Nuclear envelope dynamics and nucleocytoplasmic transport

MURRAY STEWART, SUE WHYTOCK and ROBERT D. MOIR

MRC Laboratory o f Molecular Biology, Hills Rd., Cambridge CB2 2QH, England

Summary

We have combined structural, biochemical and recombinant DNA methods to explore molecular interactions involved in nuclear envelope assembly dynamics and nucleocytoplasmic transport. Electron microscopy has established the overall architecture of the envelope and the relationship between nuclear pores, lamina fibres and pore-connecting fibrils. The lamin proteins that constitute the lamina resemble intermediate filament proteins, and assemble and disassemble during mitosis in response to phos­phorylation. Lamins have been expressed in E. coli to facilitate structural investigations and the explo­ration of interaction sites with other envelope com­ponents. Disruption of envelopes has shown that nuclear pores are constructed from a central cylin­der with cytoplasmic and nucleoplasmic rings. Examination of envelopes transporting gold-labelled nucleoplasmin has indicated that the transport pathway is complex and probably involves ring components in addition to the central cylinder. Molecular motors may be involved in changes in pore shape to enable transport and in the translocation mechanism.

Key words: lamins, nuclear pores, interactions, transport, structure.

Nuclear envelope structure

The nuclear envelope separates the chromosomes from the cytoplasm of eucaryotic cells and controls transport into and out of the nucleus. It is constructed from a double membrane perforated by nuclear pore complexes thought to be responsible for selective transport (reviewed by Newport and Forbes, 1987). A fibrous lamina, composed of lamins, lies under the nucleoplasmic face of the envelope, below the level of the pores (see Aebi et al. 1986; Stewart and Whytock, 1988). The lamins closely resemble inter­mediate filaments in sequence and structure (McKeon et al. 1986), forming fibres about 10 nm in diameter that are sometimes found in a remarkably regular basketweave pattern (Aebi et al. 1986). The lamina is thought to be important in organising the nuclear pore complexes and possibly also chromosomes within the nucleus (Gerace, 1986; Newport and Forbes, 1987). In addition to the lamina, there are also pore-connecting fibrils that link pore complexes directly to each other (Maul, 1977; Stewart and Whytock, 1988; Reichelt et al. 1990).

Shadowed nuclear envelopes from which membranes have been removed show pore complexes and lamina clearly and enable cytoplasmic and nucleoplasmic faces to be recognised (Aebi et al. 1986; Stewart and Whytock,Journal of Cell Science, Supplement 14, 79-82 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

1988). Cytoplasmic faces are dominated by nuclear pores which are often linked by connecting fibrils (PCFs). Stereo pairs show that the PCFs attach to the pores near their cytoplasmic end and micrographs of disrupted pore complexes indicate that the PCFs attach to the cytoplas­mic ring (Stewart et al. 1990). The nucleoplasmic face has a distinctively different appearance and the fibrous lamina is much more prominent. In areas where pore complexes are close together, the lamina fibres dominate the image and often form a fibrous felt-like mat that, in many places, is elevated above the support film (Fig. 1). In areas where the pores are more separated they have a distinctive star-like outline due to the attachment of lamina fibres. Although clearly present, the pore com­plexes are not a striking feature of nucleoplasmic faces. Fig. 2 shows a schematic representation of the structure of the Xenopus oocyte nuclear envelope.

Proteolysis has provided direct evidence that the 10 nm fibres seen in shadowed preparations are composed from lamins (Whytock et al. 1990). Increasing protease concen­trations progressively removed the lamina fibres with a corresponding decrease in lamin signal using fluorescence microscopy and ‘dot-blots’ of whole nuclei. PCFs were more resistant to proteolysis and remained in areas of digested samples where the lamina had been removed. Similarly, the morphology of the nuclear pore complexes was not markedly altered by the degree of proteolysis needed to remove the lamina. Some digestion of the nuclear pore complexes and PCFs was only seen when high concen­trations of trypsin were used. When nuclear envelopes were first digested with high concentrations of trypsin and then extracted with Triton X-100, the nuclear envelopes disintegrated completely. A similar effect of protease concentration was observed with the proteolysis of material that had been first extracted with Triton. These observations provide direct evidence that the lamina has a role in maintaining the structural integrity of the envelope (Whytock et al. 1990).

Lamin molecular structure and interactionLamins have been identified in a wide range of organisms (see Krohne and Benevente, 1986), with mammals expressing three lamin isoforms (A, B, and C), although lamins A and C are very similar and arise by alternative splicing (McKeon et al. 1986). When the nuclear envelope disassembles during mitosis, B-type lamins remain associ­ated with membranous vesicles, whereas A-type lamins become freely soluble in the cytoplasm (Gerace and Blobel, 1980). The precise pattern of lamin expression often varies in response to cell differentiation and development. Although B-type lamins appear to be ubiquitous, lamins A and C are usually expressed at low levels during early development but increase with cell differentiation (Lehner

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assemble with lamins. A 42-residue insert in the lamin rod domain may be associated with this behaviour. The assembly properties of both lamins and IFs appear to be altered by phosphorylation which may have a role in the dynamics of both filament systems during cell division (Peter et al. 1990).

Fig. 1. Shadowed Xenopus oocyte nuclear envelope, with its nucleoplasmic face uppermost, showing the dense canopy of lamina fibres supported by underlying nuclear pore complexes. Reproduced from Stewart and Whytock (1988). Bar, 0.5,iim.

et al. 1987; Stewart and Burke, 1987). Lamin sequences (McKeon et al. 1986) show strong homologies to intermedi­ate filaments (IFs) and indicate a three-domain model for lamins, with a central fibrous rod having principally a coiled-coil conformation, with non-helical N- and C-terminal domains (reviewed by Stewart, 1990). The lamin N-terminal domain is small compared with most other IF proteins, whereas the lamin C-terminal domain is comparatively large. Electron microscopy of shadowed single molecules and paracrystals (Aebi et al. 1986) support this model and show a rod-shaped molecule, about 52 nm long, with a globular domain at one end. Although there is substantial promiscuity in the assembly of IF proteins (see Stewart, 1990), they appear not to co-

Fig. 2. Schematic illustration of the arrangement of the principal non-membrane components of Xenopus oocyte nuclear envelopes. The cylindrical nuclear pore complexes are constructed from nucleoplasmic and cytoplasmic rings and are linked by pore-connecting fibrils. There is a coronet of eight granules on the cytoplasmic face of the pore complex, whereas the 10 nm lamina fibres attach to the nucleoplasmic face. In some areas the lamina fibres are arranged in a basket-weave pattern.

Lamin expression

Because the volume fraction of cells represented by nuclear envelope is small, difficulties can be experienced in preparing large quantities of lamins. Moreover, native material usually is modified post-transcriptionally. Ex­pression in E. coli circumvents these difficulties and also facilitates the production of fragments and modified material, which can be used to explore the molecular basis of assembly and domain structure, and complement expression studies in cultured cells. We initially expressed human lamin C cDNA using the pLcII vector (Moir et al. 1990), but this produced a fusion with part of the lambda ell protein which we were unable to remove. To circum­vent these difficulties, we instead used the T7 expression system to produce substantial quantities of recombinant human lamins A and C. In addition to expressing full length lamins A and C, we used site-directed mutagenesis to modify the lamin cDNA and so express a number of modified protein molecules that lacked the N- or C-terminal non-helical domains (or both).

All the expressed proteins had SDS-PAGE mobilities that corresponded to those predicted from their sequence. Shadowed preparations showed distinctive rod-like mol­ecular profiles which generally resembled native material (Aebi et al. 1986). When the C-terminal domain was retained, shadowed particles had a prominent globular domain at one end that frequently could be observed to bifurcate to give the typical two-headed appearance observed on native lamins (Aebi et al. 1986). In material that lacked the C-terminal, non-helical domain, these globular domains were absent. Suberimidate crosslinking increased the Mr fourfold, indicating that all the expressed fragments aggregated into four-chain ‘tetramer’ units analogous to those observed with other intermediate filament proteins.

The solubility of lamins A and C decreased by about an order of magnitude when the salt concentration was decreased from 0.4 to 0.1m and solubility also decreased when the pH was lowered. Fragments in which either the N- or C-terminal non-helical domain had been deleted were much more soluble. Lamins A and C both formed well-ordered paracrystals with an axial repeat of 22 nm, similar to those observed by Aebi et al. (1986) for a mixture of rat lamins A and C. Lamin C with its N-terminal domain deleted formed similar paracrystals. We were unable to form paracrystals from lamins lacking the C-terminal domain, which always yielded 10-nm fila­ments that closely resembled cytoplasmic IFs. The lamin rod domain formed well ordered paracrystals with a 45 nm axial repeat in which there were two 10 nm wide light bands separated by about 12 and 13 nm alternately.

Nuclear pores and nucleocytoplasmic transport

Nuclear pore complexes mediate both active and passive exchange of material between nucleus and cytcjplasm and are roughly cylindrical structures with eight internal

80 M. Stewart et al.

Fig. 3. Schematic illustration of the components of nuclear pores. Nucleoplasmic and cytoplasmic rings are linked to a central cylinder. These components can be seen most easily in disrupted nuclear envelope preparations such as that in Fig. 4. Reproduced from Stewart et al. (1990).

Fig. 4. Shadowed preparation of a disrupted Xenopus nuclear envelope showing the rings and other pore components, together with some islands of pores shed from the envelope.Bar, 0.5 f.on.

spoke-like units arranged between cytoplasmic and nu­cleoplasmic rings (see Akey, 1989; Reichelt et al. 1990; Unwin and Milligan, 1982). Fig. 3 illustrates the major structural components of nuclear pore complexes, which were most easily seen in partially disrupted envelopes produced by low ionic strength (Fig. 4). Here islands of

pores, often still connected by pore-connecting fibrils, were seen frequently, together with pores in various degrees of disintegration.

We used nucleoplasmin to investigate transport. Colloi­dal gold conjugated with nucleoplasmin (Au-NP) micro­injected into Xenopus oocytes accumulates in the nucleus over a period of hours, and sections of embedded material show many pores with colloidal gold markers located on their axis (Felhderr et al. 1984). When envelopes from nucleoplasmin-microinjected oocytes were disrupted, a substantial number of rings had centrally-located gold particles. When Au-NP was applied to envelopes after isolation, a clear pattern of decoration was not seen. This was consistent with studies that have shown that additional factors from the cell cytoplasm are required for nuclear transport to be effected in vitro (Newport and Forbes, 1987). When BSA-conjugated gold was micro­injected instead of nucleoplasmin, the density of gold particles attached to isolated nuclear envelopes was greatly reduced.

The observation of nucleoplasmin labelling of rings indicated that at least part of the active transport mechanism resided in this morphological component. However, we think it is likely that additional components located in the central body of the pores are also involved in active transport. The mechanism of transport through nuclear pores is not understood in detail and probably involves a number of steps and interactions with different pore components. Substantial structural changes appear to accompany transport (Akey and Goldfarb, 1989) and molecular motors may well be involved in this process. Molecular motors may also participate in the translo­cation of material, which has to move about 50 nm to traverse the entire nuclear envelope. Although clearly more work will be required to establish in detail the steps involved in nucleocytoplasmic transport, the labelling of rings we have observed here provides evidence for the location of some components of the transport machinery in the ring components at the pore periphery.

We thank Patrick Sadler for artwork. RDM was supported by the Alberta Heritage Foundation for Medical Research.

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