Conformational variability of nucleo-cytoplasmic transport factors

The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin beta-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin beta, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin beta, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.     

Some features of nucleo-cytoplasmic RNA transport from isolated nuclei

Messenger RNA is released preferentially from isolated rat liver nuclei in the presence of the ATP-generating system and cytosol. The release is suppressed by spermidine, while cytoplasmic RNase inhibitor was ineffective and PCMB like some other thiol-blocking agents inhibitory. Cytoplasmic SOD added to the system strongly suppressed RNA release. A similar effect could be obtained by anaerobiosis due to addition of SMP. In both cases the inhibition is reversed by cyanide.In contrast to normal liver where the generation of superoxide radicals takes place almost exclusively in microsomes and is coupled with the oxidation of NADPH, in mouse ascites hepatoma 22a the generation of superoxide radicals occurs mainly in the nuclear envelope and is coupled with the oxidation of both NADPH and NADH and inhibited by cyanide.Abbreviations PCMB p-Chloromercuri benzoate - SMP Submitochondrial particles - SOD Superoxide dismutase     

Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.

The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.     

Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport.

Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.     

Effect of colchicine on mammalian liver nuclear envelope and on nucleo-cytoplasmic RNA transport

The binding of colchicine to nuclear envelopes was studied in order to elucidate the mechanism whereby this compound inhibits nucleocytoplasmic RNA transport. The results suggest that a single class of colchicine-binding site (dissociation constant=approx. 0.7 mM, concentration=approx. 330 nmol colchicine/mg protein) is localised in the nuclear periphery (pore-lamina) and that binding to these sites effects a constriction of the pore-complexes with concomitant inhibition of RNA egress and disordering of the nuclear membrane phospholipid bilayers.     

UV-guided screening of benzodiazepine producing species in Penicillium

The benzodiazepine sclerotigenin (auranthine B) recently described as a metabolite of Penicillium sclerotigenum, has been isolated as the major metabolite from an isolate of P. commune. The structure of sclerotigenin was established by a single-crystal X-ray diffraction study and by NMR spectroscopy. UV-guided screening for benzodiazepine production by other penicillia revealed that sclerotigenin was also produced by isolates of P. clavigerum, P. lanosum, P. melanoconidium, P. sclerotigenum and P. verrucosum. Sclerotigenin was detected both intra- and extracellularly. Apparently, P. aurantiogriseum is the only auranthine producing species in genus Penicillium.     

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

Intimate contacts of mitochondria with nuclear envelope as a potential energy gateway for nucleo-cytoplasmic mRNA transport

The aim of the present study was to show that close contacts of mitochondria with nuclear envelope need not be just an accidental situation in the cell, but that such contacts could serve for flow of energy from a place of origin directly to a place of consumption. Mitochondria in close proximity to the nuclear envelope can be found virtually in all metabolically active cells. We used transmission electron microscopy to demonstrate this entity in different leukemia cells of human origin (patient's blood) and in mouse leukemia cell line. At high resolution, not only close proximity but even fusion of mitochondrial and nuclear membranes can be seen. Based on available data about mRNA transport through the nuclear pore complex and observed contacts of mitochondria with nuclei, we hypothesize that such contacts can provide a gateway for energy delivery to power mRNA export from the nucleus to the cytoplasm. Hence the lumen of the nuclear envelope can serve for transport or even storage of macroergic molecules in a manner similar to sarcoplasmic reticulum in fast-twitch skeletal muscles.     

Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells.

We established a straightforward experimental system to investigate directly the requirements for nucleocytoplasmic transport in live cells. For this purpose, substrates were created containing nuclear localization signals (NLS) or nuclear export signals (NES) linked to a chimeric protein composed of the glutathione S-transferase (GST) fused to the green fluorescent protein (GFP). The combination of GST/GFP-tagging allowed us to control protein expression in bacteria and to monitor protein purification during chromatography. Following microinjection into somatic cells, nuclear export/import of the highly fluorescent substrates could be observed directly by fluorescence microscopy. This system sets the stage to quantitate, in real time, the kinetics of nuclear import/export in living cells and to evaluate qualitative differences in various NLS/NES signals and pathways.     

Vision-based force measurement using neural networks for biological cell microinjection

This paper presents a vision-based force measurement method using an artificial neural network model. The proposed model is used for measuring the applied load to a spherical biological cell during micromanipulation process. The devised vision-based method is most useful when force measurement capability is required, but it is very challenging or even infeasible to use a force sensor. Artificial neural networks in conjunction with image processing techniques have been used to estimate the applied load to a cell. A bio-micromanipulation system capable of force measurement has also been established in order to collect the training data required for the proposed neural network model. The geometric characterization of zebrafish embryos membranes has been performed during the penetration of the micropipette prior to piercing. The geometric features are extracted from images using image processing techniques. These features have been used to describe the shape and quantify the deformation of the cell at different indentation depths. The neural network is trained by taking the visual data as the input and the measured corresponding force as the output. Once the neural network is trained with sufficient number of data, it can be used as a precise sensor in bio-micromanipulation setups. However, the proposed neural network model is applicable for indentation of any other spherical elastic object. The results demonstrate the capability of the proposed method. The outcomes of this study could be useful for measuring force in biological cell micromanipulation processes such as injection of the mouse oocyte/embryo.     

Microinjection of living adherent cells by using a semi-automatic microinjection system

Testing in vitro is an alternative to animal experimentation. The capillary pressure microinjection technique is a supporting technology for efficient in vitro testing. The main benefit of the technique is the possibility of injecting large molecules into a single living cell. The ultimate goal of the research discussed in this paper is to increase the cell survival rate in capillary pressure microinjection. A method to reliably evaluate cell survival rate is therefore needed. A three-phase evaluation process is presented in this paper. The first phase determines the success rate of the injection capillary to penetrate the cell membrane. The second phase studies the success rate of delivering the injection substance inside the cell, while the third phase studies cell survival after the microinjection. In addition to the three-phase evaluation process, this paper describes the initial results of penetration and injection tests performed by using a semi-automatic capillary pressure microinjection system developed by the research group. Three adherent cell lines, namely, retinal pigment epithelial cells, MCF-7 human breast cancer cells and SH-SY5Y neuroblastoma cells, were used in the experiments. The results of the penetration tests show that the average success rate of penetrating the cell membrane using the micromanipulator was 87%. The goal of the injection tests was to demonstrate the successful microinjection of living cells and to study the injection success rate. Fluorescein dextran was injected into MCF-7 cells, and preliminary results showed an injection success rate of 49%. In the survival tests, the neuronal cells were microinjected with KCl. During long-term observation after the microinjection, the microinjected cells first decreased their adhesion to the plate, but later adhered to the bottom of the plate and even grew some dendrites. In the next phase of the study, more tests will be performed in order to obtain a statistically reliable value for the survival rate.     

Post-meiotic nucleo-cytoplasmic interaction in Cosmos bipinnatus

An interaction involving the nuclear envelope and spherical double-membrane bound inclusions takes place in the cytoplasm of post-meiotic male microspores of Cosmos (tribe Heliantheae, sub-tribe Coreopsidinae). The identity of the spherical inclusions has yet to be fully established, but they closely resemble profiles elsewhere in the cytoplasm, themselves presumably derived from the mitochondrial population of the premeiotic pollen mother cells. Both the cytoplasmic and nucleaar-associated inclusions regularly contain a central vesicle, formed by an ingagination of their bounding membranes. The interaction, which occurs immediately prior to the deposition of the primexine of the pollen wall, involves the adhesion of the inclusions to the nuclear surface. Experiments with osmotically disrupted cells reveal that the inclusions are firmly bound to the envelope and, at the points of contact, electron opaque granules are regularly present. Frequently elements of the chromatin may be observed in juxtapostion to these points of contact, but on the inner face of the envelope. The interaction in Cosmos is proposed to constitute part of the process by which the cytoplasm and its content are realigned to the new gametophylic style of growth.