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INTRODUCTION
The Nuclear Pore Complex (NPC) is a supramolecular
assembly of approx. 125 MDa (Reichelt et al., 1990) in the
nuclear envelope (NE), and mediates the passive and active
exchange of macromolecules between the nucleus and
cytoplasm (reviewed in Rout and Wente, 1994; Davis, 1995;
Melchior and Gerace, 1995; Goldberg and Allen, 1995). It
comprises multiple copies of approx. 100 different
nucleoporins that form a scaffold for proteins involved in
binding and translocation of macromolecules across the NPC,
and may directly participate in either nucleocytoplasmic
transport or its regulation (for reviews, see Rout and Wente,
1994; Pante and Aebi, 1994, 1996).
In order to understand the role of individual NPC
components in the mechanism of molecular translocation a
detailed structural analysis of the NPC in resting and active
conformations is required, in contrast to the amphibian oocyte
models, which are transcriptionally inactive. The NPC has
eightfold rotational symmetry and consists of a multi-domain
spoke complex surrounding a central transporter, which is
framed by cytoplasmic and nucleoplasmic coaxial rings (Akey,
1995; Akey and Radermacher, 1993; Hinshaw et al., 1992).
Eight thin fibers involved in protein import (Pante and Aebi,
1996; Rutherford et al., 1997) project from the NPC
cytoplasmic ring, while a basket structure that participates in
mRNP export (Kiseleva et al., 1996) extends from the
nucleoplasmic ring (Ris, 1991; Jarnik and Aebi, 1991;
Goldberg and Allen, 1992; Akey and Radermacher, 1993).
Such asymmetrical NPC organization might be explained by
the vectorial nature of nuclear import and export, as well as by
differences in the processing of translocating molecules during
transport.
All actively transporting molecules appear to move through
the NPC central channel where the most intriguing and
controversial NPC structure, the transporter, is localised.
Depending on the isolation procedure and method of sample
preparation, this structure demonstrates a variable appearance
223
Journal of Cell Science 111, 223-236 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS4459
The Nuclear Pore Complex (NPC) regulates
nucleocytoplasmic transport by providing small channels
for passive diffusion and multiple docking surfaces that
lead to a central translocation channel for active transport.
In this study we have investigated by high resolution
scanning and transmission electron microscopy the
dynamics of NPC structure in salivary gland nuclei from
Chironomus during Balbiani ring (BR) mRNP
translocation, and present evidence of rearrangement of
the transporter related to mRNP export. Analysis of the
individual NPC components verified a strong evolutionary
conservation of NPC structure between vertebrates and
invertebrates. The transporter is an integral part of the
NPC and is composed of a central short double cylinder
that is retained within the inner spoke ring, and two
peripheral globular assemblies which are tethered to the
cytoplasmic and nucleoplasmic coaxial rings by eight
conserved internal ring filaments.
Distinct stages of BR mRNP nuclear export through the
individual NPC components were directly visualized and
placed in a linear transport sequence. The BR mRNP first
binds to the NPC basket, which forms an expanded distal
basket ring. In this communication we present stages of BR
mRNP transport through the nucleoplasmic, central and
cytoplasmic transporter subunits, which change their
conformation during mRNP translocation, and the
emegence of mRNP into the cytoplasm. We propose that
the reorganization of the basket may be driven, in part, by
an active translocation process at the transporter.
Furthermore, the images provide dramatic evidence that
the transporter functions as a central translocation channel
with transiently open discrete gates in its globular
assemblies. A model of NPC transporter reorganization
accompanied with mRNP translocation is discussed.
Key words: Nuclear Pore, Nuclear Envelope, mRNP transport,
Transporter, Chironomus, Scanning electron microscopy
SUMMARY
Active nuclear pore complexes in Chironomus: visualization of transporter
configurations related to mRNP export
Elena Kiseleva
1,3
, Martin W. Goldberg
1
, Terence D. Allen
1,
* and Christopher W. Akey
2
1
CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital National Health Service
Trust, Manchester, M20 9BX, UK
2
Department of Biophysics, Boston University School of Medicine, 80 East Concord St, Boston, Massachusetts 02118-2394, USA
3
Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, 630090, Russia
*Author for correspondence (e-mail: tallen@picr.man.ac.uk)
Accepted 10 November 1997: published on WWW 23 December 1997
224
and occupancy within the spoke complex, but is well preserved
in frozen-hydrated specimens (Akey, 1989, 1990; Akey and
Radermacher, 1993). It has been proposed that this structure
may represent the central channel complex, material caught in
transit, or both (Akey, 1990; Jarnik and Aebi, 1991; Akey and
Radermacher, 1993); however almost nothing is known about
the mechanism of transporter function during molecular
exchange.
Nucleocytoplasmic active transport is bidirectional and can
be divided into three major phases, including
targeting/docking, translocation across the central channel and
substrate release (Richardson et al., 1988, Newmeyer and
Forbes, 1988; Akey and Goldfarb, 1989). The mechanism of
substrate targeting and docking in nuclear import has been
extensively studied, resulting in recognition of nuclear
localization sequences (NLS) (Laskey et al., 1996) and specific
nuclear import receptors, including the alpha and beta
importins/karyopherins (Dingwall and Laskey, 1986; Gorlich
et al., 1996; Moroianu et al., 1995; Aitchison et al., 1996; Chi
et al., 1996). It was also shown that the active import of
proteins requires GTP hydrolysis, catalyzed in part by
Ran/TC4 GTPase (Melchior et al., 1993; Moore and Blobel,
1993).
The nuclear export of mRNPs still remains poorly defined
in comparison to nuclear import. It was found that this process
is also signal-mediated (Jarmolowski et al., 1994; Gorlich et
al., 1996), energy-dependent, can be inhibited by co-injection
of wheat germ agglutinin (WGA) (Dargemont and Kuhn, 1992;
Dargemont et al., 1995) and may utilize Ran GTPase (for
reviews, see Izaurralde and Mattaj, 1995; Fischer et al., 1996;
Koepp and Silver, 1996;). Several nuclear proteins, including
transportin/karyopherin β2, karyopherin β3/Pse1p and
karyopherin β4/Kap123p (Rout et al., 1997), exportin 1/Crm
1p (Fornerod et al., 1997), CAS/Cse1p (Kutay et al., 1997) and
possibly Sxm1p (Seedorf and Silver, 1997; Pollard et al.,
1996), some RNA-binding proteins (Michael et al., 1995; Visa
et al., 1996a; Daneholt, 1997) and a cap binding complex
(CBC), which is co-exported to the cytoplasm with mRNA
(Visa et al., 1996b), could be involved in mRNP transport
regulation.
The mRNP particles can be seen passing through the NPC
centre in electron microscope sections (Stevens and Swift,
1966; Franke and Sheer, 1974). Considerable progress in the
analysis of mRNP transport has been achieved by electron
microscope investigations of Chironomus salivary gland cells,
which actively synthesize abundant secretory proteins encoded
by Balbiani ring (BR) genes (for a review, see Mehlin and
Daneholt, 1993; Wieslander, 1994). 75S premessenger RNA is
synthesised at BR genes and is packaged with proteins into 50
nm mRNP particles (Stevens and Swift, 1966; Andersson et
al., 1980; Olins et al., 1980; Daneholt et al., 1982) after splicing
(Kiseleva et al., 1994). Three-dimensional electron microscope
tomography (Skoglund et al., 1986; Mehlin and Daneholt,
1993; Daneholt, 1997) and Field Emission In Lens Scanning
Electron Microscopy (FEISEM; Kiseleva et al., 1996)
demonstrated that the BR mRNP particle docks to the NPC
basket, which forms a distal basket ring and initiates the
unpacking of the BR particle into a linear RNP ribbon. This
RNP ribbon remains separated from the spoke-ring complex
during passage through the central NPC channel (Mehlin et al.,
1992), and the 5′ end of the BR mRNP transcript leads the way
in association with the CBC (Mehlin et al., 1995; Visa et al.,
1996b).
In this report, we present new data about the structural
organization and conformational plasticity of the NPC during
mRNP transit in Chironomus salivary gland cells by FEISEM
and TEM. In most respects, Chironomus NPCs are similar to
Xenopus oocyte NPCs, suggesting a strong evolutionary
conservation of NPC architecture between invertebrates and
vertebrates (also see Goldberg et al., 1997a). Direct
vizualization of the transporter showed that it is composed of
a double central cylinder, framed on the cytoplasmic and
nuclear sides by large globular assemblies. These are attached
to the thin co-axial rings at either surface by an array of eight
internal filaments (also see Goldberg and Allen, 1996) and
appear to play multifunctional roles in gating (the forming,
opening and closing of the peripheral gates of the transporter)
during BR mRNP translocation. Moreover, the BR mRNP has
been visualized exiting from the cytoplasmic side of the
transporter central cylinder (see Fig. 1) and then from the upper
part (cytoplasmic globular assembly) of the transporter,
thereby completing the translocation phase of nuclear export.
Additional details of basket conformation (diagonal orientation
of basket filaments around the distal ring) and the sites of the
binding of cytoplasmic filaments to the cytoplasmic NPC
particles were observed. New information has been used to
construct a 3-D model for the mRNP translocation through the
NPC.
MATERIALS AND METHODS
Chironomus thummi were cultured according to Lambert and
Daneholt (1975). Salivary glands were isolated from rapidly growing,
6-week-old, fourth instar larvae. Pilocarpin concentration (0.1 mg/ml)
and duration of treatment (5 hours) were as previously described
(Mahr et al., 1980).
Isolation of nuclei
Two approaches were used for nuclear isolation. (1) For preparation
of nuclei without detergent treatment the isolated salivary glands were
kept in ice-cold TKM buffer (100 mM KCl, 1 mM MgCl
2
and 10 mM
triethanolamine-HCl, pH 7.0) for 2-3 minutes and individual nuclei
were manually isolated from salivary gland cells using tungsten
dissection needles. (2) Cells in TKM buffer were pretreated for either
10 or 30 seconds with 2% NP-40 or Triton X-100 and nuclei isolated
by pipetting. In the latter method, four or six isolated glands were
placed in ice-cold TKM buffer, supplemented with 2% NP-40 or
Triton X-100, for 10-30 seconds and then transferred into 0.025% NP-
40 or Triton X-100 in TKM. The nuclei were isolated from glands by
pipetting. Nuclei isolated by both methods were quickly transferred
to poly-L-lysine-treated 5 mm silicon chips in TKM buffer and fixed
immediately.
Electron microscopy
For TEM analysis of NPC structure by thin sections, isolated salivary
glands were fixed in 2% glutaraldehyde and 0.2% tannic acid with
TKM buffer for 2 hours at 4°C, followed by 1% osmium tetroxide in
TKM buffer for 30 minutes at 4°C, dehydrated and embedded in Epon.
The sections were stained in aqueous saturated uranyl acetate,
followed by 0.4% lead citrate at a high pH, and then examined in a
Philips EM 400 TEM (Philips, Eindhoven, Netherlands) at 60 kV.
For FEISEM, nuclei that had been partly opened with a glass needle
were fixed as previously described (Goldberg and Allen, 1992).
Silicon chips (SEM specimen support) with attached nuclei were
E. Kiseleva and others
225Active nuclear pore complex structure
transferred to 2% (w/v) glutaraldehyde and 0.2% (w/v) tannic acid in
TKM for 10 minutes, washed in TKM and fixed for 10 minutes with
1% (w/v) OsO
4
in TKM, washed in distilled water, transferred to 1%
(w/v) uranyl acetate for 10 minutes, dehydrated through ethanol and
critical-point-dried via Arklone (ICI, Runcorn, Cheshire, UK) and
carbon dioxide. Specimens were then coated with 2 nm or 4 nm of
tantalum in an Edwards 306 cryo-pumped vacuum system with a
magnetron sputter head (Edwards High Vacuum International,
Crawley, West Sussex, UK) and viewed in the top stage of a Topcon
(ABT) ISI DS 130F field emission scanning electron microscope
(Topcon Corporation, Tokyo, Japan) at 30 kV. The average size
measurements of different NPC components were done from approx.
2000 NPCs chosen from three independent experiments. Corrections
for metal coat thickness were not made, as it is uncertain what effect
this had on sizes of objects; however, our previous measurements of
Xenopus NPCs were within 8% of measurements made by others
using different EM techniques. Therefore, our measurements are
presented as a rough range.
Treatments
Mechanical fracture was employed to remove the cytoplasmic rings
from unfixed nuclear pore complexes as described previously (Unwin
and Milligan, 1982; Goldberg and Allen, 1996). Nuclei were allowed
to adhere to the silicon chip and then gently rolled over the surface
by pushing with a glass needle, followed by spreading and fixation of
the NE. Baskets were removed from the NPCs by scraping spread NEs
with the tips of fine tweezers after fixation and critical-point drying.
Trypsin treatment (0.5 µg/ml) of isolated nuclei before fixation was
sometimes used (see Goldberg and Allen, 1993).
Statistical analysis
A statistical analysis of the frequency of different stages of mRNP
transport through the NPC was performed using both TEM and SEM
samples. NPC structure was investigated by TEM on cross sections
of nuclei that were chosen randomly from three different experiments.
All the NPCs in a NE section from each nucleus (four nuclei per
experiment) were analysed. Roughly 1200 NPCs at the nucleoplasmic
and 1200 NPCs at the cytoplasmic sides of the nuclear envelope in
SEM images were chosen randomly from three different experiments
and analyzed (approx. 400 NPCs were analysed for each experiment).
RESULTS
In the present work, we have studied the architecture of the
Chironomus NPC and the roles of different NPC components
in the transport of mRNPs through the central transporter. We
have used two complementary electron microscopy methods:
(1) high resolution scanning electron microscopy (FEISEM),
which provides good quality preservation of NPCs with high
definition surface images (see Figs 1-7) and (2) conventional
TEM of intact salivary gland cells in thin sections (see Figs 5,
7).
A model of the Chironomus NPC
A statistical analysis of the size and location of various
identifiable components within the Chironomus NPC (approx.
2000 NPCs were analysed in FEISEM images) was made and
is shown in Fig. 1, as a model of the NPC in side (Fig. 1a) and
top (Fig. 1b) elevations. The sizes of the NPC components
given indicate the variation that we observed. These sizes vary
no more than 8% from other published data, and may be related
to shrinkage of the specimen during critical-point drying and
metal coating. The model in Fig. 1 will be referred to in the
following sections, where we present the description of
detailed architecture of the Chironomus NPC.
NPC morphology at the cytoplasmic face of the NE
Chironomus NPCs are closely packed and distributed over the
NE with about 5 million NPCs per nucleus (see Fig. 2a). When
viewed from the cytoplasmic side, each NPC comprises a
cytoplasmic coaxial ring about 110 nm in diameter with eight
subunits distributed around the ring (Fig. 2b). Fragments of
cytoplasmic filaments (approx. 10 nm in diameter) are attached
to the upper part of the cytoplasmic ring subunits (Fig. 2b) and,
occasionally, polyribosomes have engaged RNPs in the process
of exiting from the NPC (see Fig. 2b). The cytoplasmic ring
Fig. 1. A diagram of the Chironomus NPC, showing average sizes of
the different NPC components presented with values measured from
FEISEM samples. (a) Side elevation. (b) Top elevation, representing
cross sections at two levels through the NPC, parallel with the plane
of the NE. The left half of b is taken at the level of the cytoplasmic
ring while the right is taken at the level of the inner and outer spoke
rings. CF, cytoplasmic filaments; GA, ‘gate’; RS, cytoplasmic ring
subunit; CR, cytoplasmic ring; IF, internal filaments; TG, transporter
globule; TC, transporter cylinder; ONM, outer nuclear membrane;
RA, radial arms; INM, inner nuclear membrane; S-spoke complex;
Vo and Vi, outer and inner vertical spoke domains, respectively; IS,
inner spoke ring; CS, central spoke domain; CC; central channel; LR,
lumenal ring; B, basket; BF, basket filaments; DR, distal basket ring.
226 E. Kiseleva and others
Fig. 2. NPC morphology at the cytoplasmic side of the NE. (a) Close packing generates hexagonal or orthogonal arrangements of the NPCs in
the NE, which may show a slight mechanical disruption during isolation. (b) Details of the NPC at high magnification. Short polysome (P), the
fragments of cytoplasmic filaments (CF) inserted at the surface of the ring subunits and a transporter globule (TG) are indicated. (c-i) Structural
organization of individual NPCs. The upper region of each of the eight triangular ring subunits consist of two halves (small arrows in c), and
attaches to the cytoplasmic ring (CR) (d,e) like a bead on a string. The cytoplasmic ring partly freed from the subunits (f) is seen after trypsin
treatment of the nucleus. (j,k) Raised (arrows) and flat (crossed arrows) configurations of the ring subunits on the cytoplasmic ring are shown,
which are also illustrated in l
1
and l
2
, respectively. Inner spoke ring, inner (in g) and outer (in h and i) vertical spoke domains as well as the
peripheral NPC channels (asterisks in g and h) are also visible. Abbreviations as in Fig. 1. Scale bars, 0.1 µm (same magnification in d-i).
227Active nuclear pore complex structure
subunits are triangular (about 20 nm across), divided into two
halves in the top region (Fig. 2c), and appear to sit on a
cytoplasmic coaxial ring like beads on a thickened string (Fig.
2d-f). Fig. 2f demonstrates the thin cytoplasmic ring partly
freed from the subunits after trypsin treatment.
The cytoplasmic ring subunits often have depressions of
approx. 5 nm diameter at their upper surface (Fig. 3a,b),
sometimes filled by small granules (Fig. 3b). They may
represent attachment sites for the cytoplasmic filaments. The
cytoplasmic coaxial ring and possibly the ring subunits are
attached to the underlying spoke-ring assembly by the inner
and outer vertical spoke domains (see Fig. 2g,h,i, respectively,
and the model in Fig. 1). Often neighbouring NPCs show a
variation in the angle at which the subunits sit on the
cytoplasmic ring, being either flattened or raised (Fig. 2j,k,
crossed and uncrossed arrows, respectively, and Fig. 2l). These
variations may depend on the functional transport state of the
NPC.
Visualization of the transporter subunits
The centres of many NPCs visualized from the cytoplasmic
surface are regularly occupied by globules of about 37 nm
diameter with a frequency of about 45% (Figs 2a-c; 4a-d).
This represents the cytoplasmic or upper part of the NPC
transporter (see models in Figs 1 and 8), and according to our
observations may be responsible for gating the central
channel. In resting NPCs these structures do not reveal a
central channel, however, but one is formed during RNP
translocation. Similar globules were observed on the
nucleoplasmic side of the NPC after removing the baskets
(Fig. 4i,j). In some instances the transporter globular
assemblies become detached during the isolation procedure,
revealing two different NPC spoke-ring morphologies. A hole
of about 35 nm diameter is seen at the NPC centre when the
transporter is completely removed (Fig. 4a, arrowhead).
When only the upper part of the transporter (globular) is
removed during preparation, an inner spoke ring with the
upper part of a short transporter cylinder is revealed (Fig. 4f-
h). The outer diameter of this cylinder is approx. 30 nm and
the inner diameter varies between approx. 10 nm (Fig. 4f-g)
and 26 nm (Fig. 4h). These data suggest that the Chironomus
NPC transporter is composed of four parts: a central short
cylinder (which resides within the inner spoke ring
completely and appears to consist of two halves, see Fig. 5f,g)
and two peripheral globular assemblies, located at the level
of the cytoplasmic and nucleoplasmic coaxial rings and
anchored to them by the internal filaments (see next section).
The deduced size and structure of the Chironomus NPC
transporter is in agreement with the Xenopus counterpart
visualized in 3-D maps of frozen-hydrated specimens (Akey
and Radermacher, 1993) and in FEISEM observations
(Goldberg and Allen, 1996).
When the cytoplasmic face of the NE is attached to a poly-
L-lysine-coated chip (SEM specimen support) and
subsequently rolled, the cytoplasmic coaxial rings, together
with their 37 nm transporter globules, remain attached to the
surface of chip (see Fig. 4k). This suggests that the upper part
of the transporter is an individual subunit and could be
dislodged from the spoke assembly and other parts of the
transporter during specimen preparation. The hypothesis that
the transporter globules represent endogenous substrates is
doubtful because the diameter of these globules is too large to
arise from large mRNPs (approx. 26 nm) or ribosomes (approx.
20 nm) caught in transit, nor do they have the appropriate
morphology (see also sections below).
Internal ring filaments link the NPC transporter
globules to the coaxial rings
One of the most interesting observations is that the transporter
globules are linked to the cytoplasmic ring subunits and
nucleoplasmic coaxial ring by internal ring filaments (see Figs
2c,i and 4c-e), which were identified in 27% of NPCs. Similar
internal ring filaments were also observed in Xenopus NPCs
within the cytoplasmic and nucleoplasmic coaxial rings
(Goldberg and Allen, 1996). The presence of these internal ring
filaments and their conservation between vertebrates and
invertebrates strongly suggests that the central globular
transporter particles represent an intrinsic part of the NPC (see
model in Fig. 1). This tethering between internal ring filaments
and transporter may be necessary as the transporter globule is
present in a number of differing vertical alignments in FEISEM
specimens (see Figs 4b,c, 7m,n), suggesting that it can move
vertically over a distance of perhaps 5 nm, also seen previously
in AFM images (Perez-Terzic et al., 1996) and in a 3-D
reconstruction of the Xenopus NPC (Akey and Radermacher,
1993).
Fig. 3. NPC structure at the
cytoplasmic side of the NE.
(a,b) Depressions (D) and (c)
small granules (G) are observed
on the top of the cytoplasmic
ring subunits. Scale bar, 0.1 µm
(in all images).
228
The spoke assembly
The spoke assembly was exposed (see Figs 5a-i, 7k-o) by
rolling the nucleus over a chip surface before fixation (see
Materials and methods). This procedure removed the upper
part of the NPC, including the coaxial ring with the transporter
globule, which adheres to the surface of the chip (see Fig. 4k),
and showed that the inner spoke ring is composed of eight
subunits of about 15 nm diameter (Fig. 5a,b; small arrows).
The outer and inner diameters of the inner spoke ring in such
samples are similar in size to the ring seen at the cytoplasmic
face of the intact NPC from a normally prepared nucleus (Figs
2g, 4b, 6b ) and are approx. 65 nm and 37 nm, respectively.
The inner and outer spoke rings (Figs 5c-i, 7k-o) were
observed from both the cytoplasmic and nucleoplasmic sides
of the NPC after removing the basket (Fig. 4i,j). Eight 10-12
nm particles localized at the distal ends of the radial arms of
the outer spoke ring have been observed using FEISEM (Fig.
5d,e) and also in the TEM in thin sections of intact
Chironomus nuclei (see Fig. 5j,k). In thin sections, the nuclear
membrane appears to contact the side of the NPC at a radius
that corresponds roughly to the outer edge of the inner spoke
ring, as seen previously in sections of isolated nuclear
membrane from Xenopus oocytes (Jarnik and Aebi, 1991).
Fig. 5e shows a side view, indicating that the outer spoke ring
has a thickness of about 10 nm and is localized about 15 nm
below the top of the inner spoke ring.
Two equivalent and symmetrical but opposite-facing halves
of the inner spoke ring are visible in Fig. 5f,g. It has been
E. Kiseleva and others
Fig. 4. The morphology of the
NPC transporter at the
cytoplasmic (a-h) and
nucleoplasmic (i and j) sides of
the nuclear envelope and after
isolation from the NPC (k) by
nuclear rolling. (a) NPCs with
central localised transporter
globules (TG) and those which
may have lost these globule
(arrowhead) during preparation.
(b,c) Different positions of the
cytoplasmic transporter globule
relative to the cytoplasmic ring
are shown. (d,e) The internal
ring filaments link the NPC
transporter globule to the
cytoplasmic ring.
(f-h) Transporter central
cylinders with small (f,g) and
large (h) inner diameters are
seen at the NPC center after
removal of the transporter
globules. (i,j) Nucleoplasmic
transporter globule, lower half of
inner spoke rings and lumenal
spoke ring are observed at the
nucleoplasmic side of the NPC
after removing the basket.
(k) Cytoplasmic rings with
transporter globules adhere to
the surface of the chip after
nuclear rolling. Abbreviations as
in Fig. 1. Scale bar, 0.1 µm (b-h,
same magnification; also i and
j).
229Active nuclear pore complex structure
established that the inner and outer ‘lumenal’ spoke rings are
connected to each other by additional spoke domains, which
include an outer vertical support that is clearly visible in Fig.
5h (see also Akey and Radermacher, 1993). Finally, the spoke
assembly is flexible and is observed either in ‘upright’ or in
more radially flattened configurations (compare NPC pairs in
Fig. 5h,i). We suggest that the spokes may undergo a radial
compaction to generate the ‘upright’ configuration or relax this
radial expansion to give the more flattened appearance, along
the lines detailed by Akey (1995) for vertebrate spoke-ring
assemblies.
Interaction of BR mRNP with the NPC during export
to the cytoplasm
Chironomus salivary gland cells produce a set of large
secretory proteins, which are coded by BR genes on
Fig. 5. The NPC core after rolling the nucleus over
the surface of the chip revealed by FEISEM (a-i),
and also by TEM (j and k). (a,b) Subunits (small
arrows) of the inner spoke ring are visible.
(c,d) Structure of the lumenal spoke rings with radial
arms. (e) Side view of the NPC core. (f,g) The
double organization of the inner and lumenal spoke
rings is shown; the upper halves of rings are marked
by arrows, lower halves by crossed arrows.
(h,i) Outer vertical spoke domains and two spoke
configurations (flat, double arrows and raised,
arrowheads) of the NPC core are indicated. (j,k) The
inner spoke ring and radial arms are observed in thin
sections of salivary gland cells. Abbreviations as in
Fig. 1. Scale bars, 0.1 µm (b-k, same magnification).
230
chromosome IV (Wieslander, 1994). To increase the functional
activity of the cells we used pilocarpin stimulation, which
increases the basal BR gene transcription activity by about
fivefold (Mahr et al., 1980). As shown previously, rapidly
isolated nuclei from such cells reveal numerous BR particles
approx. 50 nm in diameter, which first attach to the NPC
baskets and then are translocated through the terminal basket
ring (Fig. 7), which expands concomitantly with RNP
translocation (Kiseleva et al., 1996). Our present observations
show that the basket ring may rotate during BR RNP
translocation, as basket filaments that form this ring are often
diagonally distributed around the ring (Fig. 7i,j). This suggests
that the basket may be directly involved in mechanical
regulation of mRNP translocation.
In this paper, we have documented the next two phases of
the BR mRNP translocation cycle through the NPC core and
the cytoplasmic part of the NPC. At the cytoplasmic surface of
the NPC core, visible after nuclear rolling (see Materials and
methods), numerous 25-27 nm fibres emerge from the centre
of the NPC in the transporter cylinder region (see Fig. 7k-o,
arrows), while the diameter of the distal end of the mRNP
tapers down to about 8-10 nm (Fig. 7n, lower arrow). Constant
BR mRNP diameter over a vertical distance spanning the
spoke-ring complex can be observed in TEM thin sections, as
shown in Fig. 7d (also see Stevens and Swift, 1966; Mehlin et
al., 1992, 1995).
At the cytoplasmic side of an intact NPC the mRNP fibres
appear to be translocated through the transporter globule,
which forms during this process a transiently open ring approx.
35 nm in outer diameter at the base of the 26 nm mRNPs (see
Figs 6a-e; 7r,s). Occasionally, the upper transporter globule
displays an empty channel of approx. 26 nm in diameter (Fig.
7t), which may reflect fixation of the open channel shortly after
the release of the mRNP fibre at the end of translocation.
Subsequent phases in BR mRNP translocation through the
NPC are presented in Fig. 7 and as a model in Fig. 8, which
includes eight consecutive stages and demonstrates the NPC
transporter reorganization during mRNP export. Our
observations, together with previous analyses of Xenopus
oocytes NPC (Akey and Goldfarb, 1989; Akey, 1990; Akey and
Radermacher, 1993), strongly suggest that the BR mRNP fiber
is translocated through a central channel within the transporter,
which has a vertical dimension of approx. 60 nm (Akey and
Radermacher, 1993) with transiently open discrete gates in the
globular assembly.
To separate the mRNP export process into distinct steps we
have quantified the frequency of the various stages of mRNP
docking and translocation from the nucleoplasmic side of the
NPC basket, and the emergence of the mRNP from the NPC
cytoplasmic side, which are shown in Table 1. In FEISEM
samples (Fig. 7f-j), NPCs with closed, open and empty or
E. Kiseleva and others
Fig. 6. Translocating Balbiani ring mRNP fibres
visualized emerging from the transporter globules at
the cytoplasmic side of the nuclear envelope. mRNP
fibers are marked by unlabelled arrows, inner spoke
rings and transporter globules are marked by arrows
and labelled IS and TG, respectively. Scale bar,
0.1 µm (in all images).
Table 1. Frequency of different stages of mRNP transport
through the nuclear envelope (after pilocarpin stimulation
of salivary gland secretion)
NPC morphology Number % of total
SEM (rapid isolation and fixation of the nuclei)
Cytoplasmic side of the NE
NPCs without translocating mRNPs 1120 90.8
NPCs with translocating mRNPs 114 9.2
Nucleoplasmic side of the NE
Closed baskets 179 14.9
Baskets with docking or translocating mRNPs 450 37.4
Baskets without mRNP, but with persistent 576 47.7
basket ring structures
TEM (salivary gland cell sections)
NPCs without mRNP 2209 56
mRNPs in contact with the NPC basket 1415 36
NPCs with rod-like mRNPs translocating 315 8
through central channel of NPC
231Active nuclear pore complex structure
Fig. 7. A complete cycle of Balbiani ring mRNP translocation through the NPC as visualized in TEM (a-e) and FEISEM (f-t) samples. Each
vertical column of figures from left to right indicates a successive stage in transport; each horizontal row represents from top to bottom:
(a-e) TEM; (f,g) nucleoplasmic (N) face of the NE; 2× cytoplasmic (C) face of the NE. (a-e) The export of the Balbiani ring mRNP as visualized
in classical thin section studies. Initially, the mRNP binds to the distal portion of the basket (lower arrow in b) and then in a sequential manner is
translocated through the central NPC-channel in a linear form as it is unrolled on the nuclear side (see arrows in c-e). The surface images (f-t)
reveal this process in greater detail at the nuclear surface. (f) The nucleoplasmic entrance to the nuclear pore initially is closed by basket filaments
to which the mRNP particle docks (g) and initiates the distal basket ring formation. In subsequent steps the Balbiani ring mRNP is unrolled as it
physically moves through the center of the enlarged basket ring (h,i) and finally disappears (i,j). Balbiani ring mRNP fibres have been visualized
translocating through the NPC core (k-o). The inner diameter of the transporter cylinder expands from 10 nm (k) to 26 nm (l-o) during this
process. The cytoplasmic transporter globule is initially closed (p,q) but forms (r) a transiently open 35 nm ring at the base of mRNP during the
translocation. A longer portion of the mRNP is visible at later stage (s). Finally, the mRNP loss during isolation or the exit of the mRNP from the
central channel, the transporter globule is visualized (t) with a central 26 nm pore. mRNPs are marked by unlabelled arrows; other NPC
components are labeled respectively, according to the legend to Fig. 1. Scale bars, 0.1 µm (b-e, same magnification; also f-t).
232
regressing basket ring structures were observed in 62.6% of
the NPCs; docked or actively translocating mRNPs were
observed at a frequency of 37.4%. At the same time only
9.2% of the NPCs were observed with mRNPs emerging from
the cytoplasmic side. These results lead us to propose that the
transient gates formed by the cytoplasmic and the
nucleoplasmic transporter globules probably function
asynchronously with respect to basket ring formation. In
TEM thin sections (Fig. 7a-e), NPCs with no associated
mRNPs were present with a frequency of approx. 56%; NPCs
with mRNP docked to the baskets were present in 36% of
cases and the elongated rod phase of mRNP translocation
represented only 8% of the total NPCs. The latter value is
comparable to the observed frequency in FEISEM surface
images of the cytoplasmic side of the NPCs (9.2%). These
results of our quantitative analysis suggest that mRNP
translocation through the NPC core and cytoplasmic region
is rapid in comparison with mRNP docking and interaction
with the basket.
DISCUSSION
We have confirmed that overall architecture of the NPC is
conserved between invertebrates and vertebrates. We have,
however, found some new structural features: (1) depressions
in the cytoplasmic ring subunits, which may be anchor points
for cytoplasmic filaments; (2) the cytoplasmic ring consists of
a thin ring with the subunits attached like beads on a string; (3)
the transporter comprises four distinct subunits, formed by two
central cylinders and two globular assemblies. We have also
shown conformational variations that appear to relate to
transport activity: (1) expansion and contraction of the central
cylinder of the transporter; (2) opening and closing of the
central channel in the cytoplasmic and possibly nucleoplasmic
globular assemblies of the transporter; (3) different angular
positions of subunits on the cytoplasmic thin ring with respect
to the NPC centre; (4) upright and flattened spoke complex
conformations. FEISEM images of BR mRNP translocation
complexes in transit as well as the transport-related
configurations of the transporter were analysed and a model of
transporter reorganization during mRNP export is proposed
(Fig. 8). This model may be applicable to the export of other
mRNPs and, in a modified form, to the nuclear import of viral
nucleic acid complexes (e.g. Whittaker et al., 1996; Greber and
Kasamatsu, 1996) and the pathogenic Agrobacterial Ti plasmid
(Citovsky et al., 1992).
Architecture of the Chironomus NPC
FEISEM observations of Chironomus NPCs that are actively
transporting the BR mRNPs have been combined with our data
from TEM thin sections of Chironomus nuclei in vivo to build
a 3-D model of the Chironomus NPC (see Fig. 1). These results
verified C8 point group symmetry of the Chironomus NPC, and
together with previous TEM reconstructions of the NPC in
vertebrates (Akey, 1989, 1990, 1995; Unwin and Milligan,
1982; Hinshaw et al., 1992; Akey and Radermacher, 1993),
showed that the spoke assembly has two unique twofold axes
that relate to the cytoplasmic and nuclear halves of the spokes
within the plane of the NE. This is particularly apparent for the
inner and outer spoke rings and the transporter globular
assemblies, which were observed in SEM samples from both
cytoplasmic and nucleoplasmic sides of the detergent-treated
NE.
Our observations reveal possible cytoplasmic filament
attachment sites as depressions on the surface of the
cytoplasmic ring subunits, which demonstrates variability in
their positions on the thin ring, allowing the filaments to
approach the transporter entrance. In contrast to amphibian
oocytes (Goldberg and Allen, 1992), no evidence for the NE
lattice was found in rapidly fixed Chironomus NEs (see also
Ris, 1997).
Spoke-ring complex
We have directly observed 10 nm spherical particles at the tip
of each of eight radial arms of the outer spoke ring by FEISEM
and TEM in vivo, which were previously described by
computer reconstruction of rapidly frozen NPCs (Akey, 1989;
Akey and Radermacher, 1993) and in thin sections of Xenopus
oocyte isolated NE (Jarnik and Aebi, 1991). The function of
this conserved lumenal spoke domain is not known, but it may
play a role in anchoring the NPC to the nuclear membrane, and
participate in the initial phase of NPC assembly (Goldberg et
al., 1997b).
As well as positional variability in the cytoplasmic NPC
components, we have also observed variations in spoke
complexes conformations, which are either flattened or upright.
A similar but more limited spoke plasticity has been also
documented in frozen-hydrated membrane-associated NPCs in
Xenopus and Necturus (Akey, 1995). It was suggested that
spoke flexibility might mediate lumenal signals which regulate
transport (Greber and Gerace, 1992, 1995; Perez-Terzic et al.,
1996).
The NPC transporter is a conserved central channel
with transiently open discrete gates
Nucleocytoplasmic transport occurs through a central NPC
channel within the transporter (Akey, 1989, 1990; Akey and
Goldfarb, 1989) and has been visualized by TEM of
translocating large RNPs and nucleoplasmin-gold conjugates
(Stevens and Swift, 1966; Mehlin et al., 1992; Feldherr et al.,
1984). 3-D analysis of frozen-hydrated Xenopus NPCs
revealed the central transporter as a tripartite cylindrical
structure with occupancy of approx. 80%, which incorporates
globular cytoplasmic and nucleoplasmic assemblies joined by
a central cylinder (Akey and Radermacher, 1993).
We have directly visualised the different subunits of the
transporter in Chironomus. Our experiments demonstrated that
in invertebrates this NPC component has a four-unit structure
and makes direct contacts with the inner spoke ring and
translocating material, as in vertebrates (Akey and Goldfarb,
1989; Akey and Radermacher; 1993; Goldberg and Allen,
1996). The analysis of detergent-treated NPCs showed that the
transporter is composed of two globular 40 nm assemblies
separated by two smaller cylinders with variable internal
diameters (approx. 10 or 26 nm). The globular assemblies are
tethered to the cytoplasmic and nucleoplasmic coaxial rings by
eight internal ring filaments (also see Goldberg and Allen,
1996), and can be separated from the central transporter
cylinder and other NPC components together with the coaxial
rings during manual fracturing by rolling the nucleus. This
correlates with the previous TEM analysis of Xenopus oocyte
E. Kiseleva and others
233Active nuclear pore complex structure
NPCs after similar treatment (Unwin and Milligan, 1982;
Stewart et al., 1990), showing structural flexibility of the NPC
transporter. In inactive NPCs the transporter globules do not
reveal a central channel. However, as discussed in the next
section they form an approx. 26 nm channel during mRNP
translocation.
Fig. 8. A schematic model of active
transport of the Balbiani ring mRNP
through the NPC transporter. Two 3-
dimensional (Ia, closed and Ib, open)
and eight sequential cross sectional
(IIa-h) views of the NPC before and
during mRNP translocation are
presented. In Ia and Ib the vertical
dimensions of the NPC are
exaggerated compared to II, to
emphasise the conformational change
of the transporter subunits
(cytoplasmic and nucleoplasmic
globules and two central cylinders)
during transport; the transporter and
spoke-ring assembly (only partially
shown in the diagram) reveal twofold
symmetry about the plane of the
nuclear envelope. (IIa) The central
channel within the resting NPC is
closed to active transport by the
cytoplasmic and nucleoplasmic
transporter globules. (IIb) The mRNP
docks to the distal end of the basket,
initiates basket ring formation and
starts to translocate through the
enlarged distal ring. (IIc-g) The
mRNP enters deep into the basket and
subsequently translocates through the
transiently opened channel in the
nucleoplasmic transporter globule,
then through the enlarged channel in
the double transporter cylinder and
finally through the transiently opened
channel in the cytoplasmic transporter
globule.The transporter globules,
which are connected to the coaxial
rings by internal filaments, may
appear to move up and down (by
approx. 5 nm) together with the
translocating mRNP. (IIh) The central
channel within the NPC is again
closed by the cytoplasmic and
nucleoplasmic transporter globules
after the mRNP has emerged into the
cytoplasm. N, nucleus; C, cytoplasm;
other abbreviations as in Fig. 1.
234
A model for the translocation of mRNPs through the
central NPC channel
Currently, there are no explicit models for the translocation
step in nuclear import and export. However, nucleoplasmin-
gold (Feldherr et al., 1984; Rutherford et al., 1997) and the BR
mRNP (Stevens and Swift, 1966; Mehlin et al., 1992, 1995;
Daneholt, 1997) both form linear ‘queues’ as they cross the NE
through the centre of the NPC. In both cases, linear substrates
appear to be radially constrained within a vertical channel, for
a distance of approx. 20-30 nm above and below the central
plane of the spokes (also see Pante and Aebi, 1996). Special
recognition factors (Izaurralde and Mattaj, 1995; reviewed in
Fischer et al., 1996), members of the importin/karyopherin
protein family (Rout et al., 1997; Formerod et al., 1997; Kutay
et al., 1997; Seedorf and Silver, 1997) as well as some RNA-
binding proteins (Pinol-Roma and Dreyfuss, 1992; Daneholt,
1997) are involved in targeting mRNPs to the NPC and
subsequent transit through the NPC central channel.
Following up on earlier work (Stevens and Swift, 1966;
Mehlin et al., 1992, 1995; Kiseleva et al., 1996), we have used
the Chironomus salivary gland cell system to study the export
mechanism of BR mRNPs, which are transported across the
NPC into the cytoplasm, where they are translated to form a
family of large secretory proteins. Our previous and present
TEM observations (Fig. 7a-e) show that the BR mRNP first
docks to the nuclear surface of the NPC, and then proceeds to
unwind and translocate across the center of the NPC. Based on
3-D tomography (Mehlin et al., 1992, 1995), it was shown that
docking occurs with the 5′ end of the transcript leading the way
as the particle is fed through the center of the NPC with bound
cap binding complex (Visa et al., 1996). However, the nuclear
basket and the NPC-channel itself were not visualized in these
3-D TEM studies.
We have combined TEM and SEM observations of mRNP
translocation into a hypothetical sequence, as presented in Fig.
7. As shown previously, the distal ring of the NPC-basket may
initially be closed, and the BR mRNP first docks to this region
of the basket. In subsequent steps, the basket ring may enlarge
as the mRNP engages the central channel and is pulled
inwards, to complete transport leaving an empty basket
(Kiseleva et al., 1996). In this work we have visualized mRNP
translocation at the transporter central cylinder and then at the
cytoplasmic surface of the NPC, providing additional evidence
that the central transporter forms the export channel. The
cytoplasmic and nucleoplasmic globules of the transporter are
capped or closed in the resting state and tethered to the
cytoplasmic coaxial ring by the internal ring filaments. As the
mRNP is translocated through the center of the transporter
cylinder, which expands to 26 nm during this process, the
cytoplasmic ‘gate’ in the transporter globule opens, allowing
the mRNP to emerge. At a later stage a longer portion of the
exporting mRNP is visible and finally a central approx. 26 nm
channel is revealed within the cytoplasmic part of the
transporter at the end of translocation and prior to closing (Fig.
7t).
A comprehensive model for the entire process of mRNP
export is shown in Fig. 8. In this diagram, two 3-dimensional
and eight subsequent cross-sectional views of the NPC before
and during mRNP translocation are presented. In Fig. 8 IIa, the
BR mRNP is shown docked to the basket, an event that may
be mediated in part by nup153 localised in this region (Bastos
et al., 1996; Cordes et al., 1993). It was previously reported
(Visa et al., 1996b) that after docking, the 5′-CBC end of the
mRNP projects inwards towards the central transport channel.
We suggest that the distal basket ring is formed by branching
of individual basket filaments, and that the branch points may
retreat or advance, lengthening or shortening each filament and
reducing or enlarging the diameter of the distal ring (see Fig.
8Ib). The energy source for this conformational transition is
unknown, but may involve GTP hydrolysis by Ran. The
flexible basket may allow the 5′ end of the mRNP and
associated CBC/transport receptors to thread through the
nascent distal ring and engage the central transporter. The
translocation through the central channel would appear to
generate a vertical ‘inwards’ displacement of the mRNP that
may induce a further separation and enlargement of the distal
basket ring during passage of the mRNP. At a later stage the
BR mRNP is constrained within the transport channel to form
a rod with a diameter of approx. 26 nm (see Fig. 8 IIc-g) as it
is translocated through the different parts of NPC central
channel formed by transporter subunits. Our quantitative data
suggest that individual transporter subunits can be
asynchronously involved into the translocation process. It is
possible to suggest that during active translocation smaller
molecules, which are usually translocated through the specific
internal channels (Akey and Radermacher, 1993; Goldberg and
Allen, 1996), may perhaps flow past the macromolecular
transport complexes within the transporter.
Our data indicate that macromolecular transport across the
NPC is an active process that involves gating of the transporter
(see also Akey and Goldfarb, 1989). The gates are in fact
located at the nucleoplasmic and cytoplasmic ends (two
peripheral globular assemblies) of the transporter, because in
the inactive state these ends are closed rather than open, and
only occasional images show a 26 nm pore, which would
correspond to the expanded gate (see also Akey and
Radermacher, 1993). Hence, transport substrates would first
encounter a gating assembly located at the ends of the
elongated transport channel. These ideas are consistent with
the ‘macromolecular lock’ hypothesis (Akey, 1990, 1991),
which postulates that two opposite facing gates may open
asynchronously to allow substrate translocation, thereby
preventing the influx into the channel of macromolecules that
do not contain NLS/NES-receptor ‘keys’. It is currently
believed that all NPCs are capable of bidirectional transport;
however, no evidence exists for simultaneous transport in
opposing directions within a single NPC channel, although
docking can occur (see Dworetsky and Feldherr, 1988). Indeed
it would lead to head-on collisions within the central channel,
given the aggregate size of macromolecular substrates and their
co-transported receptor complexes. Therefore, it is likely that
a conformational ‘crosstalk’ mechanism exists between the
oppositely facing gates within the transporter, to prevent
substrate collisions (Akey, 1990).
What is the function of the conserved internal ring filaments
in transport? We suggest that the internal ring filaments may
function as ‘springs’ or struts, which connect the gating
globular subunits within the transporter to the cytoplasmic ring
subunits and nucleoplasmic coaxial ring; such an arrangement
would allow energy that is necessary for the active transport
utilized in the opening of the gates to be stored in these internal
filaments and coaxial rings as a conformational strain or
E. Kiseleva and others
235Active nuclear pore complex structure
distortion (see also Pante and Aebi, 1996; Rutherford et al.,
1997). Release of the energy stored in the ‘distorted’ internal
filaments and coaxial rings would allow closing of the gates,
thereby enhancing the efficency of gating. The local twofold
axis of the central transporter is displaced vertically about 5
nm, according to present data and to a 3-D map of the
vertebrate NPC (Akey and Radermacher, 1993). A similar
vertical displacement of the transporter has been reported by
AFM (Perez-Terzic et al., 1996) during the down-regulation of
diffusion. Perhaps a difference in tension between the central
transporter and the coaxial rings, which is generated by the
alternate opening and closing of oppositely facing gates, may
generate a cyclic vertical displacement of the transporter,
carrying the mNRP fibre through the NPC. An alternative
possible transport mechanism might involve a sequential
dilation and contraction of the various transporter subunits,
combined with the vertical displacement in a peristalsis-like
movement of the mRNP through the pore complex. Further
experiments are needed to investigate this possibility.
It is clear from the preceding discussion that an
understanding of the translocation phase of nucleocytoplasmic
transport will require an answer to the fundamental question:
‘Why is such a large assembly (the NPC) necessary to mediate
bidirectional transport?’. The integration of ongoing structural
analyses with biochemical studies of transport factors and the
nucleoporins may provide the answer.
The authors thank S. Rutherford for printing the photographs, G.
R. Bennion for assistance, and P. Chantry for excellent artwork. This
research was supported by the Wellcome Trust Foundation (E. K.),
the Cancer Research Campaign and HFSP (M. W. G. and T. D. A.)
and a grant from the NIH (C. W. A.).
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E. Kiseleva and others
