J. Mol. Biol. (1990) 213, 575-582
Association of Gold-labelled Nucleoplasmin with the Centres of Ring Components of Xenopus 0ocyte Nuclear Pore Complexes M u r r a y Stewart, Sue W h y t o c k
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, England and Anthony D. Mills
Cancer Research Campaign Molecular Embryology Research Group Department of Zoology, Downing Street Cambridge CB2 3E J, England (Received 5 June 1989; accepted 12 February 1990) We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoolasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60°/o), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.
plasmin, the most abundant nuclear protein in The nuclear envelope is constructed from a double Xenopus oocytes (Mills et al., 1980) is too large to membrane perforated by nuclear pore complexes diffuse passively through the pores and frequently which mediate selective transport (for reviews, see has been used to investigate active transport into Dingwall & Laskey, 1986; Newport & Forbes, 1987). the nucleus. Colloidal gold' conjugated with nucleoA fibrous lamina lies under the nucleoplasmic face of plasmin accumulates in the nucleus over a period of the envelope, below the level of the pores (see Aebi hours after being microinjected into Xenopus et al., 1986; Gerace, 1986; Stewart & Whytock, oocytes. Sections of embedded material that was 1988) and there are also pore-connecting fibrils on fixed while nucleoplasmin-gotd was being transthe cytoplasmic face of the envelope (see Stewart & ported show many pores with colloidal gold markers Whytock, 1988, and references cited therein). located centrally on the axis of the pore cylinder Nuclear pore complexes mediate both active and (Felhderr et al., 1984; Richardson et al., 1988; passive exchange of material between nucleus and Newmeyer & Forbes, 1988). A similar distribution cytoplasm and are roughly cylindrical structures in has also been observed in isolated nuclear envelopes which eight internal spoke-like units and a central examined in vitrified water (Akey & Goldfarb, 1989) granule are arranged between cytoplasmic and where the gold label is located near the centre of the nucleoplasmic rings (see Kartenbeck et al., 1971; circular pore profile seen in the primarily en face Franke, 1974; Maul, 1977; Unwin & Milligan, 1982; Akey, 1989, and references cited therein). Nucleoviews obtained with these whole mounts. These 575 © 1990AcademicPressLimited 0022-2836/90/120575-08 $03.00/0
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1
4
i
I
I
Figure 1. Schematic drawing of pore substructure consistent with the images we obtained from disrupted pores and with a broad range of data from other sources (see Franke, 1974; Maul. 1977; Unwin & Milligan, 1982). The basic morphological components are cytoplasmic (2) and nucleoplasmic (5) rings, the cylindrical body of the pore (4), pore-connecting fibrils (3) attached at the level of the cytoplasmic ring, and 8 granules (1) located on the cytoplasmic face. This Figure concentrates on the structural details seen in shadowed preparations and, because these visualized the surface of the pores and their morphological subunits, we were not able to observe their internal structure. Images obtained by both negative staining and freeze-fracture (see, e.g. Kartenbeck et al., 1971; Franke, 1974; Maul, 1977; Franke & Scheer, 1970) give information on the internal substructure of the cylindrical body of the pore and show that it contains 8 spoke-like units and a central granule. Isolated components, such as rings, granules and the central cylinders have also been observed by negative staining (Unwin & Milligan, 1982) and in unstained vitrified preparations (Akey, 1989). In addition, 8 granules have often been seen attached to the cytoplasmic face of pore complexes (Unwin & MiUigan, 1982; Stewart & Whytock, 1988). Cytoplasmic-nuclear transport appears to take place along the central axis of the pore ( . . . . . ) and may involve the central granule (Akey & Goldfarb, 1989; Unwin & Milligan, 1982) located in the cylindrical body of the pore.
data all indicate that nucleoplasmin is translocated along the central axis of the pore complex, and suggest a major role for the central granule, which has been referred to as the "transporter" (Akey & Goldfarb, 1989). To investigate the role of morpho-
logically defined subunits of the pore complex in the transport process, we have employed shadowed whole mounts of nuclear envelopes from which membranes have been removed (Aebi et al., 1986; Stewart & Whytock, 1988). Because of their high inherent contrast, such preparations are particularly suitable for examining the morphological subunits of nuclear pores produced by partial fragmentation of nuclear envelopes (Stewart & Whytock, 1988; Whytock & Stewart, 1988) and, in conjunction with gold-labelled nucleoplasmin, they enable the role of different morphological subunits of the pore complex in transport to be investigated. Nuclear pore complexes can be divided into a number of morphological components as illustrated in Figure 1. Rings are attached to both the nucleoplasmic and cytoplasmic faces of the disc-like body of the pore and, in addition, eight roughly spherical granules are attached to the ring facing the cytoplasm. Although specimens prepared in a number of ways all show these morphological components, their detailed appearance is not always the same because different techniques emphasize different aspects of nuclear pore structure. In particular, the internal distribution of material in the body of the pore complex, which has a central granule surrounded by eight spoke-like units, dominates images of frozen hydrated (Akey, 1989) or negatively stained (see, for example, Unwin & Milligan, 1982) material. A similar pattern is seen in micrographs of freeze-fractured nuclear envelopes (see, for example, Kartenbeck et al., 1971). On the other hand, images of shadowed whole mounts of nuclear envelopes show primarily the external surface. In such images, the body of the pore complex appears as a short cylinder or disc with its axis perpendicular to the plane of the envelope (Aebi et al., 1986; Stewart & Whytock, 1988). Sections of embedded envelopes usually show cross-sections, although in some instances en face views have been obtained that show the 8-fold internal substructure of the body of the pore (for reviews, see Franke, 1974; Maul, 1977). The cytoplasmic and nucleoplasmic rings can be seen in most types of preparation and often show a weak 8-fold rotational symmetry (Unwin & Milligan, 1982; Stewart & Whytock, 1988; Whytock & Stewart, 1988; Akey, 1989). However, the rings are most easily seen in preparations in which the pore structure has been partially disrupted. Our previous studies (Stewart & Whytock, 1988; Wytock & Stewart, 1988) showed that washing with low ionic strength buffer was an effective way of producing such partial disruption of the envelope and, as illustrated in Figure 2(a), generated large areas of envelopes from which pores were being fragmented and shed. Islands of pores, often still connected by pore-connecting fibrils, were seen frequently in such preparations, together with pores in various degrees of disintegration. We often observe rings (Fig. 2(a) to (i)), some of which contain a small amount of additional material at their centre (Fig. 2(g) to (i)). The amount of material seen at the centre of these rings was much
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577
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Figure 2. Disruption of Xenopus oocyte nuclear envelopes after washing in low-salt buffer. (a} A large area of envelope shedding pores, islands and rings. The large arrow at the bottom left shows a typical island in which most of the pores are joined by pore-connecting fibrils, that were most easily seen when, as in this example, the cytoplasmic face of the pore was uppermost. There were 3 main types of isolated particle present in disrupted preparations such as this. Large numbers of isolated rings were often seen (arrowheads at the top left indicate several examples} and in this Figure can be found over most of the field of view. In some places (small arrows} rings overlap partially. Isolated pores, such as that circled at the lower left (immediately above the island} were comparatively rare. These particles closely resembled the pores in islands and in whole envelopes {shown at the top right of the Figure} and were thought to be substantially intact. More common were particles such as those arrowed at the centre left which seemed to be rather thinner than the isolated pores (as judged by the shorter shadow they cast) and which probably represented the disc-like central body of the pore complexes from which rings had been removed. (b) to (i) A gallery of rings, some of which have additional material located at their centres ((g) to (i)). In some cases (f) and (g} pore-connecting fibrils could be seen, identifying cytoplasmic rings. (j) to (n} show rare edge views of pores. The particles in these images appear to have a distinct waist consistent with rings being located on both the top and bottom of the pore complex. The bar represents 1000 nm for (a) and 200 nm for (b) to (n). Nuclear envelopes were isolated and attached to polylysine-coated grids as described {Stewart & Whytock, 1988). Specimens were washed in low-salt buffer (10 mm-Hepes, 1 mm-KCl, 2 mm-MgCl2 (pH 7"5)) at room temperature for 10 min and then in 0"5°/o {v/v) Triton X-100, 0"3 m-KC1, 10% (w/v) sucrose, 2 mM-EDTA, 1 mm-dithiothreitoi, 20 mm-Tris- HCl for l0 min at room temperature to remove membranes. Specimens were shadowed with platinum/carbon after either air or freeze-drying as described (Stewart & Whytock, 1988; Whytock & Stewart, 1988), and examined at 80 kV in Philips EM301, EM400, EM420 or CMI2 electron microscopes using standard imaging conditions. Mierographs were usually recorded at nominal magnifications near 15,000 x. Nucleoplasmic and cytoplasmic faces were identified using the criteria described by Stewart & Whytock (1988). Micrographs are printed so that heavy metal and gold are black.
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less than would be anticipated for the central "transporter" granule, which appears to be located centrally in the body of the pore complex (Unwin & Milligan, 1982). Rare edge views of pores (Fig. 2(j) to (n)) were consistent with other data (see Unwin & Milligan, 1982; Akey, 1989), which indicated that pores have rings on both cytoplasmic and nucleoplasmic faces. Cytoplasmic rings could sometimes be distinguished from nucleoplasmic ones by the presence of pore-connecting fibrils between rings (Fig. 2(f) and (g)). Because these fibrils are located near the cytoplasmic face of the pores (Stewart & Whytock, 1988), rings connected by them must have been ~ytoplasmic ones. Views of disintegrating pores (Stewart & Whytock, 1988) have shown that rings also derive from the nucleoplasmic face. We did not observe cytoplasmic rings with granules attached to them, probably because the granules appear to be rather easily removed. Previous studies (Whytoek & Stewart, 1988) showed that sometimes groups of eight granules were seen when the cytoplasmic surface of the envelope attached transiently to the support film and left a "blot". The thin fiat structure marked by the long arrow in Figure 2 probably represents the disc-like central body of the pore complex that contributes so strongly to negatively stained images (see Unwin & Milligan, 1982). However, it was not always possible to decide if these putative pore bodies retained an underlying ring, as this could have been contrasted poorly by shadowing. The areas of rings and other fragments produced by low-salt washing could have arisen in two ways: material could have been released from pore complexes and then settled onto the support film or, alternatively, the fragments could represent a "blot" produced by dislodging part of the envelope that had become attached to the support film. Although obvious blots have been observed (Unwin & Milligan, 1982; Fig. 8 of Whytock & Stewart, 1988), a number of considerations indicated that the mass shedding of pores we observed in areas such as that shown in Figure 2 probably did not arise primarily in this way. First, the material in blots was usually in close apposition to the nuclear envelope, whereas the rings, isolated pores and islands seen with mass shedding, were often found over 20~m from the envelope. Second, the rings observed in blots had a very characteristic beaded appearance, in which usually eight granules could be observed (Unwin & Milligan, 1982; Whytock & Stewart, 1988), whereas the rings in the shed areas were much smoother (see Fig. 2). Third, blotted material would be expected always to have its cytoplasmic face attached to the support film, but in shed areas we frequently observed a mixture of orientations so that some of the material was oriented cytoplasmic face uppermost (away from the support film) as can be seen, for example, with the island arrowed in Figure 2 (see Stewart & Whytock (1988) for a discussion of the criteria whereby cytoplasmic and nucleoplasmic faces can be distinguished). Fourth, fragments in blots were
usually closely packed together similar to pores in intact nuclear envelopes, whereas the spacing between rings and isolated pores in shed areas was more variable. Fifth, the degree of disruption, both of pores into rings and of rings into smaller subunits, increased with the duration and severity of the low-salt wash (Stewart & Whytock, 1988). And finally, rings were sometimes seen to overlap partially (small arrows in Fig. 2), which would not be expected to be seen if they were produced by blotting. Nucleoplasmin-gold conjugates were easily recognized in shadowed preparations of nuclear envelopes isolated from Xenopus oocytes two to four hours after microinjection. Views of nucleoplasmic faces showed the labelling clearly (Fig. 3) and enabled the extent and position of labelling to be most precisely determined. Where the lamina was suspended above the support film (left side of the micrograph), the pore complexes were partially obscured, so we concentrated on areas where the lamina was attached to the substrate (right side of the micrograph). Here the pores were quite clear and the degree of labelling could be assessed reliably. In most instances the gold label was located centrally in the pore complex, with labels seen comparatively rarely at high radius or on the cytoplasmic granules or pore-connecting fibrils. However, there was also some random labelling of the envelopes which presumably derived from material not being actively transported (a substantial fraction of the microinjeered nucleoplasmin-gold was present in both the cytoplasm and nucleus and became distributed randomly over the envelopes during isolation). The systematic location of gold-labelled nucleoplasmin near the axis of the pore complexes from microinjected oocytes was consistent with observations on sections (Feldherr et al., 1984; Newmeyer & Forbes, 1988; Richardson et al., 1988; Feldherr & Dworetzky, 1988) and on frozen-hydrated material (Akey & Goldfarb, 1989) that indicated that this material was transported through the centre of pores. When envelopes from nucleoplasmin microinjected oocytes were partially disrupted by low-salt washes, a substantial number of the rings produced had centrally located gold particles (Fig. 4). Labelled rings were usually more common than unlabelled ones in these preparations, and of 288 rings examined 238 contained gold particles. The radial distribution of particles was assessed by dividing the rings into three equally spaced concentric zones. As shown in Table 1, most labels (60%) were located centrally, with only 22~o at intermediate radii and 18~/o at the periphery. A similar distribution was seen with intact pores. This distribution was very different from that expected for random labelling, where a distribution roughly in proportion to the areas of the zones would have been expected. Thus, random labelling would have been expected to label about 11 ~o of rings centrally. When gold-conjugated nucleoplasmin was applied to envelopes after isolation (Fig. 5(a)), a clear
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Figure 3. Labelling of Xenopu8 oocyte nuclear envelopes with microinjected nucleoplasmin-gold. This Figure shows a nucleoplasmic face of an intact envelope in which the lamina was stuck onto the support film and so had low contrast. However, a small area on the left shows the more usual view (Stewart & Whytock, 1988) where the lamina has become detached and has much higher contrast. Pores were labelled primarily on their axis. Because specimens were prepared by opening out an intact envelope on the grid. the most frequently observed views were of 2 overlapping envelope layers, in which the top layer had its cytoplasmic face uppermost and the lower had its nucleoplasmic face uppermost (see Stewart & Whytock, 1988). Although pores in the underlying layer were not delineated by shadowing, gold particles attached to them were still visible, and so it was difficult to assess the true extent of pore labelling or the precise position of many labels in these views. However, it was usually possible to find quite extensive areas within each isolated envelope in which there was only a single layer of pores. These single layers usually occurred where there were holes in the overlying layer or where it had been folded back. Because of this, single layers usually had their nucteoplasmic faces uppermost and single layers of envelope with their cytoplasmic face uppermost were rare. The bar represents 500 nm. Nucleoplasmin was prepared using an antibody afinity column and conjugated with colloidal gold as described (Richardson el al., 1988) and stored in the presence of 1% BSA. Gold-labelled BSA was prepared similarly. Mieroinjection of gold-conjugated proteins into stage 6 Xenopus oocytes was as described (Richardson et al., 1988).
Figure 4. Labelling of pore subunits in disrupted envelopes obtained from oocytes in which nucleoplasmin-gold had been microinjected. Many rings had a centrally located label and some examples are circled. The bar represents 250 nm.
580
M. Stewart et al. Table 1
Location of labels in pores and rings prepared in different ways Particles located in each concentric zone (%)
Number of
particles Method of applying nucleoplasmin-gold Central Intermediate Peripheral counted
Microinjected, pores
65
19
16
Microinjected, rings
60
22
8
After isolation, pores After isolation, rings
9 10
24 28
67 62
327 238 171 174
The objects (pores or rings) were divided into 3 concentric zones of approximately equal width. Consequently, if particles were distributed over them randomly, the percentages in the central, intermediate and peripheral zones should be approximately 11%, 33% and 55%. Nucleoplasmin-gold applied to isolated envelopeshad roughly this distribution, whereas microinjected nucleoplasmin-gold had a very different distribution, with a much higher proportion of label located centrally.
pattern of decoration was not seen. In our preparations of nuclear envelopes treated with nucleoplasmin-gold after isolation, the extent of labelling of pores and rings depended on the amount ' of nucleoplasmin-gold applied, but centrally labelled pores and rings were generally rare. The overall distribution of labels in both pores and rings was quite different to that observed in which the nucleoplasmin-gold was microinjected (Table 1). Consequently, it would seem unlikely that the centrally located labels observed with microinjected material resulted from a chance deposition of nucleoplasmin-gold particles that were either not being transported or which were released when the envelope was disrupted. These results indicated that the central labelling of the rings by the nucleoplasmin-gold conjugates was biologically significant. This could reflect either binding of the label to the rings prior to translocation or alternatively some participation of material at the centre of the rings in the translocation process itself. To demonstrate that the observed association of nucleoplasmin-gold with the pores required a functional nuclear targeting signal, we injected beads conjugated to BSAt, a protein that lacks nuclear targeting signals and which does not bind strongly to pores. When BSA-conjugated gold was microinjected instead of nucleoplasmin (Fig. 5(b)) the density of gold particles attached to isolated nuclear envelopes was greatly reduced. There was some variation in the density of labelling, depending on the proximity to the site of injection, but even the most densely labelled BSA-gold material was only comparable to the most lightly labelled areas seen with nucleoplasmin-gold. The low level of labelling precluded accurate quantification of the distribution of label. However, there was clearly some central labelling of pores, consistent with observations on t Abbreviations used: BSA, bovine serum albumin; WGA, wheat germ agglutinin.
sectioned embedded material (Feldherr et al., 1984) that indicated that BSA was transported much less efficiently than nucleoplasmin. We observed random background labelling similar to that seen with nucleoplasmin-gold. However, centrally labelled rings wel'e observed rarely. Other studies have indicated that transport from cytoplasm to nucleus involves at least two steps (Newmeyer & Forbes, 1988; Richardson et al., 1988): an initial binding to the nuclear envelope, followed by translocation. Only the translocation step appeared to require metabolic energy. In their pioneering study, which used a fully competent nuclear assembly extract, Newmeyer & Forbes (1988) showed that the initial binding step took place on isolated envelopes in the absence of ATP. Similar conclusions were reached by Richardson et al. (1988) using an in vivo system. In both of these experimental systems there may have been important soluble cytoplasmic factors that contributed to the binding to the nuclear pores, but recent studies (Adam et at., I989; Benditt et al., 1989) have indicated that binding of nuclear localization sequences to nuclear pore components can take place on isolated nuclear envelopes. Consequently, binding might have been expected to have been observed when we applied nucleoplasmin-gold to isolated envelopes. Our failure to observe specific binding under these conditions may have been due to the binding not being sufficiently strong, so that the nucleoplasmin-gold dissociated under the conditions of relatively high dilution we employed. Alternatively, cytoplasmic fibrils have been proposed to be sites of initial attachment (Richardson et al., 1988). These fibrils are very labile (Richardson et al., 1988) and could easily have been lost during envelope isolation. Finally, some glycoproteins involved in binding to nuclear localization sequences are removed from pores by treatment with detergents such as Triton X-100 (Adam et al., 1989; Benditt et al., 1989). Although strictly this work does not show that these glycoproteins are removed by Triton X-10O treatment when they are bound to nucleoplasmin-gold, it would seem very likely that this would be the case, and so would be consistent with our failure to observe binding of nucleoplasmin-gold in vitro. But regardless of precisely why we failed to observe labelling in vitro, the presence of labelling only in envelopes from microinjected oocytes indicated that the labels observed were probably mainly from material being translocared, since in vivo labels merely bound prior to translocation would be expected to be lost from our preparations in the same way as happened in vitro. Support for this hypothesis was obtained by microinjecting oocytes with 60 #l of a l0 g/1 wheat germ agglutinin (WGA) solution and incubating them at 20°C for 40 minutes before microinjecting nucleoplasmin-gold. Wheat germ agglutinin inhibits translocation of nucleoplasmin through nuclear pores but does not inhibit its initial binding (Newmeyer & Forbes, 1988). Thus, material obtained in this way should have nucleoplasmin-gold bound to the pores
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Figure 5. (a) Nucleoplasmin-gold applied to an envelope after isolation. Labelling is essentially random and there was little central labelling of pores or rings. (b) Disrupted envelope obtained from an oocyte that had been microinjeeted with BSA-gold. Here the overall level of labelling was much less than with nucleoplasmin, consistent with the lower level of transport, of this material (see Feldherr et al., 1984). The bar represents 250 nm.
but not being translocated through them. We observed that the extent of pore labelling by microinjected nucleoplasmin-goid was reduced substantially if WGA was microinjected before the nucleoplasmin-gotd. In the absence of WGA, pores with a central nucleoplasmin-gold label were found over most of the envelope. But if WGA was microinjected before the nucleoplasmin-gotcl, centrally labelled pores were rare and, when present, were restricted to a small region of the envelope that was probably close to the site of microinjection. This region accounted for much less than 10~/o of the total area of the envelope. The lack of labelling when WGA was microinjected before nucleoplasmin-gold supported the idea that label merely bound prior to translocation was not retained in our experimental system, and so was consistent with the proposal that the labels we did see when only nucleoplasmin-gold was microinjected derived mainly from material being translocated. However, clearly more work will be required to resolve this question completely. Electron micrographs of sections of nuclear envelopes in Xenopus oocytes that have been microinjected with nucleoplasmin-gold sometimes show strings of gold particles that extend along the axis of pores and into both the cytoplasm and the nucleus (Feldherr et al., 1984; Richardson et al., 1988). Such an observation suggests that there may be multiple sites along the pore axis at which nucleoptasmin can attach, either by direct binding or in the process of being translocated. This interpretation would be consistent with our findings, which suggests an interaction of nucleoplasmin-gold with both the body of the pore and with rings. However, if pores containing such strings of gold particles were to be disrupted by direct contact with the support film (that is, by blotting), they might
leave centrally located gold particles in any rings produced. We think this is unlikely to have been a major factor producing the labelled rings we observed. As discussed above, there were a number of indications that the majority of rings in the areas of mass shedding produced by low-salt washes were not produced by blotting. Moreover, in published electron migrographs (Feldherr et al., 1984; Richardson et al., 1988) the axial alignment of the particles in these strings appears often to be less perfect as it leaves pores, particularly on the cytoplasmic face. Therefore, one would not have expected to observe such a strong central localization of labels had most rings been produced by blotting. In addition, we did not observe strings of gold labels emanating from rings. Moreover, studies of frozen-hydrated envelopes (Akey & Goldfarb, 1989) suggest that initial binding or "docking" steps involved in the transport of nueleoplasmin-gold occur at a higher radius from the pore axis than does transloeation. Overall, therefore, the strong central localization of labels, both in isolated rings and along the axis of sectioned material, indicated that nueleoplasmin-gold at the level of the rings was being positioned there by a c()mponent of the transport machinery. This component probably corresponded to the small quantity of material that could frequently be seen at the centre of shadowed isolated rings (Fig. 2(g) to (i)). The mechanism of transport through nuclear pores is not understood in detail and probably involves a number of steps and interactions between different pore components. However, it is generally thought that the transport machinery is located primarily in the central body of the pore complex, which includes the radial spokes and the central granule often seen in negatively stained and freezefractured material (Franke, 1974; Maul, 1977;
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Unwin & Milligan, 1982; Akey & Goldfarb, 1989). Many isolated pores in disrupted preparations from oocytes mieroinjected with nucleoplasmin-gold showed central labelling (Fig. 4), and some of these appeared to have lost their rings, which would be consistent with nucleoplasmin-gold being bound to the body of the pore, most probably in the vicinity of the central granule. Although it was not possible to be absolutely certain that these isolated discs did not retain an underlying ring (containing a gold label), these data indicated that there was some nucleoplasmin-gold attached to the body of the pore. This observation is consistent with part of the translocation machinery being located in the central body of the pore. However, the observation of nueleoplasmin labelling of rings indicated that part of the transport mechanism resided in this morphological component as well and was not restricted entirely to the body of the pore complex. Since the nuclear envelope is about 30 to 50 nm thick, multiple transport steps may be necessary to move material over such a distance. Muscle crossbridges, for example, probably only move by about 5 to 10 nm (Ford et al., 1977) per cycle and so a single transporter unit in nuclear pores may not be able to move material sufficiently far. Alternatively, it could be that binding to rings or passage through them is a preliminary to transport through the body of the pore complexes. Such an hypothesis would be consistent with the presence of rings on both the cytoplasmic and nucleoplasmic faces of the pores. Although clearly more work will be required to establish in detail the steps involved in nucleocytoplasmic transport, the labelling of the central portion of the rings we observed here provides evidence for the location of some components of the transport machinery outside the body of the pore complex. We are most grateful to our colleagues in Cambridge, in particular Chris Akey, Colin Dingwall, Richard Henderson, Ron Laskey and Nigel Unwin, for their many helpful comments and criticisms of this work. We also thank Neil Grant and Brian Pashley for photography. Claudio Villa for technical assistance, Patrick Sadler for
artwork and a reviewer for suggesting the experiments in w h i c h WGA was microinjected prior to nucleoplasmin-gold. References
Adam, S. A., Lobl, T. J., Mitchell, M. A. & Gerace, L. (1989). Na~.ure (London), 337, 276-279. Aebi, U., Colin, J., Buhle, L. & Gerace, L. (1986). Nalure (London), 323, 560-564. Akey, C. W. (1989). J. Cell Biol. 109, 955-970 Akey, C. W. & Goldfarb, D. S. (1989). J. Cell Biol. 109, 971-982. Benditt, J. O., Meyer, C.. Fasold, H., Barnard, F. C. & Riedel, N. (1989). Proc. Nat. Acad. Sci., U.S.A. 86, 9327-9331. Dingwall. C. & Laskey, R. (1986). Annu. Rev. Cell Biol. 2, 365-388. Dworetzky, S. I. & Feldherr, C. M. (1988). J. Cell. Biol. 106, 575-584. Feldherr, C. M. & Dworetzky, S. I. (1988). Cell Biol. Int. Rep. 12, 791-808. Feldherr, C. M., Kallenbach, E. & Schuyltz, N. (1984). J. Cell Biol. 99, 2216-2222. Ford, L. E., Huxley. A. F. & Simmons. R. M. (1977). J. Physiol. (London), 269. 445-515. Franke, W. W. (1974). Int. Rev. Cytol. (Suppl.) 4, 71-236. Franke. W. W. & Scheer. U. (1970). J. Ultraslruct. Res. 30, 288-316. Gerace, L. (1986). Trends Biochem. Sci. 11,443-446. Kartenbeck, J., Zentgraf, H., Sheer. U. & Franke, W. W. (1971). Advan. Anat. Embryol. Cell Biol. 45, 1-55. Maul, G. G. (1977). Int. Rev. Cytol. (Suppl.) 6, 75-186. Mills, A. D., Laskey, R. A., Black, P. & DeRobertis, E. M. (1980). J. Mol. Biol. 139, 561-568. Newmeyer, D. D. & Forbes, D. J. (1988). Cell, 52, 641-653. N2wmeyer, D. D., Lucocq, J. M.. Burglin, T. R. & DeRobertis, E. M. (1986). EMBO J. 5, 501-510. Newport, J. W. & Forbes. D, J. (1987). Annu. Rev. Biochem. 56, 535-565. Richardson, W. D., Mills, A. D., Dillworth, S. M., Laskey, R. A. & Dingwall, C. (1988). Cell, 52. 655-664. Stewart, M. & Whytock, S. (1988). J. Cell Sci. 90, 409-423. Unwin, P. N. T. & Milligan, R. A. (1982). J. Cell Biol. 93, 63-75. Whytock, S. & Stewart, M. (1988). J. Microsc. 151, 115-126.
Edited by D. DeRosier
Association of Gold-labelled Nucleoplasmin with the Centres of Ring Components of Xenopus 0ocyte Nuclear Pore Complexes M u r r a y Stewart, Sue W h y t o c k
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, England and Anthony D. Mills
Cancer Research Campaign Molecular Embryology Research Group Department of Zoology, Downing Street Cambridge CB2 3E J, England (Received 5 June 1989; accepted 12 February 1990) We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoolasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60°/o), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.
plasmin, the most abundant nuclear protein in The nuclear envelope is constructed from a double Xenopus oocytes (Mills et al., 1980) is too large to membrane perforated by nuclear pore complexes diffuse passively through the pores and frequently which mediate selective transport (for reviews, see has been used to investigate active transport into Dingwall & Laskey, 1986; Newport & Forbes, 1987). the nucleus. Colloidal gold' conjugated with nucleoA fibrous lamina lies under the nucleoplasmic face of plasmin accumulates in the nucleus over a period of the envelope, below the level of the pores (see Aebi hours after being microinjected into Xenopus et al., 1986; Gerace, 1986; Stewart & Whytock, oocytes. Sections of embedded material that was 1988) and there are also pore-connecting fibrils on fixed while nucleoplasmin-gotd was being transthe cytoplasmic face of the envelope (see Stewart & ported show many pores with colloidal gold markers Whytock, 1988, and references cited therein). located centrally on the axis of the pore cylinder Nuclear pore complexes mediate both active and (Felhderr et al., 1984; Richardson et al., 1988; passive exchange of material between nucleus and Newmeyer & Forbes, 1988). A similar distribution cytoplasm and are roughly cylindrical structures in has also been observed in isolated nuclear envelopes which eight internal spoke-like units and a central examined in vitrified water (Akey & Goldfarb, 1989) granule are arranged between cytoplasmic and where the gold label is located near the centre of the nucleoplasmic rings (see Kartenbeck et al., 1971; circular pore profile seen in the primarily en face Franke, 1974; Maul, 1977; Unwin & Milligan, 1982; Akey, 1989, and references cited therein). Nucleoviews obtained with these whole mounts. These 575 © 1990AcademicPressLimited 0022-2836/90/120575-08 $03.00/0
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1
4
i
I
I
Figure 1. Schematic drawing of pore substructure consistent with the images we obtained from disrupted pores and with a broad range of data from other sources (see Franke, 1974; Maul. 1977; Unwin & Milligan, 1982). The basic morphological components are cytoplasmic (2) and nucleoplasmic (5) rings, the cylindrical body of the pore (4), pore-connecting fibrils (3) attached at the level of the cytoplasmic ring, and 8 granules (1) located on the cytoplasmic face. This Figure concentrates on the structural details seen in shadowed preparations and, because these visualized the surface of the pores and their morphological subunits, we were not able to observe their internal structure. Images obtained by both negative staining and freeze-fracture (see, e.g. Kartenbeck et al., 1971; Franke, 1974; Maul, 1977; Franke & Scheer, 1970) give information on the internal substructure of the cylindrical body of the pore and show that it contains 8 spoke-like units and a central granule. Isolated components, such as rings, granules and the central cylinders have also been observed by negative staining (Unwin & Milligan, 1982) and in unstained vitrified preparations (Akey, 1989). In addition, 8 granules have often been seen attached to the cytoplasmic face of pore complexes (Unwin & MiUigan, 1982; Stewart & Whytock, 1988). Cytoplasmic-nuclear transport appears to take place along the central axis of the pore ( . . . . . ) and may involve the central granule (Akey & Goldfarb, 1989; Unwin & Milligan, 1982) located in the cylindrical body of the pore.
data all indicate that nucleoplasmin is translocated along the central axis of the pore complex, and suggest a major role for the central granule, which has been referred to as the "transporter" (Akey & Goldfarb, 1989). To investigate the role of morpho-
logically defined subunits of the pore complex in the transport process, we have employed shadowed whole mounts of nuclear envelopes from which membranes have been removed (Aebi et al., 1986; Stewart & Whytock, 1988). Because of their high inherent contrast, such preparations are particularly suitable for examining the morphological subunits of nuclear pores produced by partial fragmentation of nuclear envelopes (Stewart & Whytock, 1988; Whytock & Stewart, 1988) and, in conjunction with gold-labelled nucleoplasmin, they enable the role of different morphological subunits of the pore complex in transport to be investigated. Nuclear pore complexes can be divided into a number of morphological components as illustrated in Figure 1. Rings are attached to both the nucleoplasmic and cytoplasmic faces of the disc-like body of the pore and, in addition, eight roughly spherical granules are attached to the ring facing the cytoplasm. Although specimens prepared in a number of ways all show these morphological components, their detailed appearance is not always the same because different techniques emphasize different aspects of nuclear pore structure. In particular, the internal distribution of material in the body of the pore complex, which has a central granule surrounded by eight spoke-like units, dominates images of frozen hydrated (Akey, 1989) or negatively stained (see, for example, Unwin & Milligan, 1982) material. A similar pattern is seen in micrographs of freeze-fractured nuclear envelopes (see, for example, Kartenbeck et al., 1971). On the other hand, images of shadowed whole mounts of nuclear envelopes show primarily the external surface. In such images, the body of the pore complex appears as a short cylinder or disc with its axis perpendicular to the plane of the envelope (Aebi et al., 1986; Stewart & Whytock, 1988). Sections of embedded envelopes usually show cross-sections, although in some instances en face views have been obtained that show the 8-fold internal substructure of the body of the pore (for reviews, see Franke, 1974; Maul, 1977). The cytoplasmic and nucleoplasmic rings can be seen in most types of preparation and often show a weak 8-fold rotational symmetry (Unwin & Milligan, 1982; Stewart & Whytock, 1988; Whytock & Stewart, 1988; Akey, 1989). However, the rings are most easily seen in preparations in which the pore structure has been partially disrupted. Our previous studies (Stewart & Whytock, 1988; Wytock & Stewart, 1988) showed that washing with low ionic strength buffer was an effective way of producing such partial disruption of the envelope and, as illustrated in Figure 2(a), generated large areas of envelopes from which pores were being fragmented and shed. Islands of pores, often still connected by pore-connecting fibrils, were seen frequently in such preparations, together with pores in various degrees of disintegration. We often observe rings (Fig. 2(a) to (i)), some of which contain a small amount of additional material at their centre (Fig. 2(g) to (i)). The amount of material seen at the centre of these rings was much
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577
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Figure 2. Disruption of Xenopus oocyte nuclear envelopes after washing in low-salt buffer. (a} A large area of envelope shedding pores, islands and rings. The large arrow at the bottom left shows a typical island in which most of the pores are joined by pore-connecting fibrils, that were most easily seen when, as in this example, the cytoplasmic face of the pore was uppermost. There were 3 main types of isolated particle present in disrupted preparations such as this. Large numbers of isolated rings were often seen (arrowheads at the top left indicate several examples} and in this Figure can be found over most of the field of view. In some places (small arrows} rings overlap partially. Isolated pores, such as that circled at the lower left (immediately above the island} were comparatively rare. These particles closely resembled the pores in islands and in whole envelopes {shown at the top right of the Figure} and were thought to be substantially intact. More common were particles such as those arrowed at the centre left which seemed to be rather thinner than the isolated pores (as judged by the shorter shadow they cast) and which probably represented the disc-like central body of the pore complexes from which rings had been removed. (b) to (i) A gallery of rings, some of which have additional material located at their centres ((g) to (i)). In some cases (f) and (g} pore-connecting fibrils could be seen, identifying cytoplasmic rings. (j) to (n} show rare edge views of pores. The particles in these images appear to have a distinct waist consistent with rings being located on both the top and bottom of the pore complex. The bar represents 1000 nm for (a) and 200 nm for (b) to (n). Nuclear envelopes were isolated and attached to polylysine-coated grids as described {Stewart & Whytock, 1988). Specimens were washed in low-salt buffer (10 mm-Hepes, 1 mm-KCl, 2 mm-MgCl2 (pH 7"5)) at room temperature for 10 min and then in 0"5°/o {v/v) Triton X-100, 0"3 m-KC1, 10% (w/v) sucrose, 2 mM-EDTA, 1 mm-dithiothreitoi, 20 mm-Tris- HCl for l0 min at room temperature to remove membranes. Specimens were shadowed with platinum/carbon after either air or freeze-drying as described (Stewart & Whytock, 1988; Whytock & Stewart, 1988), and examined at 80 kV in Philips EM301, EM400, EM420 or CMI2 electron microscopes using standard imaging conditions. Mierographs were usually recorded at nominal magnifications near 15,000 x. Nucleoplasmic and cytoplasmic faces were identified using the criteria described by Stewart & Whytock (1988). Micrographs are printed so that heavy metal and gold are black.
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less than would be anticipated for the central "transporter" granule, which appears to be located centrally in the body of the pore complex (Unwin & Milligan, 1982). Rare edge views of pores (Fig. 2(j) to (n)) were consistent with other data (see Unwin & Milligan, 1982; Akey, 1989), which indicated that pores have rings on both cytoplasmic and nucleoplasmic faces. Cytoplasmic rings could sometimes be distinguished from nucleoplasmic ones by the presence of pore-connecting fibrils between rings (Fig. 2(f) and (g)). Because these fibrils are located near the cytoplasmic face of the pores (Stewart & Whytock, 1988), rings connected by them must have been ~ytoplasmic ones. Views of disintegrating pores (Stewart & Whytock, 1988) have shown that rings also derive from the nucleoplasmic face. We did not observe cytoplasmic rings with granules attached to them, probably because the granules appear to be rather easily removed. Previous studies (Whytoek & Stewart, 1988) showed that sometimes groups of eight granules were seen when the cytoplasmic surface of the envelope attached transiently to the support film and left a "blot". The thin fiat structure marked by the long arrow in Figure 2 probably represents the disc-like central body of the pore complex that contributes so strongly to negatively stained images (see Unwin & Milligan, 1982). However, it was not always possible to decide if these putative pore bodies retained an underlying ring, as this could have been contrasted poorly by shadowing. The areas of rings and other fragments produced by low-salt washing could have arisen in two ways: material could have been released from pore complexes and then settled onto the support film or, alternatively, the fragments could represent a "blot" produced by dislodging part of the envelope that had become attached to the support film. Although obvious blots have been observed (Unwin & Milligan, 1982; Fig. 8 of Whytock & Stewart, 1988), a number of considerations indicated that the mass shedding of pores we observed in areas such as that shown in Figure 2 probably did not arise primarily in this way. First, the material in blots was usually in close apposition to the nuclear envelope, whereas the rings, isolated pores and islands seen with mass shedding, were often found over 20~m from the envelope. Second, the rings observed in blots had a very characteristic beaded appearance, in which usually eight granules could be observed (Unwin & Milligan, 1982; Whytock & Stewart, 1988), whereas the rings in the shed areas were much smoother (see Fig. 2). Third, blotted material would be expected always to have its cytoplasmic face attached to the support film, but in shed areas we frequently observed a mixture of orientations so that some of the material was oriented cytoplasmic face uppermost (away from the support film) as can be seen, for example, with the island arrowed in Figure 2 (see Stewart & Whytock (1988) for a discussion of the criteria whereby cytoplasmic and nucleoplasmic faces can be distinguished). Fourth, fragments in blots were
usually closely packed together similar to pores in intact nuclear envelopes, whereas the spacing between rings and isolated pores in shed areas was more variable. Fifth, the degree of disruption, both of pores into rings and of rings into smaller subunits, increased with the duration and severity of the low-salt wash (Stewart & Whytock, 1988). And finally, rings were sometimes seen to overlap partially (small arrows in Fig. 2), which would not be expected to be seen if they were produced by blotting. Nucleoplasmin-gold conjugates were easily recognized in shadowed preparations of nuclear envelopes isolated from Xenopus oocytes two to four hours after microinjection. Views of nucleoplasmic faces showed the labelling clearly (Fig. 3) and enabled the extent and position of labelling to be most precisely determined. Where the lamina was suspended above the support film (left side of the micrograph), the pore complexes were partially obscured, so we concentrated on areas where the lamina was attached to the substrate (right side of the micrograph). Here the pores were quite clear and the degree of labelling could be assessed reliably. In most instances the gold label was located centrally in the pore complex, with labels seen comparatively rarely at high radius or on the cytoplasmic granules or pore-connecting fibrils. However, there was also some random labelling of the envelopes which presumably derived from material not being actively transported (a substantial fraction of the microinjeered nucleoplasmin-gold was present in both the cytoplasm and nucleus and became distributed randomly over the envelopes during isolation). The systematic location of gold-labelled nucleoplasmin near the axis of the pore complexes from microinjected oocytes was consistent with observations on sections (Feldherr et al., 1984; Newmeyer & Forbes, 1988; Richardson et al., 1988; Feldherr & Dworetzky, 1988) and on frozen-hydrated material (Akey & Goldfarb, 1989) that indicated that this material was transported through the centre of pores. When envelopes from nucleoplasmin microinjected oocytes were partially disrupted by low-salt washes, a substantial number of the rings produced had centrally located gold particles (Fig. 4). Labelled rings were usually more common than unlabelled ones in these preparations, and of 288 rings examined 238 contained gold particles. The radial distribution of particles was assessed by dividing the rings into three equally spaced concentric zones. As shown in Table 1, most labels (60%) were located centrally, with only 22~o at intermediate radii and 18~/o at the periphery. A similar distribution was seen with intact pores. This distribution was very different from that expected for random labelling, where a distribution roughly in proportion to the areas of the zones would have been expected. Thus, random labelling would have been expected to label about 11 ~o of rings centrally. When gold-conjugated nucleoplasmin was applied to envelopes after isolation (Fig. 5(a)), a clear
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Figure 3. Labelling of Xenopu8 oocyte nuclear envelopes with microinjected nucleoplasmin-gold. This Figure shows a nucleoplasmic face of an intact envelope in which the lamina was stuck onto the support film and so had low contrast. However, a small area on the left shows the more usual view (Stewart & Whytock, 1988) where the lamina has become detached and has much higher contrast. Pores were labelled primarily on their axis. Because specimens were prepared by opening out an intact envelope on the grid. the most frequently observed views were of 2 overlapping envelope layers, in which the top layer had its cytoplasmic face uppermost and the lower had its nucleoplasmic face uppermost (see Stewart & Whytock, 1988). Although pores in the underlying layer were not delineated by shadowing, gold particles attached to them were still visible, and so it was difficult to assess the true extent of pore labelling or the precise position of many labels in these views. However, it was usually possible to find quite extensive areas within each isolated envelope in which there was only a single layer of pores. These single layers usually occurred where there were holes in the overlying layer or where it had been folded back. Because of this, single layers usually had their nucteoplasmic faces uppermost and single layers of envelope with their cytoplasmic face uppermost were rare. The bar represents 500 nm. Nucleoplasmin was prepared using an antibody afinity column and conjugated with colloidal gold as described (Richardson el al., 1988) and stored in the presence of 1% BSA. Gold-labelled BSA was prepared similarly. Mieroinjection of gold-conjugated proteins into stage 6 Xenopus oocytes was as described (Richardson et al., 1988).
Figure 4. Labelling of pore subunits in disrupted envelopes obtained from oocytes in which nucleoplasmin-gold had been microinjected. Many rings had a centrally located label and some examples are circled. The bar represents 250 nm.
580
M. Stewart et al. Table 1
Location of labels in pores and rings prepared in different ways Particles located in each concentric zone (%)
Number of
particles Method of applying nucleoplasmin-gold Central Intermediate Peripheral counted
Microinjected, pores
65
19
16
Microinjected, rings
60
22
8
After isolation, pores After isolation, rings
9 10
24 28
67 62
327 238 171 174
The objects (pores or rings) were divided into 3 concentric zones of approximately equal width. Consequently, if particles were distributed over them randomly, the percentages in the central, intermediate and peripheral zones should be approximately 11%, 33% and 55%. Nucleoplasmin-gold applied to isolated envelopeshad roughly this distribution, whereas microinjected nucleoplasmin-gold had a very different distribution, with a much higher proportion of label located centrally.
pattern of decoration was not seen. In our preparations of nuclear envelopes treated with nucleoplasmin-gold after isolation, the extent of labelling of pores and rings depended on the amount ' of nucleoplasmin-gold applied, but centrally labelled pores and rings were generally rare. The overall distribution of labels in both pores and rings was quite different to that observed in which the nucleoplasmin-gold was microinjected (Table 1). Consequently, it would seem unlikely that the centrally located labels observed with microinjected material resulted from a chance deposition of nucleoplasmin-gold particles that were either not being transported or which were released when the envelope was disrupted. These results indicated that the central labelling of the rings by the nucleoplasmin-gold conjugates was biologically significant. This could reflect either binding of the label to the rings prior to translocation or alternatively some participation of material at the centre of the rings in the translocation process itself. To demonstrate that the observed association of nucleoplasmin-gold with the pores required a functional nuclear targeting signal, we injected beads conjugated to BSAt, a protein that lacks nuclear targeting signals and which does not bind strongly to pores. When BSA-conjugated gold was microinjected instead of nucleoplasmin (Fig. 5(b)) the density of gold particles attached to isolated nuclear envelopes was greatly reduced. There was some variation in the density of labelling, depending on the proximity to the site of injection, but even the most densely labelled BSA-gold material was only comparable to the most lightly labelled areas seen with nucleoplasmin-gold. The low level of labelling precluded accurate quantification of the distribution of label. However, there was clearly some central labelling of pores, consistent with observations on t Abbreviations used: BSA, bovine serum albumin; WGA, wheat germ agglutinin.
sectioned embedded material (Feldherr et al., 1984) that indicated that BSA was transported much less efficiently than nucleoplasmin. We observed random background labelling similar to that seen with nucleoplasmin-gold. However, centrally labelled rings wel'e observed rarely. Other studies have indicated that transport from cytoplasm to nucleus involves at least two steps (Newmeyer & Forbes, 1988; Richardson et al., 1988): an initial binding to the nuclear envelope, followed by translocation. Only the translocation step appeared to require metabolic energy. In their pioneering study, which used a fully competent nuclear assembly extract, Newmeyer & Forbes (1988) showed that the initial binding step took place on isolated envelopes in the absence of ATP. Similar conclusions were reached by Richardson et al. (1988) using an in vivo system. In both of these experimental systems there may have been important soluble cytoplasmic factors that contributed to the binding to the nuclear pores, but recent studies (Adam et at., I989; Benditt et al., 1989) have indicated that binding of nuclear localization sequences to nuclear pore components can take place on isolated nuclear envelopes. Consequently, binding might have been expected to have been observed when we applied nucleoplasmin-gold to isolated envelopes. Our failure to observe specific binding under these conditions may have been due to the binding not being sufficiently strong, so that the nucleoplasmin-gold dissociated under the conditions of relatively high dilution we employed. Alternatively, cytoplasmic fibrils have been proposed to be sites of initial attachment (Richardson et al., 1988). These fibrils are very labile (Richardson et al., 1988) and could easily have been lost during envelope isolation. Finally, some glycoproteins involved in binding to nuclear localization sequences are removed from pores by treatment with detergents such as Triton X-100 (Adam et al., 1989; Benditt et al., 1989). Although strictly this work does not show that these glycoproteins are removed by Triton X-10O treatment when they are bound to nucleoplasmin-gold, it would seem very likely that this would be the case, and so would be consistent with our failure to observe binding of nucleoplasmin-gold in vitro. But regardless of precisely why we failed to observe labelling in vitro, the presence of labelling only in envelopes from microinjected oocytes indicated that the labels observed were probably mainly from material being translocared, since in vivo labels merely bound prior to translocation would be expected to be lost from our preparations in the same way as happened in vitro. Support for this hypothesis was obtained by microinjecting oocytes with 60 #l of a l0 g/1 wheat germ agglutinin (WGA) solution and incubating them at 20°C for 40 minutes before microinjecting nucleoplasmin-gold. Wheat germ agglutinin inhibits translocation of nucleoplasmin through nuclear pores but does not inhibit its initial binding (Newmeyer & Forbes, 1988). Thus, material obtained in this way should have nucleoplasmin-gold bound to the pores
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Figure 5. (a) Nucleoplasmin-gold applied to an envelope after isolation. Labelling is essentially random and there was little central labelling of pores or rings. (b) Disrupted envelope obtained from an oocyte that had been microinjeeted with BSA-gold. Here the overall level of labelling was much less than with nucleoplasmin, consistent with the lower level of transport, of this material (see Feldherr et al., 1984). The bar represents 250 nm.
but not being translocated through them. We observed that the extent of pore labelling by microinjected nucleoplasmin-goid was reduced substantially if WGA was microinjected before the nucleoplasmin-gotd. In the absence of WGA, pores with a central nucleoplasmin-gold label were found over most of the envelope. But if WGA was microinjected before the nucleoplasmin-gotcl, centrally labelled pores were rare and, when present, were restricted to a small region of the envelope that was probably close to the site of microinjection. This region accounted for much less than 10~/o of the total area of the envelope. The lack of labelling when WGA was microinjected before nucleoplasmin-gold supported the idea that label merely bound prior to translocation was not retained in our experimental system, and so was consistent with the proposal that the labels we did see when only nucleoplasmin-gold was microinjected derived mainly from material being translocated. However, clearly more work will be required to resolve this question completely. Electron micrographs of sections of nuclear envelopes in Xenopus oocytes that have been microinjected with nucleoplasmin-gold sometimes show strings of gold particles that extend along the axis of pores and into both the cytoplasm and the nucleus (Feldherr et al., 1984; Richardson et al., 1988). Such an observation suggests that there may be multiple sites along the pore axis at which nucleoptasmin can attach, either by direct binding or in the process of being translocated. This interpretation would be consistent with our findings, which suggests an interaction of nucleoplasmin-gold with both the body of the pore and with rings. However, if pores containing such strings of gold particles were to be disrupted by direct contact with the support film (that is, by blotting), they might
leave centrally located gold particles in any rings produced. We think this is unlikely to have been a major factor producing the labelled rings we observed. As discussed above, there were a number of indications that the majority of rings in the areas of mass shedding produced by low-salt washes were not produced by blotting. Moreover, in published electron migrographs (Feldherr et al., 1984; Richardson et al., 1988) the axial alignment of the particles in these strings appears often to be less perfect as it leaves pores, particularly on the cytoplasmic face. Therefore, one would not have expected to observe such a strong central localization of labels had most rings been produced by blotting. In addition, we did not observe strings of gold labels emanating from rings. Moreover, studies of frozen-hydrated envelopes (Akey & Goldfarb, 1989) suggest that initial binding or "docking" steps involved in the transport of nueleoplasmin-gold occur at a higher radius from the pore axis than does transloeation. Overall, therefore, the strong central localization of labels, both in isolated rings and along the axis of sectioned material, indicated that nueleoplasmin-gold at the level of the rings was being positioned there by a c()mponent of the transport machinery. This component probably corresponded to the small quantity of material that could frequently be seen at the centre of shadowed isolated rings (Fig. 2(g) to (i)). The mechanism of transport through nuclear pores is not understood in detail and probably involves a number of steps and interactions between different pore components. However, it is generally thought that the transport machinery is located primarily in the central body of the pore complex, which includes the radial spokes and the central granule often seen in negatively stained and freezefractured material (Franke, 1974; Maul, 1977;
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Unwin & Milligan, 1982; Akey & Goldfarb, 1989). Many isolated pores in disrupted preparations from oocytes mieroinjected with nucleoplasmin-gold showed central labelling (Fig. 4), and some of these appeared to have lost their rings, which would be consistent with nucleoplasmin-gold being bound to the body of the pore, most probably in the vicinity of the central granule. Although it was not possible to be absolutely certain that these isolated discs did not retain an underlying ring (containing a gold label), these data indicated that there was some nucleoplasmin-gold attached to the body of the pore. This observation is consistent with part of the translocation machinery being located in the central body of the pore. However, the observation of nueleoplasmin labelling of rings indicated that part of the transport mechanism resided in this morphological component as well and was not restricted entirely to the body of the pore complex. Since the nuclear envelope is about 30 to 50 nm thick, multiple transport steps may be necessary to move material over such a distance. Muscle crossbridges, for example, probably only move by about 5 to 10 nm (Ford et al., 1977) per cycle and so a single transporter unit in nuclear pores may not be able to move material sufficiently far. Alternatively, it could be that binding to rings or passage through them is a preliminary to transport through the body of the pore complexes. Such an hypothesis would be consistent with the presence of rings on both the cytoplasmic and nucleoplasmic faces of the pores. Although clearly more work will be required to establish in detail the steps involved in nucleocytoplasmic transport, the labelling of the central portion of the rings we observed here provides evidence for the location of some components of the transport machinery outside the body of the pore complex. We are most grateful to our colleagues in Cambridge, in particular Chris Akey, Colin Dingwall, Richard Henderson, Ron Laskey and Nigel Unwin, for their many helpful comments and criticisms of this work. We also thank Neil Grant and Brian Pashley for photography. Claudio Villa for technical assistance, Patrick Sadler for
artwork and a reviewer for suggesting the experiments in w h i c h WGA was microinjected prior to nucleoplasmin-gold. References
Adam, S. A., Lobl, T. J., Mitchell, M. A. & Gerace, L. (1989). Na~.ure (London), 337, 276-279. Aebi, U., Colin, J., Buhle, L. & Gerace, L. (1986). Nalure (London), 323, 560-564. Akey, C. W. (1989). J. Cell Biol. 109, 955-970 Akey, C. W. & Goldfarb, D. S. (1989). J. Cell Biol. 109, 971-982. Benditt, J. O., Meyer, C.. Fasold, H., Barnard, F. C. & Riedel, N. (1989). Proc. Nat. Acad. Sci., U.S.A. 86, 9327-9331. Dingwall. C. & Laskey, R. (1986). Annu. Rev. Cell Biol. 2, 365-388. Dworetzky, S. I. & Feldherr, C. M. (1988). J. Cell. Biol. 106, 575-584. Feldherr, C. M. & Dworetzky, S. I. (1988). Cell Biol. Int. Rep. 12, 791-808. Feldherr, C. M., Kallenbach, E. & Schuyltz, N. (1984). J. Cell Biol. 99, 2216-2222. Ford, L. E., Huxley. A. F. & Simmons. R. M. (1977). J. Physiol. (London), 269. 445-515. Franke, W. W. (1974). Int. Rev. Cytol. (Suppl.) 4, 71-236. Franke. W. W. & Scheer. U. (1970). J. Ultraslruct. Res. 30, 288-316. Gerace, L. (1986). Trends Biochem. Sci. 11,443-446. Kartenbeck, J., Zentgraf, H., Sheer. U. & Franke, W. W. (1971). Advan. Anat. Embryol. Cell Biol. 45, 1-55. Maul, G. G. (1977). Int. Rev. Cytol. (Suppl.) 6, 75-186. Mills, A. D., Laskey, R. A., Black, P. & DeRobertis, E. M. (1980). J. Mol. Biol. 139, 561-568. Newmeyer, D. D. & Forbes, D. J. (1988). Cell, 52, 641-653. N2wmeyer, D. D., Lucocq, J. M.. Burglin, T. R. & DeRobertis, E. M. (1986). EMBO J. 5, 501-510. Newport, J. W. & Forbes. D, J. (1987). Annu. Rev. Biochem. 56, 535-565. Richardson, W. D., Mills, A. D., Dillworth, S. M., Laskey, R. A. & Dingwall, C. (1988). Cell, 52. 655-664. Stewart, M. & Whytock, S. (1988). J. Cell Sci. 90, 409-423. Unwin, P. N. T. & Milligan, R. A. (1982). J. Cell Biol. 93, 63-75. Whytock, S. & Stewart, M. (1988). J. Microsc. 151, 115-126.
Edited by D. DeRosier
