Intranuclear Region Research Articles

Intranuclear mesoscale viscoelastic changes during osteoblastic differentiation of human mesenchymal stem cells

Cell nuclei behave as viscoelastic materials. Dynamic regulation of the viscoelastic properties of nuclei in living cells is crucial for diverse biological and biophysical processes, specifically for intranuclear mesoscale viscoelasticity, through modulation of the efficiency of force propagation to the nucleoplasm and gene expression patterns. However, how the intranuclear mesoscale viscoelasticity of stem cells changes with differentiation is unclear and so is its biological significance. Here, we quantified the changes in intranuclear mesoscale viscoelasticity during osteoblastic differentiation of human mesenchymal stem cells. This analysis revealed that the intranuclear region is a viscoelastic solid, probably with a higher efficiency of force transmission that results in high sensitivity to mechanical signals in the early stages of osteoblastic differentiation. The intranuclear region was noted to alter to a viscoelastic liquid with a lower efficiency, which is responsible for the robustness of gene expression toward terminal differentiation. Additionally, evaluation of changes in the mesoscale viscoelasticity due to chromatin decondensation and correlation between the mesoscale viscoelasticity and local DNA density suggested that size of gap and flexibility of chromatin meshwork structures, which are modulated depending on chromatin condensation state, determine mesoscale viscoelasticity, with various rates of contribution in different differentiation stages. Given that chromatin within the nucleus condenses into heterochromatin as stem cells adopt a specific lineage by restricting transcription, viscoelasticity is perhaps a key factor in cooperative regulation of the nuclear mechanosensitivity and gene expression pattern for stem cell differentiation.

Polyorganophosphazene stabilized gold nanoparticles for intracellular drug delivery in breast carcinoma cells

Abstract The synergistic combination of polymer-inorganic hybrid nanomaterials promises a new class of vehicle systems for stimuli-responsive and cell-specific drug delivery applications. Herein, we prepared a pH-responsive and charge reversal gold -poly (bis(carboxyphenoxy) phosphazene) (PCPP) nanohybrid system exhibits excellent dispersity and stability as well as the more efficient release rate at the acidic environment. As a stimuli-responsive drug delivery vehicle, PCPP-AuNPs potentially reduces the drug loss at neutral pH, whereas rapid drug release was noticed after internalization of nanoparticles by the cancer cells. Metal nanohybrid material effectively internalized into slightly acidic cell environment and even distribution of drug in the cytoplasm and intra-nuclear region due to charge reversal behavior. The cytotoxicity experiments corroborated that the nanohybrid system was more efficient than the free camptothecin (CPT) due to pH quenching charge reversal behavior. In addition, CPT@PCPP-AuNPs alters cell morphology, elevates the ROS levels, apoptosis protein expression and mitochondrial dysfunction. This work provides a simple method to fabricate smart pH-responsive AuNPs for anticancer drug delivery.

Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm.

Upf2 protein predominantly localizes to the cytoplasmic fraction, and binds to the exon junction complex (EJC) on spliced mRNA. The present study aimed to determine the cellular site where the interaction between Upf2 and EJC occurs. First, the cell lysate was fractionated into the cytoplasm and nucleoplasm, and western blotting to detect levels of Upf2 protein was performed. Upf2 was clearly detected in the cytoplasm and in the nucleoplasm. Secondly, immunostaining was performed, and the majority of Upf2 was detected in the cytoplasmic perinuclear region; a small quantity of Upf2 was detected in the intranuclear region. RNase treatment of the cells reduced the Upf2 immunostained signal. The immune-purified fractions containing nuclear and cytoplasmic Upf2 also contained one of the EJC core factors, RBM8A. These results implied the existence of Upf2 in the nucleoplasm and the cytoplasm, and it appeared to be involved in the construction of the mRNA complex. In order to verify the construction of Upf2-binding EJC in the nucleoplasm, an in situ proximity ligation assay was performed with anti-Upf2 and anti-RBM8A antibodies. These results demonstrated that their interaction occurred not only in the cytoplasmic region, but also in the intranuclear region. Taken together, these results suggested that Upf2 combines with EJC in both the cytoplasmic and the intranuclear fractions, and that it is involved in mRNA metabolism in human cells.

Characterization of a late gene, ORF75 from Bombyx mori nucleopolyhedrovirus

Open reading frame 75 (Bm-p33) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homologue of Autographa californica multiple nucleopolyhedrovirus ORF92. The gene is conserved among all baculoviruses that have been completely sequenced to date and is considered to be a baculovirus core set gene. No amino acid mutation was found in Bm-p33 sequences among six BmNPV strains differing in geography, phenotype, or host. The Bm-p33 transcript can be detected as early as 12h post infection (h p.i.) and remains detectable until 96h p.i. The Bm-p33 protein was detected in cell lysates from 18h p.i. through 96h p.i., and no positive band could be detected in budded viruses (BVs) and occlusion-derived viruses (ODVs) by western blot using anti-Bm-p33 serum. Immunofluorescence microscopy indicated that Bm-p33 accumulated in the nuclear membrane and the intranuclear region, especially near the nuclear membrane of the virus-infected cells. Bm75 RNAi significantly decreased the mRNA level. However, no obvious effects on ODV formation and BV production in BmNPV-infected cells could be detected. Bm-p33 is a BmNPV late gene encoding a nonstructural protein which may function mainly in the nucleus of the infected cells.

Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase-Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16-18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

Intracellular target for photosensitization in cancer antiangiogenic photodynamic therapy mediated by polycation liposome

Previous study indicated that antiangiogenic photodynamic therapy (PDT), laser irradiation at 15 min post-injection of photosensitizer in vivo, is effective for cancer treatment, and a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), encapsulated in polycation liposomes (PCLs), liposomes modified with cetylated polyethylenimine (cetyl-PEI), is more effective than BPD-MA encapsulated in non-modified liposomes [Cancer 97 (2003) 2027]. In the present study, we examined intracellular distribution of BPD-MA. BPD-MA encapsulated in liposomes or in PCLs was incubated with human endothelial cell line ECV304 cells or human umbilical vein endothelial cells (HUVECs), and monitored the intracellular distribution of BPD-MA by confocal laser scan microscopy. BPD-MA was taken up time-dependently into the cells and was distributed in not only cytoplasmic area but also intranuclear region. The enhanced uptake of BPD-MA was observed by the PCL formulation. Intracellular distribution of polycation was monitored by using fluorescein isothiocyanate-labeled cetyl-PEI (cetyl-PEI-FITC) and was colocalized with BPD-MA. Cytoplasmic BPD-MA distribution was partly overlapped with that of rhodamine 123, a mitochondrial fluorostaining probe, suggesting that mitochondrial photosensitization as well as nuclear photosensitization, is involved in the antiangiogenic PDT treatment.

Micro-organization and visco-elasticity of the interphase nucleus revealed by particle nanotracking.

The microstructure of the nucleus, one of the most studied but least understood cellular organelles, is the subject of much debate. Through the use of particle nanotracking, we detect and quantify the micro-organization as well as the viscoelastic properties of the intranuclear region in single, live, interphase somatic cells. We find that the intranuclear region is much stiffer than the cytoplasm; it is also more elastic than viscous, which reveals that the intranuclear region displays an unexpectedly strong solid-like behavior. The mean shear viscosity and elasticity of the intranuclear region of Swiss 3T3 fibroblasts are 520 Poise (P) and 180 dyn/cm(2), respectively. These measurements determine a lower bound of the propulsive forces (3-15 picoNewton) required for nuclear organelles such as promyelocytic-leukemia bodies to undergo processive transport within the nucleus by overcoming friction forces set by the intranuclear viscosity. Dynamic analysis of the spontaneous movements of nanospheres embedded in the nucleus reveals the presence of putative transient nuclear microdomains of mean size 290+/-50 nm, which are mostly absent in the cytoplasm. The strong elastic character and micro-organization of the intranuclear region revealed by particle nanotracking analysis may help the nucleus to preserve its structural coherence. These studies also highlight the difference between the low interstitial nucleoplasmic viscosity, which controls the transport of nuclear proteins and molecules, and the much higher mesoscale viscosity, which affects the diffusion and directed transport of nuclear organelles and re-organization of interphase chromosomes.

Presence and Activation of Nuclear Phosphoinositide 3-Kinase C2β during Compensatory Liver Growth

Highly purified liver nuclei incorporated radiolabeled phosphate into phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). When nuclei were depleted of their membrane, no radiolabeling of PtdIns(3,4,5)P(3) could be detected showing that within the intranuclear region there are no class I phosphoinositide 3-kinases (PI3K)s. In membrane-depleted nuclei harvested 20 h after partial hepatectomy, the incorporation of radiolabel into PtdIns(3)P was observed together with an increase in immunoprecipitable PI3K-C2beta activity, which is sensitive to wortmannin (10 nm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On Western blots PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas 20 h after partial hepatectomy gel shift of 18 kDa was noticed, suggesting that observed activation of enzyme is achieved by proteolysis. When intact membrane-depleted nuclei were subjected to short term (20 min) exposure to micro-calpain, similar gel shift together with an increase in PI3K-C2beta activity was observed, when compared with the nuclei harvested 20 h after partial hepatectomy. Moreover, the above-mentioned gel shift and increase in PI3K-C2beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that, in the membrane-depleted nuclei during the compensatory liver growth, there is an increase in PtdIns(3)P formation as a result of PI3K-C2beta activation, which may be a calpain-mediated event.

DNA replication in the 1-cell mouse embryo: stimulatory effect of histone acetylation.

Temporal and spatial distribution of the sites of DNA replication were examined in 1-cell mouse embryos. Embryos were labelled with bromodeoxyuridine (BrdU) at hourly intervals after fertilisation, and the incorporation of BrdU was examined by laser-scanning confocal microscopy following immunostaining with an anti-BrdU antibody. DNA replication first started uniformly in both the male and female pronuclei in the intranuclear region and then was observed in the peripheral regions of nucleus and nucleolus. These changes, however, occurred asynchronously in that the female pronucleus required a longer time to complete replication in the intranuclear region but not in the peripheral regions. Inhibiting transcription with alpha-amanitin had no effect on the temporal and spatial patterns of DNA replication. Treatment of the embryos with trapoxin, a specific inhibitor of histone deacetylase, accelerated the completion of replication in the peripheral regions but not in the intranuclear region. These results suggest that DNA replication is temporally and spatially regulated in the 1-cell embryos and that acetylation of histones, but not transcription, is involved in the regulation of DNA replication.

A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues.

A novel grid polymerase chain reaction (G-PCR) method has been developed to be used at the ultrastructural level and with a high degree of resolution. Samples applied to test the method were fresh cell lines (CaSki, SiHa) and HPV-16 DNA-containing tissues rescued from routine paraffin blocks. The specimens were embedded in Epon-Araldite and/or hydrophilic-resin LRWhite. Ultrathin sections mounted on grids were subjected to G-PCR using an HPV-16-specific primer set. The amplified products were identified by auro-immunohistochemical labelling of the biotinylated nucleotide. The results indicated successful amplification of target DNA in both cell and tissue samples, being confined to the intranuclear region. The negative controls [HeLa cells, isolated mammary carcinoma cell cultures (MCF 7, and T47-D) (ATCC) (U.S.A.), normal thyroid tissue and steroid-producing tumour tissue] failed to exhibit any amplification of the target DNA sequences. The sensitivity of the G-PCR system was evaluated by performing a parallel in situ hybridization (ISH) of serial sections. The signals obtained from G-PCR were more intense than those of ISH and more informative as to the precise subcellular localization of amplicons.

A novel grid polymerase chain reaction (G‐PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues

A novel grid polymerase chain reaction (G-PCR) method has been developed to be used at the ultrastructural level and with a high degree of resolution. Samples applied to test the method were fresh cell lines (CaSki, SiHa) and HPV-16 DNA-containing tissues rescued from routine paraffin blocks. The specimens were embedded in Epon-Araldite and/or hydrophilic-resin LRWhite. Ultrathin sections mounted on grids were subjected to G-PCR using an HPV-16-specific primer set. The amplified products were identified by auro-immunohistochemical labelling of the biotinylated nucleotide. The results indicated successful amplification of target DNA in both cell and tissue samples, being confined to the intranuclear region. The negative controls [HeLa cells, isolated mammary carcinoma cell cultures (MCF 7, and T47-D) (ATCC) (U.S.A.), normal thyroid tissue and steroid-producing tumour tissue] failed to exhibit any amplification of the target DNA sequences. The sensitivity of the G-PCR system was evaluated by performing a parallel in situ hybridization (ISH) of serial sections. The signals obtained from G-PCR were more intense than those of ISH and more informative as to the precise subcellular localization of amplicons. © 1997 John Wiley & Sons, Ltd.

Nuf2, a spindle pole body-associated protein required for nuclear division in yeast.

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.

Identification and immunocharacterization of a 25K structural protein in Autographa californica nuclear polyhedrosis virus (AcMNPV)

A 25K protein was a major component of the Triton X-100 solubilized fraction prepared for the isolation of nuclear matrices from Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus (AcMNPV). Polyclonal antisera to p25 were produced in rabbits and used in the characterization of p25. Western blots of proteins probed with the p25 antiserum demonstrated the presence of the p25 in both the polyhedral-derived and extracellular viral phenotypes. Using an indirect fluorescent antibody assay, p25 was detected within the infected host cell as early as 6 h p.i. and throughout the time course examined. The intensity in fluorescence increased from the periphery of the infected cells to within the nucleus at late times post-infection. On electron micrographs, the p25 was observed as dense gold particles in localized pockets and around virions in the nucleus at 16 and 36 h p.i., respectively. By 48 h p.i., p25 was concentrated over the surface of nucleocapsids in the intranuclear region and within polyhedra. p25 was not associated with any infected cell structures and did not appear to be involved in polyhedron formation. These studies demonstrated that p25 was a structural protein which was expressed very early during infection and displayed characteristics similar to In major capsid proteins of the baculoviruses.