The illustrates the work-flow of the project. The oocytes were de-folliculated and spun down to move the nuclei to the poles. The nucleoplasmic or cytoplasmic microinjections (a) of the biomolecules, including transgenes’ vectors, were performed. The nuclei were isolated (b), by tearing the cell membrane with surgical forceps and rinsing with the buffer. Further, the preparations were pursued along the two different routes. First (c), the nuclei were attached to the sticky carriers. For studies of the nucleoplasmic side of the nuclear envelope, they were opened to remove nucleoplasm. Occasionally, they were digested with DNase and RNase. Then, after thorough rinsing, these preparations were exposed to the modified DNA for binding assays. The nuclear envelopes, attached to the carriers, were cryo-immobilized and processed for molecular imaging and elemental analysis with the aid of the energy dispersive x-ray spectroscopy (EDXS) and electron energy loss spectroscopy (EELS). All the probes and Fvs used in this project were modified to chelate the exogenous metals, thus were unambiguously distinguished from the cell structures and specifically identified with high sensitivity. Second (d), the nuclei were rapidly cryo-immobilized and/or homogenized. The DNA, RNA, and proteins were separated for analysis with nuclear magnetic resonance spectroscopy (NMRS) or total reflection x-ray fluorescence spectroscopy (TRXFS).
The reveals the overall architecture of the ultrastructures assembled onto the nucleoplasmic side of the nuclear envelope (NE). The sample was prepared by gentle removing of nucleoplasm, rapid cryoimmobilization, freeze-drying, and cryo-coating. The image was acquired in the field emission scanning electron microscope (FESEM). It is representative of a hundred studied. The three main ultrastuctures are apparent: nuclear routing networks (NRNs), nuclear pore complexes (NPCs), and lamin networks. In the field of view, there are present numerous “baskets” of the nuclear pore complexes (NPC), which protrude from the nuclear envelope. They are “inserted” into the lamin networks. Bundles of 6 nm thin filaments project from the tops of most of the NPC “baskets”. The bundled filaments create inside-empty micro-tubes. They project into the nucleus’ interior. These bundles extend for distances from 2 micrometers to more than 10 micrometers in lengths. Short and long stretches of the filamentous bundles inter-connect with each other to form nuclear networks. In a few cases, attached fibers have all broke off the “baskets”, thus revealing the top rings only. At all distal ends of the eight fibers (projecting from the 120 nm diameter, intra-nuclear ring of the NPCs) there are the 10 nm in diameter spheres. These spheres attach to each other to form the “necklace-like” rings – as if they would be holding together the bundles of filaments. These rings are distributed along the filamentous bundles with the regular periodicity of approximately 50 nm. Collectively, these structures build the nuclear routing networks (NRNs) described in more details earlier [25
Architecture of the nuclear routing networks (NRNs)
The results of an alternative method of the sample preparation are illustrated in the . After extracting chromatin, the nuclear envelopes were cryoimmobilized, as whole mounts, freeze-substituted, embedded, and sectioned. The samples were imaged with the electron energy loss spectroscopy (EELS). The image is representative for ten preparations. The long projections of the nuclear routing networks extend from the NPCs. They have 50 nm periodicities along their length. Their architecture is identical to that revealed in the .
Ultrastructure of the nuclear routing networks (NRNs)
Studies aimed to reveal associations of the NRNs with chromatin were pursued on two ways: (1) labeling of the samples with the Fv antibodies targeting genomic DNA, while revealing the NRNs; (2) digesting all the nucleic acids from the oocytes’ preparations with the DNases and RNases followed by DNA binding assays.
To identify the structures attached to the NRNs, a panel of clones of the antibodies targeting the dsDNA was used as shown in the . All these Fvs contained the domains chelating Gd, Eu, or Tb as described earlier [78
]. The nuclear envelopes were exposed by gentle removing chromatin gel, which followed by immunolabeling with the Fvs targeting the dsDNA. Thereafter, the samples were rapidly cryo-immobilized, freeze-substituted, and cryo-sputter-coated to render them good electro-conductors and high secondary electron emitters. This figure illustrates the preparation representative of twenty different preparations. The overview of the architecture is revealed with the secondary emission as shown in the . The NRNs architecture is identical to that shown in the –. Conglomerates of amorphous structures attached to the NRNs were of particular interest. They were probed by labeling with the Gd chelated Fvs targeting the genomic dsDNA. The sample was analyzed with the EDXS as illustrated in the , shown as the binary elemental distribution map. This elemental distribution map was constructed in several steps. First, the structural overview was acquired as the reference. Second, the elemental spectrum profile was acquired for every pixel of the image to create the elemental composition data bank. Third, a map of Gd distribution in the sample was extracted from the elemental composition data bank. Since the Gd atoms were chelated within the dedicated domains of the Fvs, then the Gd distribution map was indicative of the distribution of the Fvs, thus the distribution of the genomic DNA. Fourth, superimposing the elemental Gd map from over the architecture overview revealed distribution of the Fvs against the cell structures as shown in the . After outlining areas of the oocyte compartments on the computer screen, the areas containing Gd atoms were automatically calculated by the elemental analysis software ceiled at 1 million counts. Since all and only, the anti-DNA Fv clones chelated Gd, then distribution of Gd was indicating with high specificity and high sensitivity the distribution of the gDNA. The associations of the dsDNA with the NRNs were evident. Several controls were run to validate these results. First, when the data were acquired from the unlabeled samples, then no scintillations were counted at the Gd energy peak above the sensor noise (black screen). Second, labeling with the isotypic antibodies had no counts above scintillation noise, thus not statistically significant influence on the signal recorded from the anti-dsDNA. Third, initial labeling with the elemental tag free anti-dsDNA Fvs followed by the Fvs tagged with Gd, Eu, or Tb resulted in very low scintillation counts. All these observations were validated by quantification and statistical analysis, as illustrated in the . For that purpose ten EDXS scintillation counts’ at the Gd, Tb, or Eu energy edge were acquired from ten randomly selected areas from ten different samples. A similar approach, but involving chelated Gd, Eu, or Tb coordinated in the metal chelate of the anti-biotin Fv or anti-digoxigenin resulted in the same outcome. The data were further validated with the NMRS and TRXRFS. The statistical confidence was approved with the unpaired t test at the P value < 0.0001. The variances were significantly different in the F test and approved at the P value < 0.0001. One way ANOVA analysis resulted in the variance P value < 0.0001 with the means significantly different P < 0.05. The Bartlett’s test for equal variances was calculated at the P value p < 0.0001. This was indeed the quantitative proof, that the clusters attached to the NRNs indeed contained the genomic DNA.
Associations of the gDNA with the NRNs
Statistical analysis of associations of the gDNA with the NRNs
To further identify the structures assembled onto the NRNs, the DNA and RNA were entirely removed from the samples by digestion with the DNase and RNase mixtures, which followed by the chelated DNA binding assay as shown in the . In this assay, after digestion with the DNases and RNases, and thorough rinsing, all the nucleic acids were entirely removed from the samples. Thereafter, the samples were exposed to the genomic DNA tagged with Gd, Eu, or Tb, while being suspended in a solution of the diluted nucleoplasm to retain possible interfacing molecules. Prior to this binding assay, the genomic DNA was isolated and purified from the oocytes, sheared with ultrasound, and thereafter modified by incorporation of the dNTPs analogs: Biotin-14-dATP, Biotin-14-dCTP, FITC-11-dUTP, or Digoxigenin-11-dUTP. These tags were then linked with the Fv targeting biotin, digoxigenin, or FITC, while coordinating Gd, Tb, or Eu according to the technology described earlier [78
]. This sample is representative of twenty different preparations. The structural overview of the sample acquired at the secondary emission is shown in the . The main difference between the samples, prepared by digestion with DNases and RNases versus the one, in which digestion was followed by labeling with the modified DNA, is presence of amorphous material attached to the NPCs and NRNs in the latter sample. In the field of view, there are a few NPCs with the open, empty tops. The amorphous material attached to the NRNs and NPCs was identified with the energy dispersive x-ray spectroscopy (EDXS). The elemental binary map of the added, modified DNA is shown in the . The overlay of the added DNA distribution over the nuclear structures is shown in the . Considering that only the modified DNA contained chelated metals, incorporated via dNTP analogs, the conclusion was clear, that the structures, which firmly adhered to the NRNs, were indeed the modified DNA added to the samples previously depleted of any traces of the native genomic DNA. Three control tests were performed to validate the above findings. First, the samples which were digested with DNases and RNases, but not labeled with the DNA modified with the dNTP analogs coordinating the Gd. Such samples did not generate the counts above the scintillators’ noise. Second, labeling with the Fvs anti-dsDNA without elemental tags prior to labeling with the Fvs coordinating Gd resulted in the signal counts minimally above the threshold of detection. This was indicative of the DNA binding sites being occupied. Third, labeling of the NRN preparations with the mixes of the tagged and non-tagged anti-dsDNA Fvs reduced the levels of the scintillation’s counts. The results, obtained from coordinating Eu or Tb, were identical. These observations were quantified and analyzed statistically as shown in the . For that purpose, the scintillation counts were acquired in the EDXS from ten randomly selected areas from ten different preparations of the NRNs. The statistical confidence was satisfied with the t test P value < 0.0001. The variances were significantly different in the F test with the P value < 0.0001.
Statistical analysis of the DNA binding assay
Studies aimed to determine involvement of the NRN into trafficking of biomolecules between the NPCs and chromatin were pursued using four assays: (1) SV40 LTA NLS-modified transgenes; (2) chelate-modified histones; (3) HIV Rev NES-modified transgenes; (4) chelate-modified rRNA. The results are illustrated in the –. All the experiments were quantified by measuring the final concentration of the chelated metals followed by statistical analysis at the final points of the assays. That way, all these biomolecules were quantified primarily with the EDXS, but also followed by the NMRS, TRXFS, EELS, or fluorometry. More precisely, the transgenes’ vectors were bioengineered to carry the closed, circular dsDNA (ccdsDNA) constructs [74
]. The DNA had incorporated dNTP analogs modified with biotin, digoxigenin, or fluorescein. They served to lock the vectors into the transgenes’ constructs. They also had the dNTP analogs incorporated into the strands to determine integrity of the plasmid constructs within the vectors and to report trafficking of the vectors. For intra-nuclear import, the vectors consisted of the SV40 LTA nuclear localization signal (NLS), anti-biotin domain – anchoring the dsDNA, and chelating domain – harboring Gd, Eu, Ru, or Tb. For extra-nuclear export, the vectors consisted of the HIV Rev nuclear export signal (NES), and metal binding domains. The third and fourth type of the vectors – named shuttle vectors consisted of the NES and NLS domains bioengineered either with the anti-digoxigenin or with the anti-biotin Fvs. Microinjections of these vectors and biomolecules into oocytes helped to probe the functions of the nucleo-cytoplasmic transport. At different time intervals passing from the moment of microinjections, the oocytes were cryo-immobilized. They were processed by freeze-substitution, embedding and sectioning. The studies were conducted either on sections with the EELS and EDXS. Alternatively, the studies were conducted on the whole mounts with the NMRS and TXRFS preparations, which were thawed and separated into the fractions: nucleoplasm, nuclear envelope and associated structures, cytoplasm. This procedure prevented any translocations of labeled molecules and/or antibodies during initial stages of the preparation and accuracy of the quantitative analysis. The data from ten experiments for each of the transgenes were averaged, the standard deviations calculated and normalized as the ratios between three main compartments: nucleoplasm, NRN+NPC, cytoplasm. The statistical analysis was conducted on the counts from the start and end points of experiments with the statistical significance from the t test approved at the P value < 0.01. The results are presented in the –.
Trafficking of the SV40 LTA NLS-modified transgenes’ vectors
The outcome of the cytoplasmic microinjections of the transgenes’ vectors guided by the SV40 LTA NLS is shown in the . The transgenes’ vectors concentrations into the cytoplasm slowly, but steadily, decreased. At the same time, the concentrations inside the NRNs + NPCs were initially increasing, thereafter stabilized. Microinjection of the same transgenes’ vectors, which were not modified with the NLS, resulted in their absence in the NRNs and NPCs. Gradually decreasing concentrations of the transgenes’ vectors in the areas of the injections were due to diffusion. For comparison with the rates and paths of trafficking of natural biomolecules the histones modified with the chelates were microinjected also in the perinuclear space as shown in the . The curve of the trafficking is almost identical to that of the SV40 LTA NLS-driven vectors. The concentrations in the site of microinjections gradually were decreasing, while the concentrations of the modified histones in the NRNs + NPCs were slowly increasing. Competitive transport assay, in which the histones modified with the reporters were mixed with the non-modified histones resulted in the reduced counts, due to competition for the trafficking routes. The results of the intra-nuclear microinjections of the trangenes’ vectors modified with the HIV Rev NES are illustrated in the . The concentrations of the transgenes inside the nuclei were initially rapidly decreasing, which was followed by less rapid pace until the complete depletion. The entry of the vectors into and passage through the NRNs were indicated by the increasing count values. The vectors without the NES were residing in the nuclei as reflected by the constant values over time. Clearly, they were not entering the NRNs and NPCs. For comparison, the rRNA modified with the chelates was microinjected into the nuclei as illustrated in the . Its concentrations in the NRNs and NPCs compartments were promptly increasing. At the comparable time intervals, their concentrations were decreasing in the nucleoplasm. The competition assay, which was set up by mixing the chelates-modified RNA with non-modified RNA resulted in the reduced values of counts recorded during their entry into and passage through the NPCs and NRNs.
Trafficking of the histones
Distribution of the HIV Rev NES-modified transgenes’ vectors
Altogether, the data acquired in these experiments provide the evidence for the NRNs, as the structures linking the NPCs and the genomic DNA, as we have illustrated in the . Furthermore, these data demonstrate that the NRNs guide intranuclear trafficking of the molecules between the NPCs and the gDNA.
The NRNs span between the NPCs and the gDNA