Active Nucleocytoplasmic Transport Research Articles

Reconstitution of nuclear envelope subdomain formation on mitotic chromosomes in semi-intact cells.

In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.

Balance of osmotic pressures determines the nuclear-to-cytoplasmic volume ratio of the cell

The volume of the cell nucleus varies across cell types and species and is commonly thought to be determined by the size of the genome and degree of chromatin compaction. However, this notion has been challenged over the years by much experimental evidence. Here, we consider the physical condition of mechanical force balance as a determining condition of the nuclear volume and use quantitative, order-of-magnitude analysis to estimate the forces from different sources of nuclear and cytoplasmic pressure. Our estimates suggest that the dominant pressure within the nucleus and cytoplasm of nonstriated muscle cells originates from the osmotic pressure of proteins and RNA molecules that are localized to the nucleus or cytoplasm by out-of-equilibrium, active nucleocytoplasmic transport rather than from chromatin or its associated ions. This motivates us to formulate a physical model for the ratio of the cell and nuclear volumes in which osmotic pressures of localized proteins determine the relative volumes. In accordance with unexplained observations that are a century old, our model predicts that the ratio of the cell and nuclear volumes is a constant, robust to a wide variety of biochemical and biophysical manipulations, and is changed only if gene expression or nucleocytoplasmic transport is modulated.

Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration.

Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to design cell lines stably expressing fluorophore proteins shuttling between the nucleus and cytoplasm by karyopherins of choice. To validate this protocol, we measured an ALS-associated nucleocytoplasmic transport pathway in the presence of the disease-associated peptide poly-PR. This technique allows measuring a particular active nucleocytoplasmic transport pathway in intact cells in a neurodegenerative disease-associated context. Moreover, these experiments can be performed without the need for expensive equipment and have the potential to be upscaled for high-throughput screening purposes.

C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport

Repeat expansions in the C9orf72 gene cause amyotrophic lateral sclerosis and frontotemporal dementia characterized by dipeptide-repeat protein (DPR) inclusions. The toxicity associated with two of these DPRs, poly-GR and poly-PR, has been associated with nucleocytoplasmic transport. To investigate the causal role of poly-GR or poly-PR on active nucleocytoplasmic transport, we measured nuclear import and export in poly-GR or poly-PR expressing Hela cells, neuronal-like SH-SY5Y cells and iPSC-derived motor neurons. Our data strongly indicate that poly-GR and poly-PR do not directly impede active nucleocytoplasmic transport.

Landscape of nuclear transport receptor cargospecificity.

Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo–NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.

Inhibition of active nuclear transport is an intrinsic trigger of programmed cell death in trypanosomatids

The link between nucleocytoplasmic transport and apoptosis remains controversial. Nucleocytoplasmic exchange of molecules seems indeed essential for the initiation and execution of the apoptotic programme; but inhibition of nuclear transport factors may also represent a powerful apoptotic trigger. The GTPase Ran (together with its partners), first discovered to be essential in nucleocytoplasmic transport, has multiple key functions in cell biology, and particularly in spindle assembly, kinetochore function and nuclear envelope assembly. Among the Ran partners studied, NTF2 appears to be solely involved in nucleocytoplasmic transport. Here, we localised Ran and several of its partners, RanBP1, CAS and NTF2, at the nuclear membrane in the trypanosomatid Leishmania major. Remarkably, these proteins fused to GFP decorated a perinuclear 'collar' of about 15 dots, colocalising at nuclear pore complexes with the homologue of nucleoporin Sec13. In the other trypanosomatid Trypanosoma brucei, RNAi knockdown of the expression of the corresponding genes resulted in an apoptosis-like phenomenon. These phenotypes show that Ran and its partners have a key function in trypanosomatids like they have in mammals. Our data, notably those about TbNTF2 RNAi, support the idea that active nucleocytoplasmic transport is not essential for the initiation and execution of apoptosis, and, rather, the impairment of this transport constitutes an intrinsic signal for triggering PCD.

The pheromone-induced nuclear accumulation of the Fus3 MAPK in yeast depends on its phosphorylation state and on Dig1 and Dig2.

BackgroundLike mammalian MAP kinases, the mating-specific Fus3 MAPK of yeast accumulates in the nuclei of stimulated cells. Because Fus3 does not appear to be subjected to active nucleo-cytoplasmic transport, it is not clear how its activation by mating pheromone effects the observed change in its localization. One possibility is that the activation of Fus3 changes its affinity for nuclear and cytoplasmic tethers.ResultsDig1, Dig2, and Ste12 are nuclear proteins that interact with Fus3. We found that the pheromone-induced nuclear accumulation of a Fus3-GFP reporter is reduced in cells lacking Dig1 or Dig2, whereas Fus3T180AY182A-GFP localization was unaffected by the absence of these proteins. This suggests that Dig1 and Dig2 contribute to the retention of phosphorylated Fus3 in the nucleus. Moreover, overexpression of Ste12 caused the hyper-accumulation of Fus3-GFP (but not Fus3T180AY182A-GFP) in the nuclei of pheromone-treated cells, suggesting that Ste12 also plays a role in the nuclear retention of phosphorylated Fus3, either by directly interacting with it or by transcribing genes whose protein products are Fus3 tethers. We have previously reported that overexpression of the Msg5 phosphatase inhibits the nuclear localization of Fus3. Here we show that this effect depends on the phosphatase activity of Msg5, and provide evidence that both nuclear and cytoplasmic Msg5 can affect the localization of Fus3.ConclusionOur data are consistent with a model in which the pheromone-induced phosphorylation of Fus3 increases its affinity for nuclear tethers, which contributes to its nuclear accumulation and is antagonized by Msg5.

A picornavirus protein interacts with Ran-GTPase and disrupts nucleocytoplasmic transport.

Active nucleocytoplasmic transport of protein and RNA in eukaryotes depends on the Ran-GTPase system to regulate cargo-receptor interactions. Several viruses, including the RNA picornaviruses, encode factors that alter nuclear transport with the aim of suppressing synthesis of antiviral factors and promoting viral replication. Picornaviruses in the cardiovirus genus express a unique 67-aa Leader protein (L), known to alter the subcellular distribution of IFN regulatory proteins targeted to the nucleus. We report here that L binds directly to Ran and blocks nuclear export of new mRNAs. In Xenopus egg extracts, recombinant L also inhibits mitotic spindle assembly, a RanGTP function crucial to cell-cycle progression. We propose that L inhibits nucleocytoplasmic transport during infection by disrupting the RanGDP/GTP gradient. This inhibition triggers an efflux of nuclear proteins necessary for viral replication and causes IFN suppression. To our knowledge, L is the first viral picornaviral protein to interact directly with Ran and modulate the Ran-dependent nucleocytoplasmic pathway.

Nucleocytoplasmic Traffic Disorder Induced by Cardioviruses

Some picornaviruses, for example, poliovirus, increase bidirectional permeability of the nuclear envelope and suppress active nucleocytoplasmic transport. These activities require the viral protease 2A(pro). Here, we studied nucleocytoplasmic traffic in cells infected with encephalomyocarditis virus (EMCV; a cardiovirus), which lacks the poliovirus 2A(pro)-related protein. EMCV similarly enhanced bidirectional nucleocytoplasmic traffic. By using the fluorescent "Timer" protein, which contains a nuclear localization signal, we showed that the cytoplasmic accumulation of nuclear proteins in infected cells was largely due to the nuclear efflux of "old" proteins rather than impaired active nuclear import of newly synthesized molecules. The nuclear envelope of digitonin-treated EMCV-infected cells permitted rapid efflux of a nuclear marker protein. Inhibitors of poliovirus 2A(pro) did not prevent the EMCV-induced efflux. Extracts from EMCV-infected cells and products of in vitro translation of viral RNAs contained an activity increasing permeability of the nuclear envelope of uninfected cells. This activity depended on the expression of the viral leader protein. Mutations disrupting the zinc finger motif of this protein abolished its efflux-inducing ability. Inactivation of the L protein phosphorylation site (Thr47-->Ala) resulted in a delayed efflux, while a phosphorylation-mimicking (Thr47-->Asp) replacement did not significantly impair the efflux-inducing ability. Such activity of extracts from EMCV-infected cells was suppressed by the protein kinase inhibitor staurosporine. As evidenced by electron microscopy, cardiovirus infection resulted in alteration of the nuclear pores, but it did not trigger degradation of the nucleoporins known to be degraded in the poliovirus-infected cells. Thus, two groups of picornaviruses, enteroviruses and cardioviruses, similarly alter the nucleocytoplasmic traffic but achieve this by strikingly different mechanisms.

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection.

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.

Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast.

CRM1, an evolutionarily conserved protein, was shown to be a receptor for leucine-rich nuclear export signal (NES)-dependent protein transport. In lower eukaryotes CRM1 is reported to be required for the export of mRNA, however, involvement of the NES-dependent transport pathway in mRNA export in higher eukaryotes has not been established. We have found that treatment of mammalian cells with leptomycin B (LMB), a specific inhibitor of CRM1, induces the nuclear accumulation of endogenous mRNA, probably due to the inhibition of its export. In fission yeasts, the nuclear accumulation of mRNA also occurred in cells treated with LMB or in a temperature-sensitive crm1 mutant at a restrictive temperature. A synthetic mRNA that was injected into the nucleus of mammalian cultured cells was exported from the nucleus within 5 h. This export was inhibited by both wheat germ agglutinin and a temperature of 4 degrees C. Importantly, this mRNA export was inhibited by LMB or by an excess amount of the NES peptide-conjugates. LMB treatment, on the other hand, rapidly induced the nuclear entry of RanBP1, a factor involved in the active nucleocytoplasmic transport, although the treatment did not interfere with a nuclear localization signal-dependent transport system within 7 h. These results suggest that CRM1 is involved in mRNA export in eukaryotic cells.

Nuclear Targeting in Plants

The nucleus is the site of highly active two-way macromolecular traffic between the nucleoplasm and the cytoplasm. Nuclear import and export must be highly specific processes because the content of the nucleus is distinguished compared to the cytoplasm. This is rather amazing, considering that the nuclear envelope dissolves during mitosis of animal and plant cells, necessitating reassembly of all nuclear components and reentry of nuclear proteins into the nucleus. In addition, many basic cellular processes, such as gene transcription and cell division, clearly require proper nuclear localization of regulatory and housekeeping proteins. Furthermore, experimental evidence indicates the existence of receptors and other cellular factors that mediate nuclear transport. Nuclear transport, although extensively studied in animal and lower eukaryotic systems (3, 20), has only recently been addressed in higher plants (1, 2, 5, 7, 8, 15, 24-26). This attention to protein and nucleic acid transport into the nucleus in plants was stimulated not only by a need to understand basic cell biology, but also by the desire to modulate or regulate the expression of genes via genetic engineering. The nucleus (Fig. 1) is surrounded by the nuclear envelope, which includes the inner and outer nuclear membranes and, between them, the perinuclear space. The outer nuclear membrane is contiguous with the rough ER, and its outer surface is generally studded with ribosomes. Underlying the inner nuclear membrane is the nuclear lamina, which is composed of a network of filamentous proteins. Unlike other organelles, the nuclear envelope contains NPC2, the major sites of bidirectional protein and nucleic acid traffic. Though understood on a structural level, knowledge of the biochemistry behind the NPC is limited. Only a few proteins associated with the NPC in animal systems have been identified (3); however, their actual involvement in nuclear import has not been demonstrated. Structurally, the NPC traverses the double membrane, bringing the lipid bilayers of the inner and outer membranes together around the margins of each pore. The NPC consists of eight large protein granules arranged in a circle with a large central granule or transporter that is thought to control active nucleocytoplasmic transport of large molecules (6). In addition, eight peripheral channels of a smaller diameter would allow a passive exchange of small molecules. Therefore, transport into the nucleus is fundamentally different from that into other organelles, where transported proteins pass directly through the membrane. Although most nuclear proteins carry information that simply directs them through the nuclear pore, others must continue their journey to a final subnuclear compartment (12). With regard to what is specifically responsible for proper localization of a protein inside the nucleus, very little is known. Results obtained from yeast and animal systems have led investigators to suggest some basic rules for nuclear protein targeting (3). Proteins smaller than 40 to 60 kD are thought to diffuse through the nuclear pore, though no physiologically relevant macromolecule has been shown to traverse the nuclear pore by diffusion. However, it has been shown that many proteins require at least one NLS. Two criteria are used to define these NLSs: (a) an NLS is sufficient to redirect a cytoplasmic protein to the nucleus, and (b) an NLS is necessary for directing a nuclear protein to the nucleus. However, in many cases the NLSs are not adequately characterized by both criteria. Known NLSs can be grouped into several categories (3). First, SV40-like NLSs contain a short stretch of basic amino acids (PKKKRKV). Second, MAT a2-like NLSs consist of short hydrophobic regions that contain one or more basic amino acids (KIPIK). Third, bipartite NLSs are usually a combination of two regions of basic amino acids separated by a spacer of more than four residues (SPPKAVKRPAATKKAGQAKKKKLDKEDES) (17). The first region has two basic amino acids and the second has at least three out of five basic residues. According to current speculation, the two basic regions cooperate in binding, whereas the spacer may facilitate their cooperative interaction. In addition, there are some NLSs that do not fit any of the above-mentioned groups and that are characteristic of some viral nuclear proteins (for example, the NLS of influenza ribonucleoprotein is AAFEDLRVRS). Although NLSs have been grouped into these categories, a consensus as to sequence, either between or within groups, has not been reached. This article will focus on recent advances in our understanding of NLSs recognized in plants. For earlier reviews on nuclear targeting in animal and yeast cells, see Garcia-Bustos et al. (3) and Silver (20).

Architecture and design of the nuclear pore complex

A three-dimensional analysis of the nuclear pore complex reveals the underlying, highly symmetric framework of this supramolecular assembly, how it is anchored in the nuclear membrane, and how it is built from many distinct, interconnected subunits. The arrangement of the subunits within the membrane pore creates a large central channel, through which active nucleocytoplasmic transport is known to occur, and eight smaller peripheral channels that are probable routes for passive diffusion of ions and small molecules.

Observations on the Nissl's substance, cytoplasmic filaments and the nuclear membrane of spinal ganglion cells.

Thin sections of spinal ganglia were examined with the electron microscope. The axon runs in the cytoplasm of the capsule cells which are separated from the nerve cell by a lipid sheath. Nissl’s substance occurs as discrete aggregates in ‘light’ cells, and dispersed throughout the cytoplasm in ‘dark’ cells. The unit structures of Nissl’s substance are granules of size range 50 to 200 Å. Both nerve-cell cytoplasm and axoplasm have small mitochondria and a background network of fine filaments of 20 to 50 Å diameter. The nuclear membrane consists of a multilayered structure. This membrane has distributed through it an arrangement of nodes, 850 Å in diameter. It is suggested that the nuclear membrane may undergo phasic alteration in structure and that the nodes are characteristically present during active nucleocytoplasmic transport.