Active nuclear pore complexes in Chironomus: visualization ... · INTRODUCTION The Nuclear Pore Complex (NPC) is a supramolecular assembly of approx. 125 MDa (Reichelt et al., 1990)

223Journal of Cell Science 111, 223-236 (1998)Printed in Great Britain © The Company of Biologists Limited 1998JCS4459

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

1CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital National Health ServiceTrust, Manchester, M20 9BX, UK2Department of Biophysics, Boston University School of Medicine, 80 East Concord St, Boston, Massachusetts 02118-2394, USA3Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, 630090, Russia*Author for correspondence (e-mail: [email protected])

Accepted 10 November 1997: published on WWW 23 December 1997

The Nuclear Pore Complex (NPC) regulatesnucleocytoplasmic transport by providing small channelsfor passive diffusion and multiple docking surfaces thatlead to a central translocation channel for active transport.In this study we have investigated by high resolutionscanning and transmission electron microscopy thedynamics of NPC structure in salivary gland nuclei fromChironomus during Balbiani ring (BR) mRNPtranslocation, and present evidence of rearrangement ofthe transporter related to mRNP export. Analysis of theindividual NPC components verified a strong evolutionaryconservation of NPC structure between vertebrates andinvertebrates. The transporter is an integral part of theNPC and is composed of a central short double cylinderthat is retained within the inner spoke ring, and twoperipheral globular assemblies which are tethered to thecytoplasmic and nucleoplasmic coaxial rings by eightconserved internal ring filaments.

Distinct stages of BR mRNP nuclear export through the

individual NPC components were directly visualized andplaced in a linear transport sequence. The BR mRNP firstbinds to the NPC basket, which forms an expanded distalbasket ring. In this communication we present stages of BRmRNP transport through the nucleoplasmic, central andcytoplasmic transporter subunits, which change theirconformation during mRNP translocation, and theemegence of mRNP into the cytoplasm. We propose thatthe reorganization of the basket may be driven, in part, byan active translocation process at the transporter.Furthermore, the images provide dramatic evidence thatthe transporter functions as a central translocation channelwith transiently open discrete gates in its globularassemblies. A model of NPC transporter reorganizationaccompanied with mRNP translocation is discussed.

Key words: Nuclear Pore, Nuclear Envelope, mRNP transport,Transporter, Chironomus, Scanning electron microscopy

SUMMARY

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INTRODUCTION

The Nuclear Pore Complex (NPC) is a supramolecuassembly of approx. 125 MDa (Reichelt et al., 1990) in tnuclear envelope (NE), and mediates the passive and aexchange of macromolecules between the nucleus cytoplasm (reviewed in Rout and Wente, 1994; Davis, 19Melchior and Gerace, 1995; Goldberg and Allen, 1995).comprises multiple copies of approx. 100 differenucleoporins that form a scaffold for proteins involved binding and translocation of macromolecules across the Nand may directly participate in either nucleocytoplasmtransport or its regulation (for reviews, see Rout and Wen1994; Pante and Aebi, 1994, 1996).

In order to understand the role of individual NPcomponents in the mechanism of molecular translocatiodetailed structural analysis of the NPC in resting and acconformations is required, in contrast to the amphibian oocmodels, which are transcriptionally inactive. The NPC h

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eightfold rotational symmetry and consists of a multi-domaspoke complex surrounding a central transporter, which framed by cytoplasmic and nucleoplasmic coaxial rings (Ake1995; Akey and Radermacher, 1993; Hinshaw et al., 199Eight thin fibers involved in protein import (Pante and Aeb1996; Rutherford et al., 1997) project from the NPCcytoplasmic ring, while a basket structure that participates mRNP export (Kiseleva et al., 1996) extends from thnucleoplasmic ring (Ris, 1991; Jarnik and Aebi, 1991Goldberg and Allen, 1992; Akey and Radermacher, 1993Such asymmetrical NPC organization might be explained the vectorial nature of nuclear import and export, as well as differences in the processing of translocating molecules duritransport.

All actively transporting molecules appear to move througthe NPC central channel where the most intriguing ancontroversial NPC structure, the transporter, is localiseDepending on the isolation procedure and method of sampreparation, this structure demonstrates a variable appeara

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and occupancy within the spoke complex, but is well preserin frozen-hydrated specimens (Akey, 1989, 1990; Akey aRadermacher, 1993). It has been proposed that this strucmay represent the central channel complex, material caugtransit, or both (Akey, 1990; Jarnik and Aebi, 1991; Akey aRadermacher, 1993); however almost nothing is known abthe mechanism of transporter function during molecuexchange.

Nucleocytoplasmic active transport is bidirectional and cbe divided into three major phases, includintargeting/docking, translocation across the central channelsubstrate release (Richardson et al., 1988, Newmeyer Forbes, 1988; Akey and Goldfarb, 1989). The mechanismsubstrate targeting and docking in nuclear import has bextensively studied, resulting in recognition of nuclelocalization sequences (NLS) (Laskey et al., 1996) and spenuclear import receptors, including the alpha and bimportins/karyopherins (Dingwall and Laskey, 1986; Gorlicet al., 1996; Moroianu et al., 1995; Aitchison et al., 1996; Cet al., 1996). It was also shown that the active import proteins requires GTP hydrolysis, catalyzed in part Ran/TC4 GTPase (Melchior et al., 1993; Moore and Blob1993).

The nuclear export of mRNPs still remains poorly definin comparison to nuclear import. It was found that this procis also signal-mediated (Jarmolowski et al., 1994; Gorlichal., 1996), energy-dependent, can be inhibited by co-injecof wheat germ agglutinin (WGA) (Dargemont and Kuhn, 199Dargemont et al., 1995) and may utilize Ran GTPase reviews, see Izaurralde and Mattaj, 1995; Fischer et al., 19Koepp and Silver, 1996;). Several nuclear proteins, includtransportin/karyopherin β2, karyopherin β3/Pse1p andkaryopherin β4/Kap123p (Rout et al., 1997), exportin 1/Crm1p (Fornerod et al., 1997), CAS/Cse1p (Kutay et al., 1997) possibly Sxm1p (Seedorf and Silver, 1997; Pollard et 1996), some RNA-binding proteins (Michael et al., 1995; Viet al., 1996a; Daneholt, 1997) and a cap binding comp(CBC), which is co-exported to the cytoplasm with mRN(Visa et al., 1996b), could be involved in mRNP transpregulation.

The mRNP particles can be seen passing through the Ncentre in electron microscope sections (Stevens and S1966; Franke and Sheer, 1974). Considerable progress inanalysis of mRNP transport has been achieved by elecmicroscope investigations of Chironomus salivary gland cewhich actively synthesize abundant secretory proteins encoby Balbiani ring (BR) genes (for a review, see Mehlin aDaneholt, 1993; Wieslander, 1994). 75S premessenger RNsynthesised at BR genes and is packaged with proteins intnm mRNP particles (Stevens and Swift, 1966; Anderssonal., 1980; Olins et al., 1980; Daneholt et al., 1982) after splic(Kiseleva et al., 1994). Three-dimensional electron microsctomography (Skoglund et al., 1986; Mehlin and Daneho1993; Daneholt, 1997) and Field Emission In Lens ScannElectron Microscopy (FEISEM; Kiseleva et al., 1996demonstrated that the BR mRNP particle docks to the Nbasket, which forms a distal basket ring and initiates unpacking of the BR particle into a linear RNP ribbon. ThRNP ribbon remains separated from the spoke-ring compduring passage through the central NPC channel (Mehlin e1992), and the 5′ end of the BR mRNP transcript leads the w

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In this report, we present new data about the structuorganization and conformational plasticity of the NPC durinmRNP transit in Chironomus salivary gland cells by FEISEMand TEM. In most respects, Chironomus NPCs are similarXenopus oocyte NPCs, suggesting a strong evolutionaconservation of NPC architecture between invertebrates avertebrates (also see Goldberg et al., 1997a). Direvizualization of the transporter showed that it is composeda double central cylinder, framed on the cytoplasmic anuclear sides by large globular assemblies. These are attacto the thin co-axial rings at either surface by an array of eiginternal filaments (also see Goldberg and Allen, 1996) aappear to play multifunctional roles in gating (the formingopening and closing of the peripheral gates of the transporduring BR mRNP translocation. Moreover, the BR mRNP hbeen visualized exiting from the cytoplasmic side of thtransporter central cylinder (see Fig. 1) and then from the uppart (cytoplasmic globular assembly) of the transportethereby completing the translocation phase of nuclear expoAdditional details of basket conformation (diagonal orientatioof basket filaments around the distal ring) and the sites of binding of cytoplasmic filaments to the cytoplasmic NPparticles were observed. New information has been usedconstruct a 3-D model for the mRNP translocation through tNPC.

MATERIALS AND METHODS

Chironomus thummiwere cultured according to Lambert andDaneholt (1975). Salivary glands were isolated from rapidly growin6-week-old, fourth instar larvae. Pilocarpin concentration (0.1 mg/mand duration of treatment (5 hours) were as previously describ(Mahr et al., 1980).

Isolation of nucleiTwo approaches were used for nuclear isolation. (1) For preparatof nuclei without detergent treatment the isolated salivary glands wkept in ice-cold TKM buffer (100 mM KCl, 1 mM MgCl2 and 10 mMtriethanolamine-HCl, pH 7.0) for 2-3 minutes and individual nuclewere manually isolated from salivary gland cells using tungstdissection needles. (2) Cells in TKM buffer were pretreated for eith10 or 30 seconds with 2% NP-40 or Triton X-100 and nuclei isolatby pipetting. In the latter method, four or six isolated glands weplaced in ice-cold TKM buffer, supplemented with 2% NP-40 oTriton X-100, for 10-30 seconds and then transferred into 0.025% N40 or Triton X-100 in TKM. The nuclei were isolated from glands bpipetting. Nuclei isolated by both methods were quickly transferreto poly-L-lysine-treated 5 mm silicon chips in TKM buffer and fixedimmediately.

Electron microscopyFor TEM analysis of NPC structure by thin sections, isolated salivaglands were fixed in 2% glutaraldehyde and 0.2% tannic acid wTKM buffer for 2 hours at 4°C, followed by 1% osmium tetroxide inTKM buffer for 30 minutes at 4°C, dehydrated and embedded in EpThe sections were stained in aqueous saturated uranyl acefollowed by 0.4% lead citrate at a high pH, and then examined inPhilips EM 400 TEM (Philips, Eindhoven, Netherlands) at 60 kV.

For FEISEM, nuclei that had been partly opened with a glass neewere fixed as previously described (Goldberg and Allen, 1992Silicon chips (SEM specimen support) with attached nuclei we

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Fig. 1. A diagram of the Chironomus NPC, showing average sizes ofthe different NPC components presented with values measured fromFEISEM samples. (a) Side elevation. (b) Top elevation, representingcross sections at two levels through the NPC, parallel with the planeof the NE. The left half of b is taken at the level of the cytoplasmicring while the right is taken at the level of the inner and outer spokerings. CF, cytoplasmic filaments; GA, ‘gate’; RS, cytoplasmic ringsubunit; CR, cytoplasmic ring; IF, internal filaments; TG, transporterglobule; 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.

transferred to 2% (w/v) glutaraldehyde and 0.2% (w/v) tannic acidTKM for 10 minutes, washed in TKM and fixed for 10 minutes wit1% (w/v) OsO4 in TKM, washed in distilled water, transferred to 1%(w/v) uranyl acetate for 10 minutes, dehydrated through ethanol critical-point-dried via Arklone (ICI, Runcorn, Cheshire, UK) ancarbon dioxide. Specimens were then coated with 2 nm or 4 nmtantalum in an Edwards 306 cryo-pumped vacuum system witmagnetron sputter head (Edwards High Vacuum InternationCrawley, West Sussex, UK) and viewed in the top stage of a Top(ABT) ISI DS 130F field emission scanning electron microsco(Topcon Corporation, Tokyo, Japan) at 30 kV. The average smeasurements of different NPC components were done from app2000 NPCs chosen from three independent experiments. Correctfor metal coat thickness were not made, as it is uncertain what efthis had on sizes of objects; however, our previous measuremenXenopus NPCs were within 8% of measurements made by othusing different EM techniques. Therefore, our measurements presented as a rough range.

Treatments Mechanical fracture was employed to remove the cytoplasmic rinfrom unfixed nuclear pore complexes as described previously (Unand Milligan, 1982; Goldberg and Allen, 1996). Nuclei were alloweto adhere to the silicon chip and then gently rolled over the surfby pushing with a glass needle, followed by spreading and fixationthe NE. Baskets were removed from the NPCs by scraping spreadwith the tips of fine tweezers after fixation and critical-point dryinTrypsin treatment (0.5 µg/ml) of isolated nuclei before fixation wassometimes used (see Goldberg and Allen, 1993).

Statistical analysisA statistical analysis of the frequency of different stages of mRNtransport through the NPC was performed using both TEM and Ssamples. NPC structure was investigated by TEM on cross sectof nuclei that were chosen randomly from three different experimenAll the NPCs in a NE section from each nucleus (four nuclei pexperiment) were analysed. Roughly 1200 NPCs at the nucleoplasand 1200 NPCs at the cytoplasmic sides of the nuclear envelopSEM images were chosen randomly from three different experimeand analyzed (approx. 400 NPCs were analysed for each experim

RESULTS

In the present work, we have studied the architecture of Chironomus NPC and the roles of different NPC componein the transport of mRNPs through the central transporter. have used two complementary electron microscopy metho(1) high resolution scanning electron microscopy (FEISEMwhich provides good quality preservation of NPCs with higdefinition surface images (see Figs 1-7) and (2) conventioTEM of intact salivary gland cells in thin sections (see Figs7).

A model of the Chironomus NPCA statistical analysis of the size and location of varioidentifiable components within the Chironomus NPC (appro2000 NPCs were analysed in FEISEM images) was made is shown in Fig. 1, as a model of the NPC in side (Fig. 1a) atop (Fig. 1b) elevations. The sizes of the NPC componegiven indicate the variation that we observed. These sizes vno more than 8% from other published data, and may be relato shrinkage of the specimen during critical-point drying ametal coating. The model in Fig. 1 will be referred to in th

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NPC morphology at the cytoplasmic face of the NEChironomus NPCs are closely packed and distributed over tNE with about 5 million NPCs per nucleus (see Fig. 2a). Wheviewed from the cytoplasmic side, each NPC comprisescytoplasmic coaxial ring about 110 nm in diameter with eighsubunits distributed around the ring (Fig. 2b). Fragments cytoplasmic filaments (approx. 10 nm in diameter) are attachto the upper part of the cytoplasmic ring subunits (Fig. 2b) anoccasionally, polyribosomes have engaged RNPs in the procof exiting from the NPC (see Fig. 2b). The cytoplasmic rin

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 inthe NE, which may show a slight mechanical disruption during isolation. (b) Details of the NPC at high magnification. Short polysome (P), thefragments of cytoplasmic filaments (CF) inserted at the surface of the ring subunits and a transporter globule (TG) are indicated. (c-i) Structuralorganization of individual NPCs. The upper region of each of the eight triangular ring subunits consist of two halves (small arrows in c), andattaches 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 trypsintreatment 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 l1 and l2, respectively. Inner spoke ring, inner (in g) and outer (in h and i) vertical spoke domains as well as theperipheral 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).

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subunits are triangular (about 20 nm across), divided into thalves in the top region (Fig. 2c), and appear to sit oncytoplasmic coaxial ring like beads on a thickened string (F2d-f). Fig. 2f demonstrates the thin cytoplasmic ring parfreed from the subunits after trypsin treatment.

The cytoplasmic ring subunits often have depressionsapprox. 5 nm diameter at their upper surface (Fig. 3a,sometimes filled by small granules (Fig. 3b). They mrepresent attachment sites for the cytoplasmic filaments. cytoplasmic coaxial ring and possibly the ring subunits aattached to the underlying spoke-ring assembly by the inand outer vertical spoke domains (see Fig. 2g,h,i, respectivand the model in Fig. 1). Often neighbouring NPCs showvariation in the angle at which the subunits sit on tcytoplasmic ring, being either flattened or raised (Fig. 2jcrossed and uncrossed arrows, respectively, and Fig. 2l). Thvariations may depend on the functional transport state of NPC.

Visualization of the transporter subunitsThe centres of many NPCs visualized from the cytoplasmsurface are regularly occupied by globules of about 37 diameter with a frequency of about 45% (Figs 2a-c; 4a-This represents the cytoplasmic or upper part of the Ntransporter (see models in Figs 1 and 8), and according toobservations may be responsible for gating the cenchannel. In resting NPCs these structures do not reveacentral channel, however, but one is formed during RNtranslocation. Similar globules were observed on tnucleoplasmic side of the NPC after removing the bask(Fig. 4i,j). In some instances the transporter globuassemblies become detached during the isolation procedrevealing two different NPC spoke-ring morphologies. A hoof about 35 nm diameter is seen at the NPC centre whentransporter is completely removed (Fig. 4a, arrowheaWhen only the upper part of the transporter (globular) removed during preparation, an inner spoke ring with tupper part of a short transporter cylinder is revealed (Fig. h). The outer diameter of this cylinder is approx. 30 nm athe inner diameter varies between approx. 10 nm (Fig. 4fand 26 nm (Fig. 4h). These data suggest that the ChironoNPC transporter is composed of four parts: a central shcylinder (which resides within the inner spoke rin

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completely and appears to consist of two halves, see Fig. 5and two peripheral globular assemblies, located at the leof the cytoplasmic and nucleoplasmic coaxial rings ananchored to them by the internal filaments (see next sectioThe deduced size and structure of the Chironomus NPtransporter is in agreement with the Xenopus counterpvisualized in 3-D maps of frozen-hydrated specimens (Akeand Radermacher, 1993) and in FEISEM observatio(Goldberg and Allen, 1996).

When the cytoplasmic face of the NE is attached to a poL-lysine-coated chip (SEM specimen support) ansubsequently rolled, the cytoplasmic coaxial rings, togethwith their 37 nm transporter globules, remain attached to tsurface of chip (see Fig. 4k). This suggests that the upper pof the transporter is an individual subunit and could bdislodged from the spoke assembly and other parts of transporter during specimen preparation. The hypothesis tthe transporter globules represent endogenous substratedoubtful because the diameter of these globules is too largearise from large mRNPs (approx. 26 nm) or ribosomes (appr20 nm) caught in transit, nor do they have the appropriamorphology (see also sections below).

Internal ring filaments link the NPC transporterglobules to the coaxial ringsOne of the most interesting observations is that the transpoglobules are linked to the cytoplasmic ring subunits annucleoplasmic coaxial ring by internal ring filaments (see Fig2c,i and 4c-e), which were identified in 27% of NPCs. Similainternal ring filaments were also observed in Xenopus NPwithin the cytoplasmic and nucleoplasmic coaxial ring(Goldberg and Allen, 1996). The presence of these internal rfilaments and their conservation between vertebrates ainvertebrates strongly suggests that the central globutransporter particles represent an intrinsic part of the NPC (smodel in Fig. 1). This tethering between internal ring filamenand transporter may be necessary as the transporter globupresent in a number of differing vertical alignments in FEISEMspecimens (see Figs 4b,c, 7m,n), suggesting that it can mvertically over a distance of perhaps 5 nm, also seen previouin AFM images (Perez-Terzic et al., 1996) and in a 3-reconstruction of the Xenopus NPC (Akey and Radermach1993).

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Fig. 4.The morphology of theNPC transporter at thecytoplasmic (a-h) andnucleoplasmic (i and j) sides ofthe nuclear envelope and afterisolation from the NPC (k) bynuclear rolling. (a) NPCs withcentral localised transporterglobules (TG) and those whichmay have lost these globule(arrowhead) during preparation.(b,c) Different positions of thecytoplasmic transporter globulerelative to the cytoplasmic ringare shown. (d,e) The internalring filaments link the NPCtransporter globule to thecytoplasmic ring.(f-h) Transporter centralcylinders with small (f,g) andlarge (h) inner diameters areseen at the NPC center afterremoval of the transporterglobules. (i,j) Nucleoplasmictransporter globule, lower half ofinner spoke rings and lumenalspoke ring are observed at thenucleoplasmic side of the NPCafter removing the basket.(k) Cytoplasmic rings withtransporter globules adhere tothe surface of the chip afternuclear rolling. Abbreviations asin Fig. 1. Scale bar, 0.1 µm (b-h,same magnification; also i andj).

The spoke assembly The spoke assembly was exposed (see Figs 5a-i, 7k-orolling the nucleus over a chip surface before fixation (sMaterials and methods). This procedure removed the uppart of the NPC, including the coaxial ring with the transporglobule, which adheres to the surface of the chip (see Fig. and showed that the inner spoke ring is composed of esubunits of about 15 nm diameter (Fig. 5a,b; small arrowThe outer and inner diameters of the inner spoke ring in ssamples are similar in size to the ring seen at the cytoplasface of the intact NPC from a normally prepared nucleus (F2g, 4b, 6b ) and are approx. 65 nm and 37 nm, respectivThe inner and outer spoke rings (Figs 5c-i, 7k-o) weobserved from both the cytoplasmic and nucleoplasmic si

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of the NPC after removing the basket (Fig. 4i,j). Eight 10-1nm particles localized at the distal ends of the radial armsthe outer spoke ring have been observed using FEISEM (F5d,e) and also in the TEM in thin sections of intacChironomus nuclei (see Fig. 5j,k). In thin sections, the nuclemembrane appears to contact the side of the NPC at a radthat corresponds roughly to the outer edge of the inner sporing, as seen previously in sections of isolated nuclemembrane from Xenopus oocytes (Jarnik and Aebi, 1991Fig. 5e shows a side view, indicating that the outer spoke rihas a thickness of about 10 nm and is localized about 15 below the top of the inner spoke ring.

Two equivalent and symmetrical but opposite-facing halveof the inner spoke ring are visible in Fig. 5f,g. It has bee

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Fig. 5. The NPC core after rolling the nucleus overthe surface of the chip revealed by FEISEM (a-i),and also by TEM (j and k). (a,b) Subunits (smallarrows) of the inner spoke ring are visible.(c,d) Structure of the lumenal spoke rings with radialarms. (e) Side view of the NPC core. (f,g) Thedouble organization of the inner and lumenal spokerings is shown; the upper halves of rings are markedby arrows, lower halves by crossed arrows.(h,i) Outer vertical spoke domains and two spokeconfigurations (flat, double arrows and raised,arrowheads) of the NPC core are indicated. (j,k) Theinner spoke ring and radial arms are observed in thinsections of salivary gland cells. Abbreviations as inFig. 1. Scale bars, 0.1 µm (b-k, same magnification).

established that the inner and outer ‘lumenal’ spoke rings connected to each other by additional spoke domains, whinclude an outer vertical support that is clearly visible in Fi5h (see also Akey and Radermacher, 1993). Finally, the spassembly is flexible and is observed either in ‘upright’ or more radially flattened configurations (compare NPC pairsFig. 5h,i). We suggest that the spokes may undergo a racompaction to generate the ‘upright’ configuration or relax th

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Interaction of BR mRNP with the NPC during exportto the cytoplasmChironomus salivary gland cells produce a set of larsecretory proteins, which are coded by BR genes

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Fig. 6.Translocating Balbiani ring mRNP fibresvisualized emerging from the transporter globules atthe cytoplasmic side of the nuclear envelope. mRNPfibers are marked by unlabelled arrows, inner spokerings and transporter globules are marked by arrowsand labelled IS and TG, respectively. Scale bar,0.1µm (in all images).

Table 1. Frequency of different stages of mRNP transportthrough 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 90.8NPCs with translocating mRNPs 114 9.2

Nucleoplasmic side of the NEClosed baskets 179 14.9Baskets with docking or translocating mRNPs 450 37.4Baskets 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

chromosome IV (Wieslander, 1994). To increase the functioactivity of the cells we used pilocarpin stimulation, whicincreases the basal BR gene transcription activity by abfivefold (Mahr et al., 1980). As shown previously, rapidisolated nuclei from such cells reveal numerous BR particapprox. 50 nm in diameter, which first attach to the NPbaskets and then are translocated through the terminal baring (Fig. 7), which expands concomitantly with RNtranslocation (Kiseleva et al., 1996). Our present observatishow that the basket ring may rotate during BR RNtranslocation, as basket filaments that form this ring are ofdiagonally distributed around the ring (Fig. 7i,j). This suggethat the basket may be directly involved in mechanicregulation of mRNP translocation.

In this paper, we have documented the next two phasethe BR mRNP translocation cycle through the NPC core athe cytoplasmic part of the NPC. At the cytoplasmic surfacethe NPC core, visible after nuclear rolling (see Materials amethods), numerous 25-27 nm fibres emerge from the ceof the NPC in the transporter cylinder region (see Fig. 7karrows), while the diameter of the distal end of the mRNtapers down to about 8-10 nm (Fig. 7n, lower arrow). ConstBR mRNP diameter over a vertical distance spanning spoke-ring complex can be observed in TEM thin sectionsshown in Fig. 7d (also see Stevens and Swift, 1966; Mehlinal., 1992, 1995).

At the cytoplasmic side of an intact NPC the mRNP fibrappear to be translocated through the transporter globwhich forms during this process a transiently open ring appr35 nm in outer diameter at the base of the 26 nm mRNPs Figs 6a-e; 7r,s). Occasionally, the upper transporter globdisplays an empty channel of approx. 26 nm in diameter (F7t), which may reflect fixation of the open channel shortly afthe release of the mRNP fibre at the end of translocatSubsequent phases in BR mRNP translocation through

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NPC are presented in Fig. 7 and as a model in Fig. 8, whincludes eight consecutive stages and demonstrates the Ntransporter reorganization during mRNP export. Oobservations, together with previous analyses of Xenopoocytes NPC (Akey and Goldfarb, 1989; Akey, 1990; Akey aRadermacher, 1993), strongly suggest that the BR mRNP fiis translocated through a central channel within the transporwhich has a vertical dimension of approx. 60 nm (Akey anRadermacher, 1993) with transiently open discrete gates inglobular assembly.

To separate the mRNP export process into distinct stepshave quantified the frequency of the various stages of mRdocking and translocation from the nucleoplasmic side of tNPC basket, and the emergence of the mRNP from the Ncytoplasmic side, which are shown in Table 1. In FEISEsamples (Fig. 7f-j), NPCs with closed, open and empty

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. Eachvertical 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 visualizedin 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 istranslocated 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 filamentsto which the mRNP particle docks (g) and initiates the distal basket ring formation. In subsequent steps the Balbiani ring mRNP is unrolled as itphysically moves through the center of the enlarged basket ring (h,i) and finally disappears (i,j). Balbiani ring mRNP fibres have been visualizedtranslocating through the NPC core (k-o). The inner diameter of the transporter cylinder expands from 10 nm (k) to 26 nm (l-o) during thisprocess. The cytoplasmic transporter globule is initially closed (p,q) but forms (r) a transiently open 35 nm ring at the base of mRNP during thetranslocation. 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 thecentral channel, the transporter globule is visualized (t) with a central 26 nm pore. mRNPs are marked by unlabelled arrows; other NPCcomponents are labeled respectively, according to the legend to Fig. 1. Scale bars, 0.1 µm (b-e, same magnification; also f-t).

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regressing basket ring structures were observed in 62.6%the NPCs; docked or actively translocating mRNPs weobserved at a frequency of 37.4%. At the same time o9.2% of the NPCs were observed with mRNPs emerging frthe cytoplasmic side. These results lead us to propose thatransient gates formed by the cytoplasmic and tnucleoplasmic transporter globules probably functioasynchronously with respect to basket ring formation. TEM thin sections (Fig. 7a-e), NPCs with no associatmRNPs were present with a frequency of approx. 56%; NPwith mRNP docked to the baskets were present in 36%cases and the elongated rod phase of mRNP translocarepresented only 8% of the total NPCs. The latter valuecomparable to the observed frequency in FEISEM surfaimages of the cytoplasmic side of the NPCs (9.2%). Theresults of our quantitative analysis suggest that mRtranslocation through the NPC core and cytoplasmic regis rapid in comparison with mRNP docking and interactiowith the basket.

DISCUSSION

We have confirmed that overall architecture of the NPCconserved between invertebrates and vertebrates. We hhowever, found some new structural features: (1) depressin the cytoplasmic ring subunits, which may be anchor poifor cytoplasmic filaments; (2) the cytoplasmic ring consistsa thin ring with the subunits attached like beads on a string;the transporter comprises four distinct subunits, formed by tcentral cylinders and two globular assemblies. We have ashown conformational variations that appear to relate transport activity: (1) expansion and contraction of the cencylinder of the transporter; (2) opening and closing of tcentral channel in the cytoplasmic and possibly nucleoplasglobular assemblies of the transporter; (3) different angupositions of subunits on the cytoplasmic thin ring with respeto the NPC centre; (4) upright and flattened spoke compconformations. FEISEM images of BR mRNP translocaticomplexes in transit as well as the transport-relatconfigurations of the transporter were analysed and a modetransporter reorganization during mRNP export is propos(Fig. 8). This model may be applicable to the export of othmRNPs and, in a modified form, to the nuclear import of virnucleic acid complexes (e.g. Whittaker et al., 1996; Greber Kasamatsu, 1996) and the pathogenic Agrobacterial Ti plas(Citovsky et al., 1992).

Architecture of the Chironomus NPCFEISEM observations of Chironomus NPCs that are activtransporting the BR mRNPs have been combined with our dfrom TEM thin sections of Chironomus nuclei in vivo to buila 3-D model of the Chironomus NPC (see Fig. 1). These resverified C8 point group symmetry of the Chironomus NPC, atogether with previous TEM reconstructions of the NPC vertebrates (Akey, 1989, 1990, 1995; Unwin and Milliga1982; Hinshaw et al., 1992; Akey and Radermacher, 199showed that the spoke assembly has two unique twofold athat relate to the cytoplasmic and nuclear halves of the spowithin the plane of the NE. This is particularly apparent for tinner and outer spoke rings and the transporter globu

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assemblies, which were observed in SEM samples from bocytoplasmic and nucleoplasmic sides of the detergent-treatNE.

Our observations reveal possible cytoplasmic filamenattachment sites as depressions on the surface of tcytoplasmic ring subunits, which demonstrates variability intheir positions on the thin ring, allowing the filaments toapproach the transporter entrance. In contrast to amphibioocytes (Goldberg and Allen, 1992), no evidence for the Nlattice was found in rapidly fixed Chironomus NEs (see alsRis, 1997).

Spoke-ring complexWe have directly observed 10 nm spherical particles at the tof each of eight radial arms of the outer spoke ring by FEISEMand TEM in vivo, which were previously described bycomputer reconstruction of rapidly frozen NPCs (Akey, 1989Akey and Radermacher, 1993) and in thin sections of Xenopoocyte isolated NE (Jarnik and Aebi, 1991). The function othis conserved lumenal spoke domain is not known, but it maplay a role in anchoring the NPC to the nuclear membrane, aparticipate in the initial phase of NPC assembly (Goldberg eal., 1997b).

As well as positional variability in the cytoplasmic NPCcomponents, we have also observed variations in spocomplexes conformations, which are either flattened or uprighA similar but more limited spoke plasticity has been alsodocumented in frozen-hydrated membrane-associated NPCsXenopus and Necturus (Akey, 1995). It was suggested thspoke flexibility might mediate lumenal signals which regulatetransport (Greber and Gerace, 1992, 1995; Perez-Terzic et 1996).

The NPC transporter is a conserved central channelwith transiently open discrete gatesNucleocytoplasmic transport occurs through a central NPchannel within the transporter (Akey, 1989, 1990; Akey anGoldfarb, 1989) and has been visualized by TEM otranslocating large RNPs and nucleoplasmin-gold conjugat(Stevens and Swift, 1966; Mehlin et al., 1992; Feldherr et a1984). 3-D analysis of frozen-hydrated Xenopus NPCrevealed the central transporter as a tripartite cylindricastructure with occupancy of approx. 80%, which incorporateglobular cytoplasmic and nucleoplasmic assemblies joined ba central cylinder (Akey and Radermacher, 1993).

We have directly visualised the different subunits of thetransporter in Chironomus. Our experiments demonstrated thin invertebrates this NPC component has a four-unit structuand makes direct contacts with the inner spoke ring antranslocating material, as in vertebrates (Akey and Goldfar1989; Akey and Radermacher; 1993; Goldberg and Allen1996). The analysis of detergent-treated NPCs showed that transporter is composed of two globular 40 nm assemblieseparated by two smaller cylinders with variable internadiameters (approx. 10 or 26 nm). The globular assemblies atethered to the cytoplasmic and nucleoplasmic coaxial rings beight internal ring filaments (also see Goldberg and Allen1996), and can be separated from the central transporcylinder and other NPC components together with the coaxirings during manual fracturing by rolling the nucleus. Thiscorrelates with the previous TEM analysis of Xenopus oocyt

233Active nuclear pore complex structure

nextNP

Fig. 8.A schematic model of activetransport of the Balbiani ring mRNPthrough the NPC transporter. Two 3-dimensional (Ia, closed and Ib, open)and eight sequential cross sectional(IIa-h) views of the NPC before andduring mRNP translocation arepresented. In Ia and Ib the verticaldimensions of the NPC areexaggerated compared to II, toemphasise the conformational changeof the transporter subunits(cytoplasmic and nucleoplasmicglobules and two central cylinders)during transport; the transporter andspoke-ring assembly (only partiallyshown in the diagram) reveal twofoldsymmetry about the plane of thenuclear envelope. (IIa) The centralchannel within the resting NPC isclosed to active transport by thecytoplasmic and nucleoplasmictransporter globules. (IIb) The mRNPdocks to the distal end of the basket,initiates basket ring formation andstarts to translocate through theenlarged distal ring. (IIc-g) ThemRNP enters deep into the basket andsubsequently translocates through thetransiently opened channel in thenucleoplasmic transporter globule,then through the enlarged channel inthe double transporter cylinder andfinally through the transiently openedchannel in the cytoplasmic transporterglobule.The transporter globules,which are connected to the coaxialrings by internal filaments, mayappear to move up and down (byapprox. 5 nm) together with thetranslocating mRNP. (IIh) The centralchannel within the NPC is againclosed by the cytoplasmic andnucleoplasmic transporter globulesafter the mRNP has emerged into thecytoplasm. N, nucleus; C, cytoplasm;other abbreviations as in Fig. 1.

NPCs after similar treatment (Unwin and Milligan, 198Stewart et al., 1990), showing structural flexibility of the NPtransporter. In inactive NPCs the transporter globules do

2;Cnot

reveal a central channel. However, as discussed in the section they form an approx. 26 nm channel during mRtranslocation.

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A model for the translocation of mRNPs through thecentral NPC channelCurrently, there are no explicit models for the translocatistep in nuclear import and export. However, nucleoplasmgold (Feldherr et al., 1984; Rutherford et al., 1997) and the mRNP (Stevens and Swift, 1966; Mehlin et al., 1992, 199Daneholt, 1997) both form linear ‘queues’ as they cross the through the centre of the NPC. In both cases, linear substrappear to be radially constrained within a vertical channel, a distance of approx. 20-30 nm above and below the cenplane of the spokes (also see Pante and Aebi, 1996). Sprecognition factors (Izaurralde and Mattaj, 1995; reviewedFischer et al., 1996), members of the importin/karyopheprotein family (Rout et al., 1997; Formerod et al., 1997; Kutet al., 1997; Seedorf and Silver, 1997) as well as some RNbinding proteins (Pinol-Roma and Dreyfuss, 1992; Daneh1997) are involved in targeting mRNPs to the NPC asubsequent transit through the NPC central channel.

Following up on earlier work (Stevens and Swift, 196Mehlin et al., 1992, 1995; Kiseleva et al., 1996), we have uthe Chironomus salivary gland cell system to study the expmechanism of BR mRNPs, which are transported acrossNPC into the cytoplasm, where they are translated to formfamily of large secretory proteins. Our previous and presTEM observations (Fig. 7a-e) show that the BR mRNP fidocks to the nuclear surface of the NPC, and then proceedunwind and translocate across the center of the NPC. Base3-D tomography (Mehlin et al., 1992, 1995), it was shown thdocking occurs with the 5′ end of the transcript leading the waas the particle is fed through the center of the NPC with boucap binding complex (Visa et al., 1996). However, the nuclbasket and the NPC-channel itself were not visualized in th3-D TEM studies.

We have combined TEM and SEM observations of mRNtranslocation into a hypothetical sequence, as presented in7. As shown previously, the distal ring of the NPC-basket minitially be closed, and the BR mRNP first docks to this regiof the basket. In subsequent steps, the basket ring may enas the mRNP engages the central channel and is puinwards, to complete transport leaving an empty bas(Kiseleva et al., 1996). In this work we have visualized mRNtranslocation at the transporter central cylinder and then atcytoplasmic surface of the NPC, providing additional evidenthat the central transporter forms the export channel. Tcytoplasmic and nucleoplasmic globules of the transporter capped or closed in the resting state and tethered to cytoplasmic coaxial ring by the internal ring filaments. As tmRNP is translocated through the center of the transpocylinder, which expands to 26 nm during this process, cytoplasmic ‘gate’ in the transporter globule opens, allowithe mRNP to emerge. At a later stage a longer portion of exporting mRNP is visible and finally a central approx. 26 nchannel is revealed within the cytoplasmic part of ttransporter at the end of translocation and prior to closing (F7t).

A comprehensive model for the entire process of mRNexport is shown in Fig. 8. In this diagram, two 3-dimensionand eight subsequent cross-sectional views of the NPC beand during mRNP translocation are presented. In Fig. 8 IIa,BR mRNP is shown docked to the basket, an event that mbe mediated in part by nup153 localised in this region (Bas

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et al., 1996; Cordes et al., 1993). It was previously report(Visa et al., 1996b) that after docking, the 5′-CBC end of themRNP projects inwards towards the central transport channWe suggest that the distal basket ring is formed by branchof individual basket filaments, and that the branch points mretreat or advance, lengthening or shortening each filament reducing or enlarging the diameter of the distal ring (see F8Ib). The energy source for this conformational transition unknown, but may involve GTP hydrolysis by Ran. Thflexible basket may allow the 5′ end of the mRNP andassociated CBC/transport receptors to thread through nascent distal ring and engage the central transporter. Ttranslocation through the central channel would appear generate a vertical ‘inwards’ displacement of the mRNP thmay induce a further separation and enlargement of the dibasket ring during passage of the mRNP. At a later stage BR mRNP is constrained within the transport channel to fora rod with a diameter of approx. 26 nm (see Fig. 8 IIc-g) asis translocated through the different parts of NPC centrchannel formed by transporter subunits. Our quantitative dsuggest that individual transporter subunits can basynchronously involved into the translocation process. It possible to suggest that during active translocation smamolecules, which are usually translocated through the specinternal channels (Akey and Radermacher, 1993; Goldberg aAllen, 1996), may perhaps flow past the macromolecultransport complexes within the transporter.

Our data indicate that macromolecular transport across NPC is an active process that involves gating of the transpo(see also Akey and Goldfarb, 1989). The gates are in flocated at the nucleoplasmic and cytoplasmic ends (twperipheral globular assemblies) of the transporter, becausethe inactive state these ends are closed rather than open,only occasional images show a 26 nm pore, which woucorrespond to the expanded gate (see also Akey aRadermacher, 1993). Hence, transport substrates would encounter a gating assembly located at the ends of elongated transport channel. These ideas are consistent the ‘macromolecular lock’ hypothesis (Akey, 1990, 1991which postulates that two opposite facing gates may opasynchronously to allow substrate translocation, therepreventing the influx into the channel of macromolecules thdo not contain NLS/NES-receptor ‘keys’. It is currentlybelieved that all NPCs are capable of bidirectional transpohowever, no evidence exists for simultaneous transport opposing directions within a single NPC channel, althougdocking can occur (see Dworetsky and Feldherr, 1988). Indeit would lead to head-on collisions within the central channegiven the aggregate size of macromolecular substrates and tco-transported receptor complexes. Therefore, it is likely tha conformational ‘crosstalk’ mechanism exists between toppositely facing gates within the transporter, to prevesubstrate collisions (Akey, 1990).

What is the function of the conserved internal ring filamenin transport? We suggest that the internal ring filaments mfunction as ‘springs’ or struts, which connect the gatinglobular subunits within the transporter to the cytoplasmic rinsubunits and nucleoplasmic coaxial ring; such an arrangemwould allow energy that is necessary for the active transputilized in the opening of the gates to be stored in these interfilaments and coaxial rings as a conformational strain

235Active nuclear pore complex structure

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distortion (see also Pante and Aebi, 1996; Rutherford et 1997). Release of the energy stored in the ‘distorted’ interfilaments and coaxial rings would allow closing of the gatethereby enhancing the efficency of gating. The local twofoaxis of the central transporter is displaced vertically abounm, according to present data and to a 3-D map of vertebrate NPC (Akey and Radermacher, 1993). A simivertical displacement of the transporter has been reportedAFM (Perez-Terzic et al., 1996) during the down-regulation diffusion. Perhaps a difference in tension between the centransporter and the coaxial rings, which is generated by alternate opening and closing of oppositely facing gates, mgenerate a cyclic vertical displacement of the transporcarrying the mNRP fibre through the NPC. An alternatipossible transport mechanism might involve a sequendilation and contraction of the various transporter subuncombined with the vertical displacement in a peristalsis-limovement of the mRNP through the pore complex. Furthexperiments are needed to investigate this possibility.

It is clear from the preceding discussion that aunderstanding of the translocation phase of nucleocytoplastransport will require an answer to the fundamental questi‘Why is such a large assembly (the NPC) necessary to medbidirectional transport?’. The integration of ongoing structuranalyses with biochemical studies of transport factors and nucleoporins may provide the answer.

The authors thank S. Rutherford for printing the photographs,R. Bennion for assistance, and P. Chantry for excellent artwork. Tresearch was supported by the Wellcome Trust Foundation (E. the Cancer Research Campaign and HFSP (M. W. G. and T. D.and a grant from the NIH (C. W. A.).

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