The nuclear pore complex: nucleocytoplasmic transport and beyond

Over the past two years, it has become evident that there is an unexpected link between nuclear pore complex structure and dynamics, nucleocytoplasmic transport and chromosome segregation. In addition, a tomographic three-dimensional reconstruction of native nuclear pore complexes preserved in thick amorphous ice has unveiled a number of new structural features of this supramolecular machine. These data, together with some of the elementary physical principles that underlie nucleocytoplasmic transport, will be discussed in this review.     

Modularity within the architecture of the nuclear pore complex

Transport between nucleus and cytoplasm is exclusively mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. The NPC is an enormously elaborate protein assembly, reflecting its ability to multitask by simultaneously regulating the trafficking of a diverse spectrum of substrates, ranging from microRNAs to assembled ribosomal subunits. The complexity and sheer size of the NPC have hampered efforts to elucidate its molecular architecture. However, recent studies using a battery of complementary techniques have significantly enhanced our understanding of the NPC structure. The picture of a highly dynamic and modular machine is emerging.     

Environmental change and rates of evolution: the phylogeographic pattern within the hartebeest complex as related to climatic variation

Global climate fluctuated considerably throughout the Pliocene-Pleistocene period, influencing the evolutionary history of a wide array of species. Using the phylogeographic patterns within the hartebeest (Alcelaphus buselaphus (Pallas, 1766)) complex, we evaluated the evolutionary consequences of such environmental change for a typical large mammal ranging on the African savannah. Our results, as generated from two mitochondrial DNA markers (the D-loop and cytochrome b), suggest an origin of the hartebeest in eastern Africa from where the species has colonized other parts of the continent. Phylogenetic analyses revealed an early diversification into southern and northern hartebeest lineages, an event that may be related to the formation of the Rift Valley lakes. The northern lineage has further diverged into eastern and western lineages, most probably as a result of the expanding central African rainforest belt and subsequent contraction of savannah habitats during a period of global warming. The diversification events appear to have coincided with major climatic changes and are highly correlated in time. These observations strongly suggest that large-scale climatic fluctuations have been a major determinant for the species' evolutionary history and that hartebeest evolution has mainly taken place in isolated yet environmentally favourable refugia during periods of global warming. Indications of sudden population expansion for two putative ancestral hartebeest populations provide further support for a refugia-based explanation of the diversification events. Reciprocal monophyly between southern and northern lineages may suggest that reproductive barriers exist and that the hartebeest complex comprises two different species.     

Regulated transport as a mechanism for pattern generation: Capabilities for phyllotaxis and beyond

Large-scale pattern formation is a frequently occurring phenomenon in biological organisms, and several local interaction rules for generating such patterns have been suggested. A mechanism driven by feedback between the plant hormone auxin and its polarly localized transport mediator PINFORMED1 has been proposed as a model for phyllotactic patterns in plants. It has been shown to agree with current biological experiments at a molecular level as well as with respect to the resulting patterns. We present a thorough investigation of variants of models based on auxin-regulated polarized transport and use analytical and numerical tools to derive requirements for these models to drive spontaneous pattern formation. We find that auxin concentrations in neighboring cells can feed back either on exocytosis or endocytosis and still produce patterns. In agreement with mutant experiments, the active cellular efflux is shown to be more important for pattern capabilities as compared to active influx. We also find that the feedback must originate from neighboring cells rather than from neighboring walls and that intracellular competition for the transport mediator is required for patterning. The importance of model parameters is investigated, especially regarding robustness to perturbations of experimentally estimated parameter values. Finally, the regulated transport mechanism is shown to be able to generate Turing patterns of various types.     

Plant defence as a complex and changing phenotype throughout ontogeny

Background and Aims Ontogenetic changes in anti-herbivore defences are common and result from variation in resource availability and herbivore damage throughout plant development. However, little is known about the simultaneous changes of multiple defences across the entire development of plants, and how such changes affect plant damage in the field. The aim of this study was to assess if changes in the major types of plant resistance and tolerance can explain natural herbivore damage throughout plant ontogeny.Methods An assessment was made of how six defensive traits, including physical, chemical and biotic resistance, simultaneously change across the major transitions of plant development, from seedlings to reproductive stages of Turnera velutina growing in the greenhouse. In addition, an experiment was performed to assess how plant tolerance to artificial damage to leaves changed throughout ontogeny. Finally, leaf damage by herbivores was evaluated in a natural population.Key Results The observed ontogenetic trajectories of all defences were significantly different, sometimes showing opposite directions of change. Whereas trichome density, leaf toughness, extrafloral nectary abundance and nectar production increased, hydrogen cyanide and compensatory responses decreased throughout plant development, from seedlings to reproductive plants. Only water content was higher at the intermediate juvenile ontogenetic stages. Surveys in a natural population over 3 years showed that herbivores consumed more tissue from juvenile plants than from younger seedlings or older reproductive plants. This is consistent with the fact that juvenile plants were the least defended stage.Conclusions The results suggest that defensive trajectories are a mixed result of predictions by the Optimal Defence Theory and the Growth–Differentiation Balance Hypothesis. The study emphasizes the importance of incorporating multiple defences and plant ontogeny into further studies for a more comprehensive understanding of plant defence evolution.     

Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export

Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.     

Association of purine asymmetry,strand-biased gene distribution and PolC within Firmicutes and beyond: a new appraisal

Background

The Firmicutes often possess three conspicuous genome features: marked Purine Asymmetry (PAS) across two strands of replication, Strand-biased Gene Distribution (SGD) and presence of two isoforms of DNA polymerase III alpha subunit, PolC and DnaE. Despite considerable research efforts, it is not clear whether the co-existence of PAS, PolC and/or SGD is an essential and exclusive characteristic of the Firmicutes. The nature of correlations, if any, between these three features within and beyond the lineages of Firmicutes has also remained elusive. The present study has been designed to address these issues.

Results

A large-scale analysis of diverse bacterial genomes indicates that PAS, PolC and SGD are neither essential nor exclusive features of the Firmicutes. PolC prevails in four bacterial phyla: Firmicutes, Fusobacteria, Tenericutes and Thermotogae, while PAS occurs only in subsets of Firmicutes, Fusobacteria and Tenericutes. There are five major compositional trends in Firmicutes: (I) an explicit PAS or G + A-dominance along the entire leading strand (II) only G-dominance in the leading strand, (III) alternate stretches of purine-rich and pyrimidine-rich sequences, (IV) G + T dominance along the leading strand, and (V) no identifiable patterns in base usage. Presence of strong SGD has been observed not only in genomes having PAS, but also in genomes with G-dominance along their leading strands – an observation that defies the notion of co-occurrence of PAS and SGD in Firmicutes. The PolC-containing non-Firmicutes organisms often have alternate stretches of R-dominant and Y-dominant sequences along their genomes and most of them show relatively weak, but significant SGD. Firmicutes having G + A-dominance or G-dominance along LeS usually show distinct base usage patterns in three codon sites of genes. Probable molecular mechanisms that might have incurred such usage patterns have been proposed.

Conclusion

Co-occurrence of PAS, strong SGD and PolC should not be regarded as a genome signature of the Firmicutes. Presence of PAS in a species may warrant PolC and strong SGD, but PolC and/or SGD not necessarily implies PAS.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-430) contains supplementary material, which is available to authorized users.     

Arthropods within the woody element of hedgerows and their distribution pattern

1 The diversity and abundance of arthropods within hedgerows was investigated using insecticide fogging. In total, 13 390 arthropods were collected from 181 m³ of hedge (2% of the total volume). The taxonomic diversity of the total sample included 51 families in 13 orders, all within the phylum Arthropoda. Five orders accounted for 90% of all arthropods: Araneae, Coleoptera, Diptera, Hemiptera and Hymenoptera. The predators were the dominant functional group accounting for 40% of the total sample. 2 The linear distribution of hedgerow arthropod assemblages was investigated by sampling arthropods in 13 hedges at seven equidistant points along each hedge. Abundance of most arthropod taxa and the four functional groups (predators, parasitoids, herbivores and scavengers) consistently showed a clumped distribution along hedges, with high numbers at both ends and in the middle section. Hedge ends were defined either as a node, where the hedge intersected with another hedge, or as a gateway prior to an adjacent landscape feature such as a wood. Aggregation of arthropods at the nodes may reflect preferable microclimatic conditions at hedgerow junctions, whereas aggregation at a gap suggests the gap acted as a barrier to movement. The aggregation at the centre remains unexplained but may be a manifestation of the movement of individual arthropods along the hedgerow. 3 The great abundance and diversity of arthropods found in the present study emphasizes the status of hedges as one of the most important noncrop habitats on farmland. The arthropods that they contain may act as food for other farmland species, aid pest control and contribute to crop pollination.     

Caregiving within and beyond the family is associated with lower mortality for the caregiver: A prospective study

Grandparenting has been proposed as an ultimate evolutionary mechanism that has contributed to the increase in human life expectancy (see the grandmother hypothesis). The neural and hormonal system – originally rooted in parenting and thus grandparenting – that is activated in the process of caregiving has been suggested as a potential proximate mechanism that promotes engagement in prosocial behavior towards kin and non-kin alike. Evidence and theory suggest that activating this caregiving system positively impacts health and may reduce the mortality of the helper. Although some studies have found grandparental care to have beneficial effects on grandparents' health outcomes, most studies have focused on the detrimental health consequences of providing custodial care for grandchildren. Little is known about how non-custodial grandparental and other forms of caregiving relate to mortality hazards for the care provider. Using an evolutionary framework, we examined whether caregiving within and beyond the family is related to mortality in older adults. Survival analyses based on data from the Berlin Aging Study revealed that mortality hazards for grandparents who provided non-custodial childcare were 37% lower than for grandparents who did not provide childcare and for non-grandparents. These associations held after controlling for physical health, age, socioeconomic status and various characteristics of the children and grandchildren. Furthermore, the effect of caregiving extended to non-grandparents and to childless older adults who helped beyond their families. Potential ultimate and proximate mechanisms underlying these effects are discussed.     

RIG-I: a multifunctional protein beyond a pattern recognition receptor

It was widely known that retinoic acid inducible gene I (RIG-I) functions as a cytosolic pattern recognition receptor that initiates innate antiviral immunity by detecting exogenous viral RNAs. However, recent studies showed that RIG-I participates in other various cellular activities by sensing endogenous RNAs under different circumstances. For example, RIG-I facilitates the therapy resistance and expansion of breast cancer cells and promotes T cell-independent B cell activation through interferon signaling activation by recognizing non-coding RNAs and endogenous retroviruses in certain situations. While in hepatocellular carcinoma and acute myeloid leukemia, RIG-I acts as a tumor suppressor through either augmenting STAT1 activation by competitively binding STAT1 against its negative regulator SHP1 or inhibiting AKT-mTOR signaling pathway by directly interacting with Src respectively. These new findings suggest that RIG-I plays more diverse roles in various cellular life activities, such as cell proliferation and differentiation, than previously known. Taken together, the function of RIG-I exceeds far beyond that of a pattern recognition receptor.     

Domain topology of nucleoporin Nup98 within the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.     

A new family of yeast nuclear pore complex proteins

We have identified a novel family of yeast nuclear pore complex proteins. Three individual members of this family, NUP49, NUP100, and NUP116, have been isolated and then characterized by a combination of molecular genetics and immunolocalization. Employing immunoelectron and immunofluorescence microscopy on yeast cells, we found that the binding of a polyspecific monoclonal antibody recognizing this family was predominantly at the nuclear pore complexes. Furthermore, the tagging of NUP49 with a unique epitope enabled the immunolocalization of this protein to the nuclear pore complex by both fluorescence and electron microscopy. DNA sequence analysis has shown that the amino-terminal regions of NUP49, NUP100, and NUP116 share repeated "GLFG" motifs separated from each other by glutamine, asparagine, serine and threonine rich spacers. All three proteins lack a repetitive domain found in the two precisely described yeast nuclear pore complex proteins. Only NUP49 is essential for cell viability. NUP116-deficient cells grow very slowly and are temperature sensitive, whereas the lack of NUP100 has no detectable phenotype. NUP100 and NUP116 are homologous over their entire lengths. Interestingly, NUP100 and NUP116 are both flanked by a histidine tRNA gene and a transposon element suggesting that they may have arisen by gene duplication. We propose that subfamilies of pore complex proteins can be defined by their characteristic combinations of different modular domains.     

The antizyme family: polyamines and beyond

The family of antizymes functions as regulators of polyamine homeostasis. They are a class of small, inhibitory proteins, whose expression is regulated by a unique ribosomal frameshift mechanism. They have been shown to inhibit cell proliferation and possess anti-tumor activity. Antizymes bind ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. They inhibit its enzymatic activity and promote the ubiquitin-independent degradation of ODC by the 26S proteasome. In addition, they also negatively regulate polyamine transport. Antizyme-mediated, ubiquitin-independent degradation of ODC is conserved from yeast to man. But recent data suggest that this degradation pathway might not be restricted to ODC alone and could involve newly discovered antizyme binding partners. Interestingly, antizyme proteins have been strictly preserved over a vast evolutionary timeframe. Antizymes consequently represent an important class of proteins that regulate cell growth and metabolism by a diverse set of mechanisms that include protein degradation, inhibition of enzyme activity, small molecule transport and other, potentially not yet discovered properties.     

The cancer nuclear microenvironment: interface between light microscopic cytology and molecular phenotype

A definitive diagnosis of cancer may be rendered by microscopic assessment of only a few cells in an appropriate clinical setting due to the distinctive nuclear structure of most cancer cells in comparison to nuclei of normal human cells. The molecular architecture of non-neoplastic human nuclei--of the nuclear matrix and of matrix-associated proteins and nucleic acids--is being characterized in exquisite molecular detail. What is missing is the application of the findings and tools of molecular biology to understanding the cytological structure of cancer nuclei. This article delves into the basis of nuclear structure at different levels of resolution--light microscopic, electron microscopic, and molecular.     

Insertion of a long KpnI family member within a mitochondrial-DNA-like sequence present in the human nuclear genome

Structural analysis of a phage lambda Charon 4A clone carrying one of the human nuclear mitochondrial(mut)-DNA-like sequences revealed that a KpnI-family member (KpnI 5.5-kb DNA) is inserted within this sequence. The inserted KpnI 5.5-kb DNA contains several possible polyadenylation signal sequences followed by an A-rich sequence at its 3' end and is flanked by perfect 13-bp direct repeats of the duplicated mtDNA-like sequences. These structures strongly suggest that the KpnI 5.5-kb DNA is a mobile element. Comparison of the 5' terminal sequences of the KpnI 5.5-kb DNA and four other long KpnI-family DNAs so far examined, using the predicted general promoter sequence for eukaryotic tRNAs, indicates that they contain the consensus sequences for the split internal RNA polymerase III control region.     

Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex.     

Conservation of folding and stability within a protein family: the tyrosine corner as an evolutionary cul-de-sac

What are the selective pressures on protein sequences during evolution? Amino acid residues may be highly conserved for functional or structural (stability) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are using an experimental "fold approach" to the study of protein folding. This compares the folding and stability of a number of proteins that share the same fold, but have no common amino acid sequence or biological activity. The fold selected for this study is the immunoglobulin-like beta-sandwich fold, which is a fold that has no specifically conserved function. Four model proteins are used from two distinct superfamilies that share the immunoglobulin-like fold, the fibronectin type III and immunoglobulin superfamilies. Here, the fold approach and protein engineering are used to question the role of a highly conserved tyrosine in the "tyrosine corner" motif that is found ubiquitously and exclusively in Greek key proteins. In the four model beta-sandwich proteins characterised here, the tyrosine is the only residue that is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucleate folding in the immunoglobulin superfamily, whereas it is involved to some extent in early structure formation in the fibronectin type III superfamily. The tyrosine corner is important for stability, mutation to phenylalanine costs between 1.5 and 3 kcal mol(-1). We propose that the high level of conservation of the tyrosine is related to the structural restraints of the loop connecting the beta-sheets, representing an evolutionary "cul-de-sac".