Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation.

DNA (from bacteriophage lambda or Xenopus) is assembled into nucleus-like structures when mixed with an extract from Xenopus eggs. Electron microscopy shows that these in vitro-reconstituted nuclei possess complete double membranes; some, but not all, nuclei have pore complexes. Extracts depleted of their endogenous ATP (by addition of ATPases) cannot assemble nuclear envelopes visible by phase-contrast microscopy. Once synthetic nuclei are assembled, however, they are stable when ATP is subsequently depleted, although their chromatin becomes condensed. About one-fourth of the nuclei assembled in vitro from lambda DNA accumulate nuclear proteins such as nucleoplasmin. ATP depletion blocks nucleoplasmin accumulation both in vitro, in pre-assembled synthetic nuclei, and in vivo, in the nucleus of microinjected oocytes. However, nucleoplasmin previously accumulated by reconstituted nuclei or by the germinal vesicle in microinjected oocytes is retained after ATP depletion.     

Nucleocytoplasmic protein traffic in single mammalian cells studied by fluorescence microphotolysis

Fluorescence microphotolysis was employed to measure in single living cells the kinetics of nucleocytoplasmic transport and the coefficients of intracellular diffusional mobility for the nuclear non-chromosomal protein nucleoplasmin. Nucleoplasmin was isolated from Xenopus ovary and labeled fluorescently. By injection into Xenopus oocytes it was ascertained that fluorescent labeling did not interfere with normal nuclear accumulation. Upon injection into the cytoplasm of various mammalian cell types nucleoplasmin was rapidly taken up by the nucleus. In rat hepatoma cells the half-time of nuclear uptake was approx. 5 min at 37 degrees C; the nucleocytoplasmic equilibrium concentration ratio had a maximum of 6.5 +/- 1.4 and depended on the injected amount. Upon co-injection of ATPases or reduction of temperature to 10 degrees C a nucleocytoplasmic equilization but no nuclear accumulation was observed. Equilization was fast (time constant 65 s at 23 degrees C), similar to that of 10-kDa dextran permeating the nuclear envelope by simple diffusion through functional pores. Nucleoplasmin (160 kDa), however, is too large to permeate passively the nuclear envelope, which is apparent from the fact that its tryptic 'core' fragment (100 kDa) could not permeate the nuclear envelope. On the other hand, a large fluorescent protein, phycoerythrin (240 kDa), was targeted to the nucleus by conjugation with nucleoplasmin. In the nucleus-to-cytoplasm direction the nuclear envelope was completely impermeable to nucleoplasmin, independently of temperature or ATP depletion. Nucleoplasmin, its core fragment, phycoerythrin and the phycoerythrin-nucleoplasmin conjugate were mobile in both cytoplasm and nucleus.     

Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin

At fertilization, sperm chromatin decondenses in two stages, which can be mimicked in extracts of Xenopus eggs. Rapid, limited decondensation is followed by slower, membrane-dependent decondensation and swelling. Nucleoplasmin, an acidic nuclear protein, occurs at high concentration in Xenopus eggs and has a histone-binding role in nucleosome assembly. Immunodepleting nucleoplasmin from egg extracts inhibits the initial rapid stage of sperm decondensation, and also the decondensation of myeloma nuclei, relative to controls of mock depletion and TFIIIA depletion. Readdition of purified nucleoplasmin recues depleted extracts. A physiological concentration of purified nucleoplasmin alone decondenses both sperm and myeloma nuclei. We conclude that nucleoplasmin is both necessary and sufficient for the first stage of sperm decondensation in Xenopus eggs.     

Heterogeneity in nuclear transport does not affect the timing of DNA synthesis in quiescent mammalian nuclei induced to replicate in Xenopus egg extracts

Intact G0 nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in Xenopus egg extracts, despite exposure to the same concentration of replication factors. This indicates that individual nuclei differ in their ability to respond to the inducers of DNA replication. Since the induction of DNA synthesis requires the accumulation of replication factors by active nuclear transport, any variation in the rate of transport among nuclei could contribute to the variability of DNA replication. Using the naturally fluorescent protein allophycocyanin (APC) coupled with the nuclear localization sequence (NLS) of SV40 T antigen, as a marker of nuclear uptake, we show here that individual G0 nuclei differ in their rate of transport over a range of more than 20-fold. Surprisingly, this variation has no direct influence on the timing or extent of DNA synthesis. Similar results were obtained by monitoring the uptake of nucleoplasmin, a nuclear protein present at high levels in egg extracts. These experiments show that the initiation of DNA synthesis is not driven merely by the accumulation of replication factors to some threshold concentration. Instead, some other explanation is needed to account for the timing of initiation.     

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.     

DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.     

Mutation of the yeast epsilon-COP gene ANU2 causes abnormal nuclear morphology and defects in intracellular vesicular transport.

Previously we reported an original method of visualizing the shape of yeast nuclei by the expression of green fluorescent protein (GFP)-tagged Xenopus nucleoplasmin in Saccharomyces cerevisiae. To identify components that determine nuclear structure, we searched for mutants exhibiting abnormal nuclear morphology from a collection of temperature-sensitive yeast strains expressing GFP-tagged nucleoplasmin. Four anu mutant strains (anu1-1, 2-1, 3-1 and 4-1; ANU=abnormal nuclear morphology) that exhibited strikingly different nuclear morphologies at the restrictive temperature as compared to the wild-type were isolated. The nuclei of these mutants were irregularly shaped and often consisted of multiple lobes. ANU1, 3 and 4 were found to encode known factors Sec24p, Sec13p and Sec18p, respectively, all of which are involved in the formation or fusion of intracellular membrane vesicles of protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus. On the other hand, ANU2 was not well characterized. Disruption of ANU2 (delta anu2) was not lethal but conferred temperature-sensitivity for growth. Electron microscopic analysis of anu2-1 cells revealed not only the abnormal nuclear morphology but also excessive accumulation of ER membranes. In addition, both anu2-1 and delta anu2 cells were defective in protein transport between the ER and the Golgi, suggesting that Anu2p has an important role in vesicular transport in the early secretory pathway. Here we show that ANU2 encodes a 34 kDa polypeptide, which shares a 20% sequence identity with the mammalian epsilon-COP. Our results suggest that Anu2p is the yeast homologue of mammalian epsilon-COP and the abrupt accumulation of the ER membrane caused by a blockage of the early protein transport pathway leads to alteration of nuclear morphology of the budding yeast cells.     

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.     

Movement of a karyophilic protein through the nuclear pores of oocytes

It has recently been shown that large karyophilic proteins are transported across the nuclear envelope in amphibian oocytes. In consideration of this, the present experiments were performed to identify the specific sites within the envelope through which transport occurs and determine if molecular size is a limiting factor in the transport process. The following experimental procedure was employed: Colloidal gold particles, varying in size from approximately 20 to 170 A in diameter were coated with nucleoplasmin, a 165,000-mol-wt karyophilic protein, which is known to be transported through the envelope. The coated gold particles were microinjected into the cytoplasm of Xenopus oocytes, and the cells were fixed 15 min and 1 h later. The intracellular localization of the gold was then determined with the electron microscope. It was found that nucleoplasmin-coated particles readily enter the nucleus. On the basis of the distribution of the particles associated with the envelope, we concluded that transport occurs through the nuclear pores. Furthermore, the size distributions of the gold particles present in the nucleus and cytoplasm were not significantly different, indicating that the envelope does not discriminate among particles with diameters ranging from 50 to 200 A (the dimensions including the nucleoplasmin coat). Colloidal gold coated with trypsin-digested nucleoplasmin (which lacks the polypeptide domain required for transport) or exogenous polyvinylpyrrolidone were largely excluded from the nucleus and showed no evidence of transport.     

Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.     

The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo

The GTPase Ran is known to regulate transport of proteins across the nuclear envelope. Recently, Ran has been shown to promote microtubule polymerization and spindle assembly around chromatin in Xenopus mitotic extracts and to stimulate nuclear envelope assembly in Xenopus or HeLa cell extracts. However, these in vitro findings have not been tested in living cells and do not necessarily describe the generalized model of Ran functions. Here we present several lines of evidence that Ran is indispensable for correct chromosome positioning and nuclear envelope assembly in C. elegans. Embryos deprived of Ran by RNAi showed metaphase chromosome misalignment and aberrant chromosome segregation, while astral microtubules seemed unaffected. Depletion of RCC1 or RanGAP by RNAi resulted in essentially the same defects. The immunofluorescent staining showed that Ran localizes to kinetochore regions of metaphase and anaphase chromosomes, suggesting the role of Ran in linking chromosomes to kinetochore microtubules. Ran was shown to localize to the nuclear envelope at telophase and during interphase in early embryos, and the depletion of Ran resulted in failure of nuclear envelope assembly. Thus, Ran is crucially involved in chromosome positioning and nuclear envelope assembly in C. elegans.     

The function of the nuclear envelope in nuclear protein accumulation

The mechanism by which proteins accumulate in the cell nucleus is not yet known. Two alternative mechanisms are discussed here: (a) selective unidirectional entry of karyophilic proteins through the nuclear pores, and (b) free diffusion of all proteins through the nuclear pores and specific binding of nuclear proteins to nondiffusible components of the nucleoplasm. We present experiments designed to distinguish between these alternatives. After mechanical injury of the Xenopus oocyte nuclear envelope, nuclear proteins were detected in the cytoplasm by immunohistochemical methods. In a second approach, nuclei from X. borealis oocytes were isolated under oil, the nuclear envelopes were removed, and the pure nucleoplasm was injected into the vegetal pole of X. laevis oocytes. With immunohistochemical methods, it was found that each of five nuclear proteins rapidly diffuses out of the injected nucleoplasm into the surrounding cytoplasm. The subsequent transport and accumulation in the intact host nucleus could be shown for the nuclear protein N1 with the aid of a species-specific mAb that reacts only with X. borealis N1. Purified and iodinated nucleoplasmin was injected into the cytoplasm of Xenopus oocytes and its uptake into the nucleus was studied by biochemical methods.     

A lamin-independent pathway for nuclear envelope assembly

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.     

Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIPalpha but not importin alpha.

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism. RPA consists of three subunits (70, 33 and 14 kDa). We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA. XRIPalpha homologues are found in human and in Drosophila but not in yeast. XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta. We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA. Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate. RPA import can be restored by addition of recombinant XRIPalpha. Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA. GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro. Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.     

Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin

The lectin wheat germ agglutinin (WGA), which has been reported to inhibit nuclear protein uptake in vitro by isolated nuclei (Finlay et al. (1987) J. Cell Biol. 104, 189), also blocks, on microinjection into living cells, the migration of proteins into the cell nucleus. Radioactively labeled nuclear proteins were injected into the cytoplasm of Xenopus oocytes and their reentry into the nucleus was analyzed in the presence or absence of WGA by two-dimensional gel electrophoresis. In another set of experiments, fluorescently labeled nucleoplasmin was injected, alone or together with WGA, into the cytoplasm of rat hepatoma cells, and its nucleocytoplasmic distribution was studied by quantitative laser fluorescence microscopy. The results indicate that WGA inhibits the uptake of karyophilic proteins in general, independent of their sizes. Since the nucleocytoplasmic flux of a dextran with Mr 10,000 was not affected it can be excluded that WGA acts by a general blockade or constriction of the functional pore channel. At reduced WGA concentrations, the rate but not the final extent of nuclear protein accumulation was decreased. These findings support the concept that the O-glycosidically bound carbohydrates of certain nuclear pore complex proteins are exposed to the pore interior and that these regions are probably involved in nucleocytoplasmic translocation processes.     

Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase- arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.     

Metaphase protein phosphorylation in Xenopus laevis eggs.

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.     

Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes

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 cytoplasmic 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%), 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.