIDIRECTIONAL TRANSPORT OF MOLECULES BETWEEN THE NUCLEUS
AND THE CYTOPLAM
Passive diffusion
Ions and small proteins lacking a nuclear localising signal (NLS) are able to passively diffuse
through the NPC. Macromolecules below the size of 90-100 Å or approximately 60 kDa
equilibrates between the nucleus and the cytoplasm with rates inversely proportioned to their
size. The actual transport channel for passive diffusion across the pore is not defined but two
alternatives have been suggested. First, the central transporter may function as the transit
element for both active transport and diffusion (Feldherr and Akin, 1997). Second, Hinshaw
and his colleagues reported 8 peripheral channels located within the NPC spoke complex that
potentially also could mediate diffusion of small permeants (Hinshaw et al., 1992).
Active import
Whereas the nuclear pore permit passage of relatively small molecules by diffusion, particles
with a diameter as large as 230-250 Å can enter the nucleoplasm or cytoplasm by signal
mediated transport (Feldherr and Akin, 1990). This transport requires specific interaction
with either the NPC or with members of a conserved super family of soluble guiding
receptors referred to as b-karyopherins. b-karyopherins can be classified into importins or
exportins depending on the direction in which they transport a cargo. Fourteen transport
receptors in the yeast Saccharomy cererevisiae and over 20 in higher vertebrates have been
identified (reviewed in Strom and Weis, 2001). Some classes of cargo contain signals that
bind directly to a cognate receptor while other types of signals bind indirectly, via adaptor
proteins belonging to the karyopherin-a, or importin-a family. Human and mouse posses at
least 5 isoforms of importin-a (a 1-5) that recognize different cargo target sequences
(reviewed in Jans et al., 2000). They all interact specifically with importin-b1 through a
conserved importin-b binding (IBB) domain (Kohler et al., 1999; Cingolani et al., 1999;
Gorlich et al., 1996a). The existence of multiple receptors that specifically recognise one or a
set of cargoes result in a still unknown number of different transport pathways, each
regulating the localisation of one or several macromolecules. The most classical and probably
most widely used import pathway is a multi-step process that can be divided into three major
steps (Fig. 2A). First, proteins containing a nuclear localising signal (NLS) are recognised in
the cytoplasm by a importin-a. Variations in NLS occur but the best defined are those of the
SV40 Large T antigen and nucleplasmin. They consist of one (monopartite) or two stretches
of basic amino acids (bipartite), respectively (Robbins et al., 1991; Kalderon et al., 1984).
Other nuclear import signal-receptor system has been described. For example, tranportin
(karyopherin-b2) mediates the nuclear import of a subset of hnRNP proteins bearing an M926
type NLS that is rich in glycine and aromatic residues (Siomi and Dreyfuss, 1995).
Transportin binds directly to the M9-containing cargo (Nakielny et al., 1996). Importin-a
forms a complex with importin-b1 (karyopherin-b1), which target the heterotrimer to the
cytoplasmic face of the NPC where it binds to components of the cytoplasmic fibres that
radiate from NPC into the cytoplasm (Pante and Aebi, 1996; Delphin et al., 1997).
Interestingly, cytoplasmic fibres seems not to be essential for nuclear import indicating that
adjacent nucleo pore domains also is able to mediate the docking phase (Walther et al., 2002).
In the second step, proteins are, in a still poorly understood fashion, translocated in an energy
dependent manner along the axis of the NPC into the nucleoplasm. Finally, translocation is
followed by displacement of the cargo from the carrier. Cargo release is restricted to the
nucleus by the interaction of the importin a/b-cargo heterotrimer with Ran (Gorlich et al.,
1996b; Moore and Blobel, 1993), a small Ras related GTPase. Ran binds to GTP or GDP, and
its nucleotide-bound state is regulated by several cofactors. The conversion of RanGDP into
RanGTP requires Ran guanine exchange factor (RanGEF or RCC1), that predominantly is
found in the nucleus, bound to chromatin (Bischoff and Ponstingl, 1991). GTP hydrolysis by
Ran occurs exclusively in the cytoplasm and is initiated by the RanGTPase activating
protein-1 (RanGAP) and co stimulated by the Ran-binding proteins 1 and 2 (RanBP1 and
Nup358/RanBP2) (Bischoff and Gorlich, 1997; Bischoff et al., 1994; Yaseen and Blobel,
1999). Nuclear transport factor 2 (NTF2), a RanGDP-binding protein, facilitates nuclear
accumulation of Ran and is responsible for the accumulation of Ran in the Nucleus at cellular
steady state (Ribbeck et al., 1998). The result of these actions is a sharp gradient where the
GTP-bound form of ran is found in the cytoplasm and the GDP-bound form is in the
nucleoplasm. This asymmetry is crucial for at least major transport pathways in both
directions (Izaurralde et al., 1997). It has been suggested that directionality of the
bidirectional transport process is maintained through interactions with RanGTP/GDP and
indeed, an artificial reversal of the RanGTP/GDP gradient has been shown to run transport
backwards (Nachury and Weis, 1999). After releasing their cargo into the nucleus importin-b
is returned to the cytoplasm in a heterodimeric form with RanGTP. Importin-a is translocated
back through interaction with the exportin CAS in a RanGTP dependent manner and has
thereby completed one import cycle (Kutay et al., 1997).
27
Active export
Nuclear export of proteins and RNAs is closely analogous to nuclear import and involves
distinct targeting signals, importin homologs, and nucleoporin binding sites, as well as Ran
and its modifying factors (Fig. 2B). Nearly all nucleus-encoded RNAs require export to the
cytoplasm as part of their maturation process or biosynthetic function. RNAs are transported
as ribonucleoprotein (RNP) complexes and receptors have been identified that conduct
mRNAs, tRNAs and untranslated small nuclear RNAs (snRNAs) to the cytoplasm, each by
largely independent pathways. The giant Balbani ring transcripts in Chironomus tentans offer
a unique model system for visualising assembly and transport of RNP particles (Kiesler et al.,
2002; reviewed in Daneholt 2001).
The best understood export signal in proteins is the leucine-rich nuclear export signal
(NES), first identified in the immunodeficiency virus (HIV)-1 Rev protein (Fischer et al.,
1995). Crm1 (exportin1), a protein that shares sequence similarities to the karyopherin b
family appears to act as a export receptor for proteins containing a NES, including a cisacting nuclear export signal that interacts with a dimeric protein complex binding to the 5'
monomethylated cap structure of spliceosomal U snRNAs (Izaurralde et al., 1995). Initially,
it was shown in S, pombe, that Crm1 is a target for the antifungal drug leptomycin B (Fukuda
et al., 1997). When leptomycin B was found to inhibit nuclear export of the HIV-1 protein
Rev, Crm1 was suggested to be the karyopherin that exports Rev from the nucleus (Wolff et
al., 1997). The human homolog of Crm1 is a soluble protein that interacts with Nup214/CAN
(Fornerod et al., 1997). Crm1 seems to interact directly, without adaptor molecules, to NES
elements (Ossareh-Nazari et al., 1997). The role of RanGTP during export is in contrast to the
import process not to dissociate the receptor-cargo complex but to stabilise it. This has been
confirmed by in vitro studies where the addition of Crm1 and RanGTP but not Crm1 alone to
digitonin-permeabilised cells promotes the accumulation of export cargo on the cytoplasmic
face of the nuclear pore complex (Black et al., 2001). Thus, Crm1 and RanGTP are necessary
and sufficient for assembly of transport complexes in which the cargo contains a NES, as
well as their targeting and translocation through the NPC. Following the translocation, the
export complex reaches the cytoplasmic side of the NPC where its disassembly releases all
units into the cytoplasm. This step includes RanGAP dependent hydrolysis of RanGTP to
RanGDP, probably in co-operation with RanBP1 and RanBP2. Since Crm1 has a low affinity
for RanGDP this conversion efficiently makes the disassembly process irreversible. An
additional molecule that has been shown to be essential for the export cargo release is NXT1
(p15) (Black et al., 2001), a RanGTP binding factor that is able to shuttle between the nucleus
and the cytoplasm (Black et al., 1999). As mentioned, several other export receptor molecules
have been identified, each with a distinct or in some cases redundant binding and transport
capacity. For example, mature tRNA is directly recognized by a karyopherin b-like export
28
receptor, exportin-t, in a RanGTP-dependent manner (Arts et al., 1998; Kutay et al., 1998).
Less is known about mechanisms responsible for mRNA export but Ran-dependent pathways
involving different soluble proteins likely coexist and converge to the NPC. Heterogeneous
nuclear ribonucleoporins (hnRNPs), associating with mRNA during or just after transcription
could provide NESs. In particular, hnRNP A1, which contains a NES termed M9 is thought
to stimulate mRNA export (Michael et al., 1995). Recent investigations have proposed
human TAP (also known as NXF1) as a mRNA export receptor. This protein is distinct from
the karyopherin-b family and belongs to a conserved group of putative receptor molecules
known as the nuclear export factor (NXF) family (Herold et al., 2000). Tap interacts with
components of the NPC, as well as to three proteins implicated in the export of cellular
mRNAs: RAE1/hGle2, E1B-AP5 and U2AF (Bachi et al., 2000; Zolotukhin et al., 2002), and
has been shown to promote export of viral mRNAs bearing a constitutive transport element
(CTE) (Gruter et al., 1998). Furthermore, a TAP homolog in C. Elegans and Mex67p, the
yeast homolog of hTAP, are both essential for the sequence non-specific export of bulk
polyadenylated RNAs to the cytoplasm in each species, respectively (Tan et al. 2000; SantosRosa et al., 1998). Accumulating data suggests that the small NXT1/p15 protein may function
as critical cofactor for TAP mediated mRNA export. It has been shown that NXT1 regulates
the ability of TAP to interact with components of the nuclear pore (Wiegand et al., 2002).
Nucleocytoplasmic traffic is thus regulated at multiple sites in the cytoplasm, nucleus and the
NPC. Variations in the molecular compositon of the NPC and the pore membrane could
significantly influence transport, but has not beem much studied.
29
Figure 2. Molecular interactions during nucleocytoplasmic transport. (A) Import receptors
bind cargo molecules in the cytoplasm and mediate their transport to the nucleus through the
nucleopore complex. Binding of RanGTP to the import complex in the nucleus causes cargo
and importin-a release. Recycling and disassembly of the RanGTP-karyopherin b complex
upon RanGTP hydrolysis terminates the import cycle. (B) The export cycle is highly similar
to the import cycle but uses different transport receptors. Furthermore, the RanGTP promote
the receptor-cargo interaction. (Adapted from Kuersten et al., 2001).
30
RESULTS AND DISCUSSION
All organ development involves co-ordinated growth and differentiation of epithelial and
mesenchymal tissues. A unique feature of the kidney is that the mesenchyme converts into
epithelium. The model system can be used to define markers for epithelial polarization. In the
first study (I), our aim was to identify such components. By screening of a mouse cDNA l
phage library representing the induced state of metanephrogenic mesenchyme and using
cDNA probes prepared from either induced or uninduced mesenchymes we were able to
identify a gene that was up regulated during this development. Unexpectedly, this gene
turned out to be gp210, an integral membrane pore protein that previously had been described
in rat (Wozniak et al., 1989), Drosophila melangoaster (Berrios et al., 1995) and Xenopus
laevis (Gajewski et al., 1996). mRNA in situ hybridisation revealed that the transcript of
gp210 was confined to epithelial tissues and to the condensing mesenchyme in the
developing kidney. This epithelial pattern was demonstrated in other embryonic organs,
including skin, lung, liver, brain, gut and pancreas, whereas no or little expression could be
detected in stromal compartments or in mesenchymal tissues such as cartilage. In vitro, we
could demonstrate that regulation of the gp210 gene is not dependent on cell proliferation.
Thus, regulatory pathways separated from this cellular process activate the expression of
gp210 (I).
Nuclear pore complexes are critical for nucleocytoplasmic transport and as such
fundamental for cell survival. Present models suggest that a single NPC is able to mediate
both nuclear import and export of material with no variations in the NPC configuration exists
between different cells types. It is therefore striking that several cell types appear to lack
gp210. An extensive examination of the relative mRNA expression of several characterised
nucleoporins in different organs and at discrete developmental stages revealed no dramatic
pattern variations. By using a non-radioactive northern blot system with deoxyginine labed
probes directed to Nup98/96, Nup62, Nup107, Nup54, Nup84, Nup153 and gp210, we where
able to observe detectable signals for each individual nucleoporin in brain, liver and kidney.
Developmental stages for each organ ranged from embryonic day 12 to adult stages (data not
shown). In sharp contrast to all other nups analysed, no gp210 transcripts could be detected in
four different cell lines analyzed (II). This finding was confirmed at the protein level using a
polyclonal antibody raised to the COOH-terminal end of gp210 (II). Furthermore, a very
restricted expression of gp210 was seen in developing mouse embryos, in sharp contrast to
the distributon of Nup358, Nup214, Nup153 and Nup62, four nucleoporins collectively
targeted by mAb214 (II). In conclusion, it seems that the expression of mouse gp210 is not
synchronised with nucleoporins in general. In addition, the apparent absence of the gp210
protein in several specific compartments of the developing embryo and in certain cell lines
31
indicates that gp210 is a major polypeptide of the NPC of only some cell types. Hence, it
could be that gp210 is not essential for NPC anchoring and pore formation. The data suggest
cell-type specific functions.
It is well known that the establishment and maintenance of polarity of epithelia is
essential for functions of epithelial sheets. A major issue in cell biology has been to
characterize components required for cell polarization (Drubin and Nelson, 1996). Spatially
and functionally distinct protein scaffolds, assembled from transmembrane and cytoplasmic
proteins, provide clues for polarization. Components known to control polarity include
laterally located cell adhesion molecules, basally located receptors for basement membranes
(Durbeej et al., 1995), and apically located cytoplasmic proteins (Knust, 2000). The current
work on gp210 is descriptive, but introduces the new concept that epithelial cell polarity
correlates with expression of a nuclear pore membrane protein. Gp210 could be involved in
the development and maintenance of epithelial cell polarization.
In order to get insight into mechanisms driving the expression of the gp210 gene, we
analysed the promoter region for human gp210. Since neither TATA nor CAAT box elements
could be identified in the DNA sequence preceding the ATG start codon, transcription start
sites where determined experimentally. Two start sites where found, one major at position
–26 and one minor at position –18. We could further demonstrate several putative binding
elements for tissue specific transcription factors that where conserved in the corresponding
mouse genomic region, including clustered consensus sequences associated with Sp1 and
Wt1 (III). Experimentally, we tested whether or not Wt1 was involved in the regulation of
gp210. This was done in osteosarcoma (SAOS-2) cells using a model system in which Wt1
expression is controlled by tetracyclin. A modest downregulation of gp210 mRNA expression
by Wt1 was noted. This means that the putative regulators of the gp210 gene remain to be
identified (III).
For a long time, gp210 and POM121 were the only known transmembrane proteins that
localised to the pore membrane domain of the nuclear envelope. However, the identification
and characterisation of the POMFIL1, POMFIL2 and POMFIL3 enlarged the group of
integral membrane proteins associated with the nuclear pore (Coy et al., 2002). These novel
proteins are regulated in a tissue and developmental specific manner. It is thus gradually
becoming evident that variations of NPC configuration occur in metazoans, depending on cell
type. To our knowledge, this possibility was first suggested based on the mRNA data for
gp210 (I).
Receptor mediated transport of proteins can be targeted for regulation at several steps
in their movement across the nuclear envelope. In most known cases, the cargo itself is
modified in a way that affects its interaction with import or export receptors. Tethering of the
cargo-receptor complex to immobile cellular components or regulation of the soluble
32
transport machinery are other examples of how the cell is able to control a variety of
fundamental processes (for a comprehensive review on this subject, see Kaffman and O'Shea,
1999). Modifications of the NPC by cell-type specific nucleoporins could confer an
additional level of regulation of protein localization.
Antibodies directed to the cytoplasmic tail of gp210 arrests pore formation at an early
stage when added to Xenopus nuclear assembly extracts (Drummond and Wilson, 2002). The
apparent lack of gp210 protein in many cell types does not fit with the view of gp210 as a
crucial mediator of one or several discrete steps in early NPC biogenesis.
The abundance of gp210 has been calculated to 16-25 copies per nuclear pore
representing 5-6 Mda in some cell types (Gerace et al., 1982). Based on these calculations, it
has been suggested that this protein may be a major component of the lumenal spoke domain
of the nuclear pore complex (Wozniak et al., 1989; Yang et al., 1998). In vitro, it was shown
that gp210 is able to self-associate into dimers and that these dimers have the capability to
form higher orders of oligomerisation. Large arrays of gp210 could in vivo provide a basic
framework that organises into a circular lumenal skeleton for the pore membrane (Favreau et
al., 2001). Since our data (I, II) indicate high amounts of gp210 in epithelial tissues compared
to non-detectable levels in surrounding cells, it is tempting to speculate that the extent of the
lumenal skeleton vary in a cell type specific manner. This variation could in turn influence
both the pore and the NPC itself in multiple ways.
Inhibition of signal mediated transport of proteins into the nucleus as well as passive
diffusion through the pore complex upon depletion of calcium from the lumen of the
endoplasmic reticulum and nuclear envelope has been reported (Greber and Gerace, 1995).
Similar effects on passive and active transport have been shown by introducing an antibody,
targeting a luminal epitope of gp210, into the perinuclear cisterna (Greber and Gerace, 1992).
These results provide direct functional evidence for conformational flexibility of the pore
complex. Since the amino-terminal of gp210 contains a putative EF hand Ca2+-binding
domain (Greber et al., 1990), it has been speculated that variations in calcium levels may
trigger rearrangements within the lumenal domain and result in conformational changes in the
inner spoke ring and central transporter (Perez-Terzic et al., 1997; Perez-Terzic et al., 1996).
At present, the physiological relevance of conformational changes within the NPC is
unknown but they could play an important role in regulation of specific states of the cell
cycle or differentiation (Feldherr et al., 1994; Feldherr and Akin, 1993). Our findings
revealed that the expression of gp210 is most prominent in epithelial cells of the mouse
embryo (I, II). These data do not directly address the function of gp210. However, an
intriguing possibility is that an enhanced expression of gp210 induces a reversible or nonreversible conformational change in the NPC with subsequent effects on cell differentiation.
The transition of progenitor metanephric mesenchymal cells into cells committed to
terminal differentiation is accompanied by a distinct up regulation of gp210. Gene regulation
33
of other nucleoporins is not well documented but has in a few cases revealed interesting
information. Nup88 has been shown to be a marker for metastaticy in cutaneous melanoma
cell lines (Zhang et al., 2002), and to be selectively over expressed, as compared to nup214
and nup153, in several other malignant tumours (Gould et al., 2000; Gould et al., 2002).
Nup88 may, therefore, be considered as a putative marker for tumour growth and is probably
related to increased cell cycling. Apart from being structural components of the NPC, nup96
and nup98 are also associated with the nuclear interior. Both proteins are expressed from a
single gene, which are activated by INF-g in a STAT1 dependent manner. Nup98 is a target
of the vesicular stomatitis virus matrix protein-mediated inhibition of mRNA nuclear export
and it has been reported that up regulation of 98, induced by INF-g constitutes a mechanism
that reverses M protein-mediated inhibition of gene expression (Enninga et al., 2002). These
examples clearly demonstrate that studies of mechanisms that regulate nucleoporin genes
could provide functional aspects of this group of proteins. The promoter characterisation of
the human and mouse gp210 genes (III) was a first step towards an identification of one or
several transcription factor utilised in the activation of this gene.
34
FUTURE PERSPECTIVES
Our data presented in paper I-III do not rule out the possibility that all cells in the mouse
express some gp210, but it is evident that the expression levels vary drastically depending on
the cell type in multicellular organisms. It would therefore be of interest to examine if gp210
is fundamental for cell survival. This could be performed by experimental overexpression, or
by gene targeting experiments in mice.
Expression of gp210 is drastically increased when mesenchymal cells are converted to
epithelial cells. This regulation might reflect a dynamic change of the NPC as the cells
acquire more specialised functions. Is gp210 merely a marker for epithelial cells, or does it
have functions not needed for other cell types? One approach that eventually could answer
this question is to induce high expression of gp210 in a cell line, which shows low levels of
this protein. The role of gp210 in nucleocytoplasmic transport, NPC conformation, and the
cellular phenotype could be analyzed.
The promoter sequence of gp210 contains several putiative binding sites for cell specific
regulator elements. By using reporter gene assays, gel mobility shift assays and antibodies
directed to putative regulators, it would be possible to clarify the role of such factors.
POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA
Det nukleära höljet består av ett yttre och ett inre membran. Tillsammans begränsar de den
nukleära regionen i kärnförsedda eukaryota celler och ger upphov till en funktionell miljö för
dess kromosomer. Separationen av DNA-replikation och RNA-transkription i kärnan från den
cytoplasmatiska protein-translationen ger eukaryota celler en mer sofistikerad kontroll över
genernas uttryck än vad som är möjlig hos prokaryoter. Kontrollen förutsätter dock att
proteiner och RNA har möjlighet att passera den barriär membranen utgör. Nukleära
porkomplex (NPK) är stora proteinkomplex (125 mDa) som i elektronmikroskopi uppvisar en
åttafaldig symmetri runt en, i förhållande till det nukleära höljet, vertikal axel. De ligger
inbäddade i de nukleära membranen och bildar rörliknande strukturer som förbinder
cytoplasma och kärna. Kanalerna är den enda kända förbindelselänken mellan dessa cellulära
rum. NPK har även påträffats i cytoplasmatiska membran (s.k. Annulate lamellae), men där
är deras funktion ännu inte känd. Man har uppskattat att det i porkomplexen ingår 30 olika
protein (nukleoporin, nup), varav ett tjugotal är karakteriserade. Det pågår en intensiv
forskning för att utreda den specifika funktionen för vart och ett och för att identifiera nya.
35
Import av proteiner till kärnan styrs av ett antal signalsekvenser varav den klassiska nukleära
lokaliseringssignalen (NLS) och importsignalen M9 är de bäst karaktäriserade. På
motsvarande sätt styrs RNA ut i cytosolen. Den nukleära export signalen (NES) är en
leucinrik aminosyra-sekvens som ofta återfinns på RNA bindande protein. Lösliga receptorer
tillhörande genfamiljen b-kayoferiner binder till signalerna och lotsar därefter substratet fram
till och igenom porkomplexen. Den här processen är energikrävande.
Vissa protein transporteras inte konstitutivt, utan är beroende av posttranslationella modifieringar för sin cellulära distribuering. Selektiv transport av proteiner
genom NPK är på så vis ett sätt för cellen att kontrollera genuttryck, och därmed en av flera
mekanismer som gör det möjligt för cellen att reglera exempelvis proliferation och
differentiering. Den nu rådande modellen beskriver NPK som en homogen, statisk struktur,
utan direkt betydelse för selektiviteten under den nukleocytoplasmatiska trafiken. Det finns
dock rapporter som tyder på motsatsen och visar att det nukleära porkomplexet är mer
dynamiskt än vad som tidigare antagits. Nukleoporin gp210 (glykoprotein 210) är ett integralt
membranprotein med en mindre cytoplasmatisk karboxyterminal del, en transmembranregion
samt en stor lumenal aminoände. Proteinet är väl karakteriserat och dess funktion har länge
associerats med biogenes och stabilitet av NPK. Mina data visar att gp210 uppregleras under
en tidig fas av njurutvecklingen. Expressionsstudier på musfoster påvisade även ett begränsat
uttrycksmönster, som förutom i njure även i hud, lever och lunga sammanföll med
framväxande epitelcellers. Studien genomfördes initialt på RNA-nivå men konfirmerades på
proteinnivå med hjälp av en antikropp riktad mot den cytoplasmatiska domänen av gp210.
Vidare tyder våra in-vitro data på att ett stort antal celltyper helt saknar gp210. Jämförelser
med andra karakteriserade nukleoporiner tyder på att distributionen av gp210 är unik. Den
begränsade utbredningen av gp210 under den embryonala utvecklingen och i cellinjer visar
att det existerar olika typer av NPK. Gp210 kan alltså ha en annan funktion än den tidigare
föreslagna.
Länge var gp210 tillsammans med POM121 (POre Membrane protein 121) de
enda karakteriserade membranprotein som var lokaliserade till NPK. Så är inte längre fallet.
Nyligen isolerades en familj (POMFIL1, POMFIL2 och POMFIL3) av gener med tydliga
homologier till POM121. Presenterade data tyder på att åtminstone POMFIL1 och POMFIL2
är membranbundna nukleoporin med ett, liksom gp210, differentiellt uttrycksmönster under
embryogenesen. Dessa resultat styrker våra teorier om att det förekommer variationer i
sammansättningen av NPK, och att dessa variationer är vävnads och/eller celltyps beroende.
Denna variation kan vara av stor betydelse för den molekylära trafiken mellan kärnan och
cytoplasman.
36
ACKNOWLEDGMENTS
Most of this work was carried out at the Department of Cell and Molecular Biology, Uppsala
University and at the Department of Cell and Molecular Biology, Lund University. I would
like to express my gratitude to all those that have helped me, in so many different ways,
while working on this thesis. Thank you!
Especially I would like to like to thank:
Professor Peter Ekblom, my supervisor, for your scientific guidance and a constant moral
support. Thank you for everything!
Tord Hjalt, Donald Gullberg, Erik Forsberg and Ann-Marie Olofsson for taking their time to
educate me in different aspects of molecular biology.
Marja Ekblom, for letting me inherit ”clone 5” and for being such a helpful and friendly
person.
My co-authors.
Siw Medborg, Agneta Ekman-Lundell and Sigrid Petterson, past and present secretaries. True
competence in the interface with the real world. I wish you all luck!
Madde, Teet, Calle, Kati, Maria R., Katta, Lotta, Jan, Elke, Ulrika, Hong, Yu-Chen, Hao,
Yang, Maria W., Alexander, Fredrik E., Wilhelm, Kinga and Wilma, my colleagues through
the years, for all memorable moments. It has been a pleasure to work with you all.
Susanne and Maria F., my roommates. The two of you improved Lund!
Mats, for years of great friendship and encouragement.
Peter and Elik, my good friends, for sharing pain and pleasure during the BMC years, and for
being such good losers in badminton.
All other old ”biological” friends, especially Henrik Pärn, to whom I also owe an excuse. I
couldn’t wait any longer!
Erik Lindqvist and his family, for their generous hospitality.
My family, always reliable.
And Sara, for your love and support.
37
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