Cell, Vol. 69, 1133-1141,

June

26, 1992, Copyright

0 1992 by Cell Press

Architecture and Design of the Nuclear Pore Complex Jenny E. Hinshaw,’ Bridget 0. Carragher,t and Ronald A. Mllligan’ *Department of Cell Biology The Scripps Research Institute La Jolla, California 92037 tMicroscopy and Imaging Resource University of California San Diego La Jolla, California 92093

Summary A three-dimensional analysis of the nuclear pore complex reveals the underlying, highly symmetric framework of this supramolecular assembly, how it is anchored in the nuclear membrane, and how It is built from many distinct, interconnected subunits. The arrangement of the subunits within the membrane pore creates a large central channel, through which active nucleocytoplasmlc transport is known to occur, and eight smaller peripheral channels that are probable routes for passive diffusion of Ions and small molecules. Introduction The nuclear pore complex (NPC) is a supramolecular assembly that straddles the inner and outer nuclear membranes of all eukaryotes. It is ~0.13 pm in diameter, ~0.07 urn thick, and has a relative molecular mass of over 100 megadaltons (md) (Akey, 1989; Reichelt et al., 1990). The NPC has two main functions: it allows passive diffusion of ions and small molecules, and it controls active nucleocytoplasmic transport of large molecules and ribonucleoprotein particles. Active transport takes place through a large channel located in the center of the NPC, and it is generally assumed that this is also the route for diffusion (Dworetzky et al., 1988; Feldherr and Akin, 1990; Paine et al., 1975). Despite an increasing understanding of the kinetics and regulation of active nucleocytoplasmic transport (reviewed in Gerace and Burke, 1988; Silver, 1991) the NPC is poorly understood both biochemically and structurally. This is largely due to the lack of an isolation procedure. Thus, very little is known about the protein composition of the NPC, nor is it clear whether it contains structural nucleic acid, as do a number of other large assemblies. It is estimated that the proteins identified so far may account for less than 10% of the mass of the NPC (Gerace and Burke, 1988). One protein, gp210, is a transmembrane glycoprotein most of whose mass is located within the lumen of the nuclear envelope adjacent to the pores (Greber et al., 1990; Wozniak et al., 1989). In addition, several soluble proteins containing O-linked N-acetylglucosamine (GlcNAc) residues (Davis and Blobel, 1986; Finlay et al., 1987; Holt et al., 1987; Park et al., 1987; Snow et al., 1987) and a number of proteins that bind nuclear localization signals (NLSs) (Adam and Gerace, 1991;

Imamoto-Sonobe et al., 1990; Lee et al., 1991; Li and Thomas, 1989; Pandey and Parnaik, 1991; Silver et al., 1989; Yamasaki et al., 1989) have been described. Both the O-linked glycoproteins and the NLS-binding proteins seem to be required for active transport (Adam et al., 1990; Adam and Gerace, 1991; Finlay and Forbes, 1990). Early structural investigations on thin-sectioned material demonstrated that the NPC has 8-fold rotational symmetry and established the continuous nature of the inner and outer nuclear membranes at the pore border (see Franke et al., 1981; Kessel, 1973 and references therein). However, poor preservation of the NPC in embedded and sectioned material limited the amount of structural information that could be obtained. More recently, the development of a rapid isolation Procedure for amphibian oocyte nuclear envelopes, together with the use of negative staining or frozen hydrated preservation techniques and computer-aided analysis of electron images, has allowed more detailed structural analyses (Akey, 1989; Milligan, 1986; Reichelt et al., 1990; Unwin and Milligan, 1982). Investigations to date have concentrated on two-dimensional (2D) analysis of the en face view of the NPC. Some of this work suggested that the pore complex has an underlying structure conforming to 822 symmetry (Akey, 1989, 1990; Milligan, 1986; Unwin and Milligan, 1982) whereas other results argued in favor of a wholly asymmetric structure (Reichelt et al., 1990). A low resolution threedimensional (3D) analysis of a single NPC showed mass concentrated on the cytoplasmic face of the assembly but did not reveal any details of the underlying architecture (Unwin and Milligan, 1982). There is also evidence for the existence of cage-like structures attached to the nuclear face of the NPC (Aebi et al., 1990; Ris, 1989, 1991) and fibers or fibrils projecting from the cytoplasmic face or forming links between adjacent NPCs on the cytoplasmic side (Aebi et al., 1990; Richardson et al., 1988; Ris, 1969, 1991; Stewart and Whytock, 1988). Here we have used electron microscopy coupled with image analysis to calculate 2D and 3D maps of detergentreleased NPCs. The maps provide compelling evidence for the existence of an underlying highly symmetric NPC framework that is constructed according to 822 symmetry. When the nuclear membranes are positioned in the 3D map, it is clear that part of the assembly lies within the lumen of the nuclear envelope. The map shows that each of the eight spokes seen in en face views of the NPC is built from four morphological features, termed annular, column, ring, and lumenal subunits. There are two copies of each subunit in a spoke. Within a single spoke, there is an intricate network of connections between the subunits. In contrast, there are few links between spokes. Within the pore formed by the membranes, the structural elements of the NPC are arranged to form a large central channel, through which active nucleocytoplasmic transport is known to occur, and eight peripheral channels of a diameter that would allow passive exchange of small molecules.

Cell 1134

Figure

1. Components

Released

from the Nuclear

Envelope

by Detergent

(a) En face views of NPCS (lower right), rings (upper left), and intermediate structures (lower left) in the same field of view, demonstrating their characteristic morphologies and staining characteristics. (b) Image of a deep pool of stain in which six edge views (two of which are indicated by arrows) are seen together with two oblique views (arrowheads) and a number of en face views of NPCs. Scale bar = 0.5 urn.

Results Images To prepare NPCs for electron microscopy, we dissected macronucleifrom matureXenopuslaevisoocytes, washed them in a low salt buffer, and spread the nuclear envelopes on a carbon support film on an electron microscope grid. The grids were then washed to remove excess nucleoplasm, incubated in a solution containing 0.1% Triton X-100 to remove the membranes and to release components of the nuclear envelope, and negatively stained with uranyl acetate (see Experimental Procedures). The entire isolation procedure takes less than 5 min. In the microscope, large sheets of extracted envelope showing the nuclear lamina and attached NPCs were easily identified (Unwin and Milligan, 1982). These lamina-attached NPCs were generally poorly preserved, appearing as distorted circles in which &fold symmetry could be discerned only with difficulty (data not shown). Presumably the NPCs were distorted during spreading of the NPC-lamina meshwork on the grid. Careful examination of areas of the grid peripheral to the extracted envelope sheets revealed a number of interesting structures that could be divided into four distinct groups on the basis of their morphology and staining characteristics. First, there were structures resembling ro-

settes consisting of eight spokes (Figure la, lower right) (Milligan, 1988; Reichelt et al., 1990; Unwin and Milligan, 1982). The center of the rosette was sometimes occupied by a mass of variable appearance. The dark contrast of these rosettes suggests that they are quite thick. On the basis of their appearance, we identified these structures as en face views of NPCs that had been released, presumably from the envelope, by detergent. Second, there were rectangular triple-layered structures whose lengths were similar to the diameters of the NPCs (Figure 1b, arrows). These structures were very heavily contrasted by the stain, suggesting that they are thicker than the NPC. We identified these structures as edge views of NPCs related to the en face views by a 90° rotation (Akey, 1989; Unwin and Milligan, 1982). This identification was supported by occasional images of intermediate rotations (Figure 1 b, arrowheads) and by the results of tilting experiments (data not shown). Third, we identified rings whose outer diameters were a little smaller than those of the NPCs (Figure la, upper left). These rings were very weakly contrasted by stain, suggesting that they are very thin (Akey, 1989; Reichelt et al., 1990; Unwin and Milligan, 1982). Weak inward pointing features in the rings suggest that they have 8-fold symmetry and thus are related to the NPCs. Structures belonging to the fourth group were similar in diameter to NPCs (Figure la, lower left), but they had obvious distin-

Architecture 1135

Figure

and Design

2. Projection

Maps

of the Nuclear

Obtained

Pore Complex

by Averaging

Images

of Each of the Four

Structures

Identified

in Figure

1

Regions where biological material is concentrated are darker and are enclosed by contours; regions where the negative stain is concentrated are lighter. Eight-fold symmetry was enforced in (a), (c), and (d). Two-fold and mirror symmetry were imposed in (b). (a) Average of 166 en face images (n = 166) of detergent-released NPCs. The white line indicates a putative mirror plane. A, CR, and L are the stain-excluding regions discussed in the text. Arrowhead indicates a radial arm (Akey, 1969). (b) Edge view of detergent-released NPC. n = 48. (c) Ring, n = 400. Note that the upper portion of the ring map has been low-pass filtered to a resolution of -100 A to facilitate comparison with Figure 3e. (d) Intermediate structure, n = 23. Scale bar = 500 A.

guishing characteristics: their contrast was intermediate between NPCs and rings (they are thinner than NPCs, but thicker than rings) and they seem to be very disordered, lacking obvious 8-fold symmetry. We refer to members of this fourth group as intermediate structures. These four groups are the subject of the present investigation. 2D Maps To reduce noise in the images and to reveal the structural details characteristic of each group, we calculated average 2D projection maps. Within each group, images were selected, rotationally and translationally aligned, and averaged (Frank et al., 1981a; Saxton et al., 1979; Stoops et al., 1991; van Heel and Keegstra, 1981) (see Experimental Procedures). The final averages (Figure 2) of the en face NPC, the edge view, the ring, and the intermediate structure were calculated from 168, 48, 400, and 23 images, respectively. In the en face view each of the eight spokes comprising the NPC can be divided into three stain-excluding regions. There is a reproducibly bilobed region centered at a radius of ~310 A (A in Figure 2a) that joins with its neighbors to form a prominent inner annulus encircling a m420 A diameter hole. There is a larger central region at a radius of ~410 A (CR in Figure 2a) and an outer region at a radius of -525 A (L in Figure 2a). Weak radial arms (Akey, 1989, 1990; arrowhead in Figure 2a) link the outer regions of adjacent spokes circumferentially. Including the radial arms, the NPC has an overall diameter of ml330 A (1130 A excluding the arms). Maps of well-preserved en face views are very nearly mirror symmetric about lines drawn radially through the spokes, suggesting that the NPC is composed of two equal, but oppositely facing, halves (Unwin and Milligan, 1982). A comparison about the putative mirror plane in Figure 2a gives a correlation coefficient of 0.99. The observed small deviations from perfect mirror symmetry are not unexpected for a negatively stained

preparation on a single support film (Finch and Klug, 1965; Unwin and Zampighi, 1980). Analysis of the edge views of the NPC provides additional support for the proposed 2-fold relationship between the NPC halves. Rotational harmonic analysis of 79 edge views showed that the 2-fold harmonic accounted for SlVo (f 13%) of the total azimuthal power in the images. In addition, we found, using a correlation approach, that the variation in image appearance between the two halves in a single edge view was comparable with the variation found between halves from different edge views (data not shown). These findings demonstrate that except for minor deviations in the en face views caused by staining, the two sides of the detergent-released NPCs are equivalent and the assembly conforms to 822 symmetry at the resolution seen here. Image averages of the edge views (Figure 2b) also provide a reliable measure of the separation of the two rings of the NPC (-520 A). Alignment and averaging of 400 ring images gave the 2D map in Figure 2c. The rings have weak 8-fold symmetry and consist of a ~150 A-wide circle of mass centered at a radius of about ~450 A. Each of the eight subunits making up the ring seems to consist of two similar domains that are related by a local quasi 2-fold axis. We speculate that a single ring may therefore be built from 16 similar domains. Intermediate structures were very rare. An average of 23 images shows that they have 8-fold symmetry and have a strong handedness, each of the eight units having roughly an “S” shape (Figure 2d). They are reminiscent of the NPC in that each S motif has prominent stainexcluding regions at roughly the same radii as the three major stain-excluding regions of the NPC spoke. 3D Map To elucidate the relationship between the rings, the intermediate structures, and the NPCs, we carried out a full 3D

Cdl 1136

Figure

3. Sections

from the 3D Map of the Detergent-Released

NPC Calculated

by the Random

Conical

Tilt Method

Assuming

Only e-Fold Symmetry

The lower right panel is a side view of the rendered 3D map and indicates the approximate z location of each section shown in (a-e). Sections are viewed from the top of the 3D map. Biological material is concentrated in the darker areas, which are enclosed by contours. The lowest contour line is at the density level that was used to calculate the rendered surface in the lower right panel. Only one half of each section is shown, the other half being related by symmetry. (a) and (e) are sections through the rings on the bottom and top of the 3D map, respectively (z = e 270 A). We attribute differences here to unequal staining of the two sides of the NPC. Note the good correspondence between the features in (e) and the projection map of the ring in Figure 2c. (c) is the central section (z = 0 A); the dashed arc indicates the location of the membrane border of the pore (Unwin and Milligan, 1982). (b) and (d) are sections through the map midway between the rings and the central section (z = k 110 A). The unenforced P-fold relationship between the two sides of the map is most evident when these two sections are compared. The same S-shaped motif is apparent in both sections, but with opposite handedness. Note the similarity between these sections and the projection map of the intermediate structure shown in Figure 2d. A. C. L. and R refer to the annular, column, lumenal, and ring subunits, respectively, which are discussed in the text. Scale bar = 500 A.

analysis of the en face detergent-released NPCs by the random conical tilt method (Radermacher, 1988) (see Experimental Procedures). In all, we calculated five totally independent 3D maps using data from grids tilted 34.4O (number of images, n = 90) 47.3O (n = 198) 48.8O (n = 44) 49.9O (n = 84) and 51.4O (n = 70) to the incident electron beam. Comparisons of the maps revealed reproducible features in the 80 A-100 A resolution range. The features to be discussed in the following paragraphs were present in all five maps. However, the most extensive data set, consisting of 198 47O images covering the 45O (380°/8) azimuthal asymmetric unit, was used to calculate the 3D map presented here (see Experimental Procedures). A full description of all the data will be published elsewhere. We first calculated a map assuming only 8-fold symmetry and examined the two halves of the NPC for evidence of the expected P-fold relationship (Figure 3). Bearing in mind that there is unequal staining of the two sides, the correspondence between the two halves was very good. Predictably, the P-fold character was most pronounced close to the central plane of the NPC and less exact, but obviously present, further away. There was a stainexcluding ring on each side (Figures 3a and 3e). On one side (presumably the better preserved side), the ring showed details that were strikingly similar to the details of the 2D projection map of the rings described earlier (compare top half of Figure 2c and Figure 3e). We conclude that the rings are integral components of the detergent-released NPC. Comparisons of sections of the 3D map midway between the rings and the central plane provide additional evidence for a 2-fold relationship between the two halves.

These sections show very similar details but have opposite handedness (Figures 3b and 3d). Furthermore, the density distribution is remarkably similar to the details in the 2D maps of the intermediate structures (compare Figure 2d with Figures 3b and 3d). The intermediate structures must therefore be composed of some or all of the components making up one half of the NPC. The central section of the 3D map (Figure 3c) shares a number of features with the 2D projection map of the en face views: inner spoke regions linked together to form an annulus, central regions in each spoke, and spoke regions at high radius, Stain penetration between the center and outer regions in each spoke, at a radius of ~465 A, correlates closely with the known radial position of the membrane (r = ~450 A) (Unwin and Milligan, 1982). The stain-excluding region at high radius must therefore lie within the lumen of the nuclear envelope. Subunit Structure of the NPC A careful examination of the 2D and 3D maps together with the evidence for 822 symmetry leads to the conclusion that each of the eight spokes seen in the en face views is built from two copies of each of four morphologically distinct features. We refer to these as annular, column, ring, and lumenal subunits. Annular subunits make up the inner annulus at a radius of -310 A (A in Figures 2a and SC). Column subunits are vertjcally elongated regions of density at a radius of ~410 A (C in Figures 3b, 3c, and 3d), lying between the inner annulus and the membrane. They are capped at the top and bottom of the assembly by ring subunits (R in Figures 3a and 3e). In the 2D map, the column and ring subunits comprise the central stainexcluding region (CR in Figure 2a). The rings are not visu-

k$itecture

Figure

and Design

4. Cylindrical

The top the other mass of subunits of mass

of the Nuclear

Sections

through

Pore Complex

the Final, Fully Symmetrized

3D Map of the NPC

view of the rendered 3D map is shown in the lefthand panel. The dashed arcs (a, b, and c) show the paths of the cylindrical sections in three panels. The solid arc shows the position of the membrane border. Section a: at a radius of 310 A, passes through the centers of the inner annular subunits (the A region in Figure 2a). Section b, at a radius of 410 A, passes through the centers of mass of the column (the CR region in Figure 2a). These subunitsOare joined to the rings on the top and bottom of the NPC. Section c passes through the centers of the lumenal subunits at a radius of 525 A (the L region in Figure 2a).

alized between the spokes in the en face view, suggesting that they are very thin. This conclusion is consistent with their appearance in negative stain (see Figure la). In the central plane of the 3D map, the lumenal subunits, at a radius of -525 A, lie within the lumen of the nuclear envelope (L in Figures 2a and 3~). To reveal the relationships between the subunits more clearly, we imposed the full 822 symmetry on the 3D map. As described above, three observations justify the imposition of full symmetry: near mirror symmetry in the en face views, strong P-fold relationships in the edge views, and strong e-fold features in a 3D map calculated assuming only &fold symmetry. To improve the relative weighting of features in the vertical (2) direction of the map, we included data from edgeviews of the NPC (see Experimental Procedures). We first examine the disposition of the subunits with respect to the vertical axis of the NPC using cylindrical sections (Figure 4). A cylindrical section at a radius of 310 A viewed from the 8-fold axis slices through the inner annulus and shows an elongated region in each spoke that is tilted ~23~ clockwise from the vertical axis (Figure 4a). Evidence that this region is indeed composed of two annular subunits is apparent in the higher resolution 2D projection map (Figure 2a), where two peaks are clearly resolved. The angle of tilt from the 3D map and the peak separation measured from the 2D map allow us to estimate that the centers of mass of annular subunit layers from each side of the NPC are -150 A apart in the vertical direction. The connection between annular subunits in adjacent spokes is diagonal, linking the top annular subunit in one spoke to the bottom annular subunit in the adjacent spoke. A cylindrical section at a radius of 410 A (Figure 4b), shows that the column subunits are tilted, *loo anticlockwise (relative to the vertical) and are linked only by the ring subunits at the top and bottom of the assembly. A cylindrical section at higher radius (r = 525 A) cuts through the centers of the lumenal subunits (Figure 4~). They are tilted ~18~ clockwise, the same sense as the annular subunits. Thus, the three concentric circles of sub-

units near the central plane of the NPC (namely, the annular, column, and lumenal subunits) are arranged with lefthand, righthand, and lefthand twists, respectively. Connections between Subunits The NPC is stabilized by an intricate network of connections between the subunits. As described above, a a-fold axis relates the four subunits in the top half of each spoke to those in the bottom half. In thecentral plane the connections are a result of dimerization of the subunits, i.e., the column subunit from the top half interacts, head to head, with the column subunit from the bottom half, and similarly for the annular and lumenal subunits (Figure 5). By contrast, within each half of the spoke, stability must be conferred by heterologous associations. Here the column sub unit is the key structural element (tan color in Figure 5). It is located centrally and is attached to the ring (Figure 5, yellow) on one side of the NPC, to the annular subunit (Figure 5, green) at low radius, and to the lumenal subunit (Figure 5, blue) at high radius, presumablyviaatransmembrane linkage. At the resolution of this study we cannot rule out the possibility of additional connections between the annular and ring subunits or between the lumenal and ring subunits. There seem to be only three types of connection linking the eight spokes together to form the NPC. At very high radius, weakly contrasted radial arms are seen in the 2D en face projection map (see Figure 2a). These arms are not clearly resolved in the 3D map, but some very weak features in the map suggest that the arms lie within the lumen of the nuclear envelope and link adjacent lumenal subunits circumferentially. The rings form the second type of connection, linking the column subunits circumferentially at the top and at the bottom of the structure and providing stability in each half of the NPC (Figures 5b and 5~). The third type of connection (i.e., diagonal links between annular subunits in adjacent spokes) would seem to be the most important one, as the inner annulus is one of the most prominent features of the NPC. Interestingly, these connections have a dual function. Not only do they

Cell 1135

serve to hold adjacent spokes together, but, by virtue of their diagonal nature, they also hold the top and bottom halves of the NPC together (Figures 5c and 5d). Peripheral Channels When the nuclear membranes are positioned in the 3D map, eight peripheral channels are created in the NPC (Figure 5d). In the 2D and 3D maps, the contrast in this region is high, demonstrating that the putative channels can be penetrated easily by the heavy metal salts used for negative staining. The identification of these features as channels is also supported by details in published images of thin-sectioned nuclear envelopes; electron translucent regions of about the correct dimensions are clearly seen between the positively stained spokes of the NPC in these preparations (see for example Figure 3c in Dwyer and Blobel, 1976 and Figures 18b and 18c in Franke and Scheer, 1974). Our results show that the channels are bounded at low radius by the inner annulus of the NPC, at high radius by the membrane, and on the two remaining sides by the column subunits in adjacent spokes. The channels have an approximately oval cross-section with an average diameter of ~100 A. Discussion The results presented here provide compelling evidence that the NPC has an underlying highly symmetric framework that is constructed according to 822 symmetry. At the resolution of the analysis each asymmetric unit of the framework (half of a spoke) is seen to be composed of four morphologically distinct units, termed the annular, column, ring, and lumenal subunits. As the total molecular mass of the framework is 112 f 11 md (Reichelt et al., 1990) each subunit has roughly a mass of 1.75 md, the column subunit mass being rather greater than this and the ring subunit mass being substantially less. Irrespective of the exact figures, a single subunit is clearly of sufficient size to accommodate around 20 average-sized proteins and perhaps nucleicacid molecules as well. It is, however, unlikely that the framework visualized here is built from the O-linked glycoproteins that are involved in active nucleocytoplasmic transport for three reasons. First, the existence of active vectorial nucleocytoplasmic exchange demands an asymmetric machine, so the symmetric

-

Figure

5. Renderings

En face (a), oblique

of the 52PSymmetrized

NPC Map

(b), and edge view (c) of the 3D map. In (a), the

dotted arc is at the radius of the membrane border. In (d), the front half of the edge view has been cut away along a diagonal passing through two of the peripheral channels. Shaded areas indicate the cut ends of the rings and the inner annulus. The path of the nuclear membranes is indicated by a dashed line. Probable routes for passive nucleocy toplasmic exchange through one of the channels are indicated by double-headed arrows. Subunits discussed in the text are identified by colors as follows: annular subunits are green, rings are yellow, and lumenal subunits are blue. The remaining tan colored parts of the 3D map enclose the column subunits. The central plane of the 3D map is indicated by a slight ridge that divides the assembly into two symmetry-related halves. Manipulation and display of the map were done with the programs SUPRIM (Stoops et al., 1991) and SYNU (Hessler et al., 1992).

Architecture 1139

and Design

of the Nuclear

Pore Complex

assembly described here is unlikely to be capable of carrying out this function. Second, the published immunolabeling studies on thin-sectioned material show scant evidence of labeling in regions that would be occupied by the framework. Rather, antibodies to the O-linked glycoproteins seem to bind close to the 6-fold axis and some distance away from the central plane of the NPC (for example, see images in: Davis and Blobel, 1966; Finlay et al., 1967; Park et al., 1967; Snow et al., 1967). Furthermore, some antibodies bind to only one side of the NPC. These results suggest that the O-linked glycoproteins are clustered within the funnel formed by the inner annulus and the ring. Third, recognizable but nonfunctional NPCs are formed in nuclear reassembly systems that have been depleted of O-linked glycoproteins (Finlay and Forbes, 1990) suggesting that the basic architectural features are insufficient for function. We therefore favor the idea that the symmetric assembly revealed by our analysis plays an indirect role in active nucleocytoplasmic transport, serving as a framework for the organization and coordination of the transport apparatus proper. This apparatus, which is not visualized here, may include such asymmetrically located structures as particles (Unwin and Milligan, 1962) fibrils and filaments (Aebi et al., 1990; Richardson et al., 1966; Ris, 1969, 1991; Stewart and Whytock, 1966) reported to be associated with the cytoplasmic side, cages associated with the nuclear side (Aebi et al., 1990; Ris, 1969, 1991) and the central plug or transporter (Akey, 1969,199O; Akey and Goldfarb, 1969; Franke and Scheer, 1970; Unwin and Milligan, 1962) as well as recently described glycoprotein complexes (Finlay et al., 1991) and NLS-binding proteins (Adam and Gerace, 1991; Imamoto-Sonobe et al., 1990; Lee et al., 1991; Li and Thomas, 1969; Pandey and Parnaik, 1991; Silver et al., 1969; Yamasaki et al., 1969). The NPC framework does, however, have other roles that are inherent in its design. First, it maintains the integrity of the pore across the two nuclear membranes, locking the membranes between the column and lumenal subunits in the central plane where they are fused. The lumenal subunits can be thought of as anchors, keeping the structural elements within the pore associated with the membranes. Correlation of results from immunolabeling experiments (Greber et al., 1990) with the 3D map described here suggests that the lumenal subunits contain the large N-terminal domain of gp210, a transmembrane glycoprotein isolated from nuclear envelopes (Wozniak et al., 1969). The small C-terminal domain of gp210, located on the other side of the membrane, presumably interacts with a component of the column subunit forming at least a part of the association between the lumenal subunit and the rest of the assembly. Second, by virtue of the organization of the structural elements within the membrane pore, the NPC framework creates, at least in part, a spatial separation between the active and the passive transport functions. The very large central channel, which normally contains what is referred to as the plug or transporter (Akey, 1969), appears to be the exclusive route for active nucleocytoplasmic transport. Gold particles coated with molecules that are known to be actively transported always traverse the NPC close to its

central axis and never at the periphery (Dworetzky et al., 1966; Feldherr and Akin, 1990). Clearly passive diffusion of small molecules may take place through the central channel, but it seems likely that the peripheral channels are also routes for diffusion, as their apparent diameter is sufficient to allow passage of the largest molecules that diffuse across the NPC (Paine et al., 1975). Thus, our findings indicate that passive nucleocytoplasmic exchange of small molecules is unlikely to be compromised by heavy macromolecular traffic through the NPC or by potential occlusion of the central channel due to lectin or antibody binding. The picture of the NPC that emerges from these data is one of an extremely large and complex machine whose construction is based on the highly symmetric framework visualized here. Inherent in the design of the framework are structural elements that anchor it in the membrane and elements whose arrangement specifies the basic features required of the machine: two types of channel. The active function of the machine (vectorial transport) must be provided by additional structural elements such as the fibers, particles, plugs, and cages described by others, many of which probably have masses in the megadalton range and which are disposed asymmetrically on the underlying framework. Experimental

Procedures

Specimen Preparatlon Mature oocytes were surgically removed from adult Xenopus females and stored in Barth’s solution (Gurdon, 1976). Large oocytes were manually enucleated and the nucleus transferred to low salt buffer (LSB is 1 mM triethanolamine-HCI, 0.5 mM MgCb [pH 7.51) to remove adhering cytoplasm. The nuclear envelope was then manually spread on a carbon film supported on an EM grid. The grid, with the adhering envelope, was washed in LSB to remove nucleoplasm and incubated in LSE containing 0.1% Triton X-100 for l-2 min (Unwin and Milligan, 1962). The grids were then negatively stained with 2% uranyl acetate and air dried. For examination and imaging, a Philips CMlPT electron microscope was used at 100 kV with a 70 urn objective aperture. The low dose kit was used to record images 4 .O pm underfocus, at a magnification of 22,000. 2D Image Analysis Selected fields on micrographs were converted to optical density arrays with a scanning microdensitometer using spot and step sizes equivalent to 11.36 A at the specimen. Rotational harmonic analysis was carried out on a MicroVax computer using software developed at MRC Laboratory of Molecular Biology, Cambridge, England (Crowther and Amos, 1971). Correlation averaging (Frank et al., 1961a; Saxton et al., 1979; Stoops et al., 1991; van Heel and Keegstra, 1961) was done on SGI Iris computers using the SUPRIM suite of programs (J.-P. Bretaudiere and J. Schroeter, University of Texas, Dallas). The former approach has been described in detail elsewhere (Crowther and Amos, 1971; Unwin and Milligan, 1962). For correlation averaging, individual structures were circularly masked and floated into 256 x 256 arrays. The average density of the perimeter of the mask was used as the float value. Auto-correlation and cross-correlation functions were used to align each particle rotationally and translationallyto a reference image. To minimize the influence of the initial reference on the final result, at least 3 cycles of alignment were carried out. At the end of each cycle an average was calculated and then used as reference for the next cycle. Results from correspondence analysis and hierarchical ascendent classification (Frank, 1990) of en face NPCs show that detergentreleased NPCs consist of a homogeneous population, and en face images cannot be distinguished from their mirror images (data not shown). These data support the conclusion that the two sides of the

Cell 1140

NPC are related by a-fold symmetry. The resolution of the final averages was determined by both ring correlation and phase residual methods (Frank et al., 1981b; van Heel and Stoffler-Meilicke, 1985). Based on results with thetwo methods, we estimate that the resolution is ~80 A for the en face views, -150 A for the edge views, -50 A for the rings, and -140 A for the intermediate structures.

marked “advertisement” in accordance solely to indicate this fact.

30 Image Analysis The 3D analysis was carried out essentially as described in Radermacher (1988) using the SUPRIM suite of programs. In brief, low dose image pairs of the same field were recorded, a high tilt image first (in this case 47O), followed by a O” image. Areas where the NPCs were well preserved, i.e., had obvious e-fold symmetry and were wellseparated from each other, were chosen for further analysis. Densitometry was done with spot and step sizes equivalent to 11.38 A at the specimen. Processing was carried out on SGI Power series and Personal Iris computers. Cray supercomputers at the San Diego Supercomputer Center and The Scripps Research Institute were sometimes used for large jobs. The tilt angle and position of the tilt axis were determined from the foreshortening in a network of points, which could be located accurately on both O” and 47O image pairs. The calculated tilt angle differed by only ~1 o from the microscope goniometer reading. First, individual NPCs from the O” images were examined by correlation methods. Those with the best B-fold symmetry were identified and the rotations necessary to bring these selected NPCs to a common phase origin were determined. The NPCs were then identified on the 47O image and were interactively masked off, contrast normalized, and rotated so that they axis and the tilt axiswere coincident. The defocus gradient across the tilt micrographs was used to keep track of the tilt direction. Translations necessary to bring all the rotated 47O images to the same phase origin were determined by a procedure involving cosine stretching of each image followed by alignment with its corresponding O” image. Since the rotation of the 0’ image relative to a reference image was already known from the correlations described above, all the parameters relating the 47O NPCs to a common phase origin (namely, tilt angle, position of tilt axis, rotation, and translation relative to a reference) were known. The final data set consisted of 198 images of tilted NPCs. The rotational relationships were such that the 45O asymmetric unit was finely sampled. R-weighted back projections were calculated and combined to yield the 3D map. Each image was included eight times, consistent with e-fold rotational symmetry. The 3D map was corrected for flattening during staining by expanding the vertical dimension by a factor of 1.4 to fit the dimensions of the NPC seen in the edge views. The map was rendered at a density level that enclosed a structure of relative molecular mass, M, = 112,000 (Reichelt et al., 1990) and examined using the program SYNU (Hessler et al., 1992). 822 symmetry was imposed by averaging two maps: the map shown in Figure 3 and the same map rotated 180° about its putative P-fold axis. Weighting of features in the z direction was then improved by including data from the edge views of the NPC. A 3D map was calculated by summation of 360 (1 o azimuth intervals) r-weighted back projections of the average edge view (Figure 2b). This approximation resulted in a 30 map that had the gross features of the 3D map from conical tilt reconstruction (i.e., rings of density on either side of a large annulus of density in the central plane) and provided a reliable measure of the mass distribution in the z direction. The edge-view map and the 822eymmetrized map were summed with relative weights of 1:8, respectively, reflecting the number of images used to calculate each map.

Adam, S. A., and Gerace, L. (1991). Cytosolic proteins bind nuclear location signals are receptors for nuclear 837-847.

Acknowledgments We thank Dave Hessler (University of California, San Diego), John Schroeter (University of Texas, Dallas), and Mike Whittaker (Scripps Institute) for their help and Larry Gerace for helpful comments on the manuscript. This research was supported by grants from the National Institutesof Health (AR39155and GM44932). R. A. M. isa Pew Scholar in the Biomedical Sciences. J. E. H. is an American Cancer Society fellow. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

Received

March

3, 1992;

revised

with

18 USC

Section

1734

May 1, 1992.

References

Adam, S. A., Marr. R. S., andGerace, in permeabilized mammalian cells tors. J. Cell Biol. 717, 807-816.

that specifically import. Cell 66,

L. (1990). Nuclear protein import requires soluble cytoplasmic fac-

Aebi, U., Jarnik, M., Reichelt, R., and Engel, A. (1990). Structural analysis of the nuclear pore complex by conventional and scanning transmission electron microscopy. EMSA Bull. 20, 89-76. Akey, C. W. (1989). Interactions complex revealed by cryo-electron 970.

and structure of the nuclear pore microscopy. J. Cell Biol. 109. 955-

Akey, C. W. (1990). Visualization of transport-related configurations the nuclear pore transporter. Biophys. J. 58, 341-355.

of

Akey, C. W., and Goldfarb, D. S. (1989). Protein import through the nuclear pore complex is a multistep process J. Cell Biol. 709, 971982. Crowther, R. A., and Amos, L. A. (1971). Harmonic analysis of electron microscope images with rotational symmetry. J. Mol. Biol. 60, 123 130. Davis, L. I., and Blobel, G. (1988). Identification and characterization of a nuclear pore complex protein. Cell 45, 899-709. Dworetzky, S. I., Lanford, R. E., and Feldherr, C. M. (1968). The effects of variations in the number and sequence of targeting signals on nuclear uptake. J. Cell Biol. 707, 12791287. Dwyer, N., and Blobel, G. (1976). A modified procedure of a pore complex-lamina fraction from rat liver nuclei. 581-591. Feldherr, toplasmic

for the iSolation J. Cell Biol. 70,

C. M., and Akin, D. (1990). EM visualization transport processes. Electron Microsc. Rev.

Finch, J. T., and Klug, A. (1985). The structure papilloma-polyoma type. J. Mol. Biol. 73, 1-12.

of nucleocy3, 73-86.

of viruses

of the

Finlay, D. A., and Forbes, D. J. (1990). Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored. Cell 60, 17-29. Finlay, D. R.. Newmeyer, D. D., Price, T. M., and Forbes, D. J. (1987). Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J. Cell Biol. 104, 189-200. Finlay, D. R., Meier, E., Bradley, P., Horecka, J., and Forbes, D. J. (1991). A complex of nuclear pore proteins required for pore function. J. Cell Biol. 774, 189-183. Frank, J. (1990). Classification of macromolecular assemblies as ‘single particles’. Quart. Rev. Biophys. 23, 281-329. Frank, J., Shimkin, B., and Dowse, software system for electron image 343-358.

H. (19818). processing.

studied

Spider-a modular Ultramicroscopy 6.

Frank, J., Verschoor, A., and Boublik, M. (1981 b). Computer averaging of electron micrographs of 40s ribosomal subunits. Science 214, 1353-l 355. Franke. W. W., and Scheer, U. (1970). The ultrastructureof the nuclear envelope of amphibian oocytes: a reinvestigation. J. Ultrastruct. Res. 30, 268-316. Franke, nuclear

W. W., and Scheer, U. (1974). Structures envelope. Cell Nucleus 7. 219-347.

and functions

Franke, W. W., Scheer, U., Krohne, G., and Jarasch, nuclear envelope and the architecture of the nuclear Biol. 97, 39s-50s.

of the

E.-D. (1981). The periphery. J. Cell

Gerace. L., and Burke, B. (1966). Functional organization clear envelope. Annu. Rev. Cell Biol. 4, 335-374.

of the nu-

Greber, U. F., Senior, A., and Gerace, L. (1990). A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with

~;cq:itecture

and Design

a large lumenal domain 1502.

of the Nuclear

and asmall

Pore Complex

cytoplasmic

tail. EM60

J. 9,1495-

Gurdon, J. 8. (1976). Injected nuclei in frogoocytes: fate, enlargement, and chromatin dispersal. J. Embryol. Exp. Morphol. 36, 523-540. Hessler, D., Young, S. J., Carragher, 8. O., Martone, M., Hinshaw, J. E., Milligan. R. A., Masliah, E., Whittaker, M., Lamont, S., and Ellisman, M. H. (1992). SYNU: softwareforvisualization of 3dimensional biological structures. In Microscopy: The Key Research Tool, C. E. Lyman, L. D. Peachey, and R. M. Fisher, eds. (Milwaukee: EMSA), pp. 73-82. Holt, G. D., Snow, C. hf., Senior, A., Haltiwanger, R. S., Gerace, L., and Hart, G. W. (1987). Nuclear pore complex glycoproteins contain cytoplasmically disposed G-linked N-acetylglucosamine. J. Cell Biol. 104, 1157-1164. Imamoto-Sonobe, N., Matsuoka, Y., Semba, T., Okada, Y., Uchida, T., and Yoneda, Y. (1990). A protein recognized by antibodies to aspasp-asp-glu-asp shows specific binding activity to heterogeneous nuclear transport signals. J. Biol. Chem. 265, 16504-16508. Kessel, R. G. (1973). Structure and function of the nuclear envelope and related cytomembranes. Prog. Surface Membr. Sci. 6243-329. Lee, W.-C., Xue, Z., and M&l&e, T. (1991). The NSRI gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs. J. Cell Biol. 173, 1-12. Li, R., and Thomas, J. 0. (1989). interactswith nuclear localization

Identification of a human protein that signals. J. Cell Biol. 709,2623-2632.

Milligan, R. A. (1966) A structural model for the nuclear pore complex. In NucleocytoplasmicTransport, R. Peters and M. Trendelenberg, eds. (Berlin: Springer-Verlag), pp. 113-122. Paine, P. L., Moore, L. C., and Horowitz, permeability. Nature 254, 109-l 14. Pandey, S., and Parnaik, V. K. (1991). tion of nuclear location signal-binding Biochim. Biophys. Acta 7063, 81-89.

S. B. (1975). Nuclear

envelope

Identification and characterizaproteins in nuclear envelopes.

Park, M. K., D’Onofrio, M., Willingham, M. C., and Hanover, J. A. (1987). A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant. Proc. Natl. Acad. Sci. USA 84, 6462-6466. Radermacher, M. (1988). Three-dimensional reconstruction of single particles from random and nonrandom tilt series. J. Electron Microsc. Tech. 9,359-394. Reichelt, R., Holzenburg, A., Buhle, E. L, Jr., Jarnik, M., EngeLA., and Aebi, U. (1990). Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J. Cell Biol. 770, 883-884. Richardson, W. D., Mills, A. D., Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1988). Nuclear protein migration involvestwo steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52, 655-664. Ris, H. (1989). high resolution

Three-dimensional imaging of cell ultrastructure with low-voltage SEM. Inst. Phys. Conf. Ser. 98, 657-662.

Ris, H. (1991). The three-dimensional structure of the nuclear pore complex as seen by high voltage electron microscopy and high resolution low voltage scanning electron microscopy. EMSA Bull. 21.54-56. Saxton, cessing: Silver,

W. O., Pitt, T. J., and Horner, M. (1979). Digital image the Semper system. Ultramicroscopy 4, 343-354. P. A. (1991).

How proteins

enter the nucleus.

pro-

Cell 84,489-497.

Silver, P. A., Sadler, I., and Osborne, hf. A. (1969). Yeast proteinsthat recognize nuclear localization sequences. J. Cell Biol. 109, 983-989. Snow, C. M., Senior, A., and Gerace, L. (1987). Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Bid. 104,1143-1156. Stewart, M., and Whytock, S. (1988). The structure and interactions of components of nuclear envelopes from Xenopus oocyte germinal vesiclesobserved by heavy metal shadowing. J. Cell Sci. 90,409-423. Stoops, J. K., Schroeter, J. P., Bretaudiere, J.-P., Olson, N. H., Baker, T. S., and Strickland, 0. K. (1891). Structural studiesof human a,-macroglobulin: concordance between projected views obtained by

negativestain 178.

and cryoelectron

microscopy.

J. Struct.

Biol. 106,172-

Unwin, P. N. T., and Milligan, R. A. (1982). A large particle associated with the perimeter of the nuclear pore complex. J. Cell Biol. 93, 63-75. Unwin, P. N. T., and Zampighi, G. (1980). Structure between communicating cells. Nature 283, 545-549. van Heel, M., and Keegstra, W. (1981). friendly image analysis software system.

of the junction

IMAGIC: a fast, Ultramicroscopy

flexible and 7,113-130.

van Heel, M., and Stiiffler-Meilicke, M. (1965). Characteristic views of E. co/i and 6. steerotherrnophilus 30s ribosomal subunits in the electron microscope. EMBO J. 4, 2369-2395. Wozniak, R. W., Bartnik, E., and Blobel, G. (1989). Primary structure analysis of an integral membrane glycoprotein of the nuclear pore. J. Cell Biol. 108, 2083-2092. Yamasaki, L., Kanda, P., and Lanford, R. E. (1989). Identification of four nuclear transpolt signal-binding proteins that interact with diverse transport signals. Mol. Cell. Biol. 9, 3028-3036.