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PubMed1997

A novel class of RanGTP binding proteins.

The importin-alpha/beta complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of approximately 20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-beta. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-beta, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-beta, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.

Amino Acid Sequence↗
PubMed2000

Isolation of a novel 190 kDa protein from tobacco BY-2 cells: possible involvement in the interaction between actin filaments and microtubules.

Interaction between actin filaments (AFs) and microtubules (MTs) has been reported in various plant cells, and the presence of a factor(s) connecting these two cytoskeletal networks has been suggested, but its molecular entity has not been elucidated yet. We obtained a fraction containing MT-binding polypeptides, which induced bundling of AFs and of MTs. A 190 kDa polypeptide which associated with AFs was selectively isolated from the fraction. This polypeptide was thought to have an ability to bind to both AFs and MTs. We raised a monoclonal antibody against the 190 kDa polypeptide. Immunostaining demonstrated the association of the 190 kDa polypeptide with AF bundles and with MT bundles formed in vitro. Immunocytochemical studies throughout the cell cycle revealed that the 190 kDa polypeptide was localized in the nucleus before nuclear envelope breakdown, and in the spindle and the phragmoplast during cell division. After the re-formation of the nuclear envelope, the 190 kDa polypeptide was sequestered to the daughter nuclei. Using the antibody, we succeeded in cloning a cDNA encoding the 190 kDa polypeptide.

Actin Cytoskeleton↗
PubMed1990

Premature mitosis induced in mammalian cells by the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine.

The protein kinase inhibitors 2-aminopurine (2-AP) and 6-dimethylaminopurine (6-DMAP) were used to examine the effects of protein dephosphorylation on the control of mitosis in mammalian cells. Both 2-AP and 6-DMAP induced premature mitosis in hamster fibroblasts that were arrested in S phase. This response was characterized by changes in cell morphology, breakdown of the nuclear envelope, and premature chromosome condensation. Premature mitosis was followed by a return to interphase morphology and reformation of the nuclear envelope around decondensed and fragmented chromatin to form numerous micronuclei. The activity of both compounds was dependent upon new protein synthesis but not new RNA synthesis. 2-AP and 6-DMAP acted cooperatively with each other and with caffeine, suggesting a common mechanism of action. In exponentially growing cells, 2-AP and 6-DMAP did not induce premature mitosis but did increase the frequency of binucleated cells by blocking cytokinesis. These findings support a role for protein dephosphorylation in the control of mitosis and indicate that cell cycle perturbations can modify this regulation.

2-Aminopurine↗
PubMed1994

Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes.

The Bcl-2 protein blocks programmed cell death and becomes overproduced in many follicular non-Hodgkin's lymphomas as the result of t(14; 18) translocations involving the Bcl-2 gene. Mcl-1 is a recently discovered gene whose encoded protein has significant homology with Bcl-2 but whose function remains unknown. In this study, we compared the in vivo patterns of Bcl-2 and Mcl-1 protein production in normal and neoplastic lymph node biopsies by immunohistochemical means using specific polyclonal antisera. Intracellular Mcl-1 immunoreactivity was located primarily in the cytosol in a punctate pattern and was also seen in association with the nuclear envelope in many cases, similar to the results obtained for Bcl-2, which resides in the outer mitochondrial membrane, nuclear envelope, and endoplasmic reticulum. In 4 of 4 reactive tonsils and 28 of 28 nodes with reactive follicular hyperplasia, reciprocal patterns of Bcl-2 and Mcl-1 protein expression were observed. Bcl-2 immunostaining was highest in mantle zone lymphocytes and absent from most germinal center cells, whereas Mcl-1 immunoreactivity was highest in germinal center lymphocytes and absent from mantle zone lymphocytes. Mcl-1 was also expressed in some interfollicular lymphocytes, particularly those that had the appearance of activated lymphocytes. Similar to the patterns of Bcl-2 and mcl-1 expression seen in reactive nodes, Mcl-1 protein was largely absent from the malignant cells in 2 of 2 mantle cell lymphomas, whereas strong Bcl-2 immunostaining was found in these cells. In contrast to normal nodes, however, the neoplastic follicles of t(14;18) containing follicular non-Hodgkin's lymphomas immunostained positively for both Bcl-2 and Mcl-1 in 24 of 27 cases. Intense immunostaining for Mcl-1 was also observed in Reed-Sternberg cells in 2 of 2 cases of Hodgkin's disease but Bcl-2 immunoreactivity was present at much lower levels. These findings demonstrate that the levels of Mcl-1 and Bcl-2 proteins are differentially regulated in normal and neoplastic cells in lymph nodes and thus suggest different roles for these proteins in the control of cell life and death in these tissues.

Amino Acid Sequence↗
PubMed1993

Regulation of CTP:phosphocholine cytidylyltransferase in HeLa cells. Effect of oleate on phosphorylation and intracellular localization.

The mechanism by which oleate induced the translocation of CTP:phosphocholine cytidylyltransferase from the soluble to particulate fraction was studied in HeLa cells. Addition of 0.5 mM oleate caused a 10-fold stimulation of [methyl-3H]choline incorporation into phosphatidylcholine. The stimulation of phosphatidylcholine synthesis was correlated with a decrease in phosphocholine and an increase in CDP-choline, indicating that cytidylyltransferase is regulatory under these conditions. The stimulation of cytidylyltransferase activity upon oleate treatment was concomitant with the translocation of this enzyme from the soluble to particulate fraction. Immunoblot analysis revealed the soluble form as multiple slowly migrating bands, and the particulate form as a faster migrating, single band. The slowly migrating form could be converted to the fast migrating form by phosphatase treatment, suggesting that the mobility difference was due to phosphorylation. In vivo 32P labeling showed that the soluble cytidylyltransferase was highly phosphorylated and the particulate form was much less phosphorylated. The kinetics of the translocation showed that the conversion of the majority of cytidylyltransferase from the soluble to the particulate form occurred within about 15 min following addition of oleate and that the translocation showed a good correlation with dephosphorylation. In contrast, the relocation of cytidylyltransferase from the particulate to the soluble fraction after the removal of oleate occurred within 1 min, and extensive phosphorylation was not required for cytidylyltransferase to leave the membrane. The relocated, soluble cytidylyltransferase was phosphorylated at a much slower rate than that at which the enzyme left the membrane. Immunolocalization showed that the cytidylyltransferase translocated to the nuclear envelope instead of the endoplasmic reticulum. These results indicate that activation of cytidylyltransferase by dephosphorylation and translocation to the nuclear envelope may be a general mechanism occurring in many cell types. Moreover, in HeLa cells, multiple mechanism exist for regulating cytidylyltransferase activity.

Biological Transport↗
PubMed2006

tRNA, new aspects in intracellular dynamics.

The nuclear envelope divides a eukaryotic cell into two compartments, the nucleus and the cytoplasm. Transcription and maturation of RNAs encoded on nuclear chromosomes are carried out in the nucleus, while the proteins coded by these RNAs are translated and processed in the cytoplasm. Cytosolic tRNAs, essential factors for translation, are transcribed in the nucleus and undergo extensive processing before reaching functionality. It had previously been believed that tRNAs have only one-way tickets to pass through the nuclear envelope after maturation in the nucleus, and that the small amounts of mature tRNAs found in the nucleus are biosynthetic intermediates. However, two reports from our lab and Anita Hopper's group recently demonstrated that tRNAs have multi-round commuter tickets to shuttle between the nucleus and the cytoplasm in the yeast Saccharomyces cerevisiae. In fact, various tRNA species, including aminoacylated full-length tRNAs and 3' end-shortened tRNAs, are actively imported into the nucleus under various conditions. These findings force a reconsideration of our view of intracellular dynamics of tRNAs, and re-evaluations of the physiological meanings of the nuclear mature tRNAs.

Animals↗
PubMed1992

Asymmetric mitotic segregation of the yeast spindle pole body.

The yeast KAR1 gene is required for spindle pole body (SPB) duplication and nuclear fusion. We determine here that KAR1-beta-galactosidase hybrid proteins localize to the outer face of the SPB. Remarkably, after SPB duplication, the hybrid protein was found associated with only one of the two SPBs, usually the one that enters the bud. Using an ndc1 mutant, which forms a defective SPB at the nonpermissive temperature, we found that the hybrid was exclusively associated with the "new" SPB. Two regions of KAR1 contribute to its localization; an internal 70 residue region was necessary and sufficient to localize hybrids to the SPB, and the hydrophobic carboxyl terminus localized proteins to the nuclear envelope. The localization domains correspond to two functional domains required for SPB duplication. We suggest that KAR1 is anchored to the nuclear envelope and interacts with at least one other SPB component during the cell cycle.

Cell Compartmentation↗
PubMed1995

Translocation and leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat basophilic leukemia cells and alveolar macrophages.

Leukotriene (LT) synthesis involves the translocation of enzymatically active 5-lipoxygenase (5-LO) from a soluble site to a bound site, where it interacts with 5-lipoxygenase-activating protein (FLAP). In human polymorphonuclear leukocytes (PMNs), 5-LO moves from the cytosol to the nuclear envelope (NE) to interact with FLAP. However, 5-LO has recently been found within the nucleus, as well as the cytosol, of rat basophilic leukemia (RBL) cells and alveolar macrophages (AMs). To assess whether nuclear 5-LO can contribute to LT synthesis in these cells, we investigated whether this enzyme pool 1) translocates upon cell activation, 2) colocalizes with FLAP, and 3) is enzymatically active. By cell fractionation followed by immunoblotting, both cytosolic and nuclear soluble 5-LO decreased dramatically in RBL cells following activation with the calcium ionophore A23187. Concurrently, 5-LO increased in the pelletable nuclear pool, where FLAP was also detected. The loss of both cytosolic and nuclear soluble 5-LO, with concomitant increase exclusively at the NE, as well as co-localization with FLAP, were confirmed by indirect immunofluorescent and confocal microscopy. In AMs, the nuclear soluble pool of 5-LO moved to the NE, where FLAP was also found; however, the cytosolic 5-LO pool did not translocate. Application of these methods to PMNs confirmed that cytosolic 5-LO moved to the nuclear envelope and co-localized with FLAP. By cell-free assay, nuclear soluble proteins from both RBL cells and AMs, but not PMNs, were able to generate 5-LO products from arachidonate, and this was inhibited by the direct 5-LO inhibitor zileuton. Cytosolic proteins from all cell types also showed cell-free 5-LO activity. These results demonstrate three distinct patterns of 5-LO translocation that are specific for each cell type: translocation of only a cytosolic pool in PMNs, of only a nuclear pool in AMs, and of both cytosolic and nuclear pools in RBL cells. By virtue of its enzymatic activity and ability to translocate, nuclear 5-LO has the potential to contribute to LT synthesis in RBL cells and AMs. Finally, these results provide a foundation for considering the individual functions of discrete pools of 5-LO in future studies.

Animals↗
PubMed1997

Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts.

We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 microM. However, a high concentration of daunomycin (50 microM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 microM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than the required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems.

Animals↗
PubMed2005

The HIV-Tat protein induces chromosome number aberrations by affecting mitosis.

To analyze the effects of the HIV-Tat-tubulin interaction, we microinjected HIV-Tat purified protein into Drosophila syncytial embryos. Following the Tat injection, altered timing of the cortical nuclear cycles was observed; specifically, the period between the nuclear envelope breakdown and anaphase initiation was lengthened as was the period between anaphase initiation and the formation of the next nuclear envelope. These two periods correspond to kinetochore alignment at metaphase and to mitosis exit, respectively. We also demonstrated that these two delays are the consequence of damage specifically induced by Tat on kinetochore alignment and on the timing of sister chromatid segregation at anaphase. Furthermore, we show that the expression of Tat in Drosophila larvae brain cells produces a significant percentage of polyploid and aneuploid cells. The results reported here indicate that Tat impairs the mitotic process and that Tat-tubulin interaction appears to be responsible for the observed defects. The presence of polyploid and aneuploid cells is consistent with a delay or arrest in the M phase of a substantial fraction of the cells expressing Tat, suggesting that mitotic spindle checkpoints are overridden following Tat expression.

Anaphase↗
PubMed1995

Protein-protein interactions between human nuclear lamins expressed in yeast.

Protein-protein interactions between the nuclear lamins are responsible for the assembly of the nuclear lamina, a meshwork of intermediate filaments associated with the nuclear envelope inner membrane. We have used the yeast two-hybrid system to examine the interactions between the predominant human nuclear lamins expressed as GAL4 fusion proteins in Saccharomyces cerevisiae. Lamin A, prelamin A, lamin B1, and lamin C were able to form homodimers as well as heterodimers. Analysis of the different structural domains of lamin B1 demonstrated that the second half of coil 2 of the rod domain was necessary for the formation of the most stable homodimers. The results show that the yeast two-hybrid system can be used to study the interactions between structural proteins and their domains.

Cloning, Molecular↗
PubMed2001

Electrophoretic plugging of nuclear pores by using the nuclear hourglass technique.

The nuclear hourglass technique (NHT) was recently introduced as a novel technique that measures the electrical nuclear envelope (NE) conductance of isolated Xenopus laevis oocyte nuclei. The main conclusion drawn from NHT work so far is that nuclear pore complexes (NPCs) of oocytes are in an electrically open state under physiological conditions, with a mean conductance of 1.7 nS per NPC. Since nuclear patch-clamp data indicate that usually NPCs are electrically closed, our work has been challenged by the notion that NHT cannot assure a high resistance seal ("gigaseal") between glass wall and NE like that required for patch-clamp experiments. Thus, NHT could have dramatically underestimated NE electrical resistance. Here we demonstrate that NHT does not require a gigaseal for accurate NE conductance measurements. In addition, we present experimental conditions where mean single NPC electrical conductance is reduced 26-fold due to electrophoretic plugging by negatively charged nucleoplasmic macromolecules. In addition, data indicate that under physiological conditions (i.e., when macromolecules are offered in the cytosolic solution) the nuclear surface is heavily folded, underestimating "true" NE surface by a factor of 2.6. When "true" NE surface area is taken into consideration, modified values of mean single NPC conductances of 654 pS for electrically open conditions and 25 pS for electrically plugged conditions can be calculated. We conclude that the large overall NE conductance detected with the nuclear hourglass technique in intact Xenopus laevis oocyte nuclei can be explained by the sum of single NPC conductances in the pS range, as long as open probability is high. This confirms previous patch-clamp work concerning single NPC conductance, but disagrees with the view that mean open probability of NPC channels is usually low.

Active Transport, Cell Nucleus↗
PubMed1999

Assembly and disassembly of the peripheral architecture of the plant cell nucleus during mitosis

The architecture of the nuclei of higher plants includes a structure similar to the nuclear lamina of vertebrates. Changes in this structure were monitored during mitosis in carrot (Daucus carota L.) and celery (Apium graveolens L.) cells by immunofluorescence microscopy using an antibody that recognized the nuclear-matrix protein NMCP1. This protein has been shown to be localized exclusively at the periphery of the nucleus (K. Masuda et al. 1997, Exp Cell Res 232: 173-187). Immunofluorescence was recognized throughout cells in mitotic metaphase, although it was distributed predominantly in the mitotic spindle zone. At late anaphase or telophase, the immunofluorescence was localized around each set of daughter chromosomes. Immunofluorescence in newly formed daughter nuclei was restricted to the periphery of nuclei. This behavior was very similar to that of the nuclear lamina of vertebrates, suggesting that the structure located between the nuclear envelope and the chromosomes in plants disassembles and assembles in parallel with the disintegration and re-formation of the nuclear envelope.

Journal Article↗
PubMed1988

Comparison of photodynamic inactivation of experimental stomach tumors sensitized by acridine orange or hematoporphyrin derivatives.

The photodynamic inactivations of Walker carcinosarcoma 256 stomach tumors by the concomitant use of acridine orange (AO) and argon laser, and by the combined use of hematoporphyrin derivatives (HPD) and dye laser were compared. Wistar rats bearing stomach tumors of 4-6 mm in diameter 5-10 days after their implantation were injected intraperitoneally with 40 mg/kg body weight of AO or HPD, and 24 h later their stomach tumors were exposed to argon laser at 488 nm or dye laser at 630 nm, respectively, at an intensity of 15 mW/cm2 for 20 min. Temperature rise was less than 3 degrees C during irradiation. Seven days after irradiation, complete or partial necrosis with sparing of the surrounding mucosa was seen histologically in all rats treated by the two therapy methods. Phase contrast and electron microscopy showed nuclear pyknosis and damage to the inner layer of the nuclear envelope in tumor cells treated with AO and argon laser, while cytotoxicity involved damage to the outer layer of the nuclear envelope and intracytoplasmic organelles in tumor cells treated with HPD and dye laser. This difference between AO and HPD is considered to be a difference in their intracellular localization in tumor cells.

Acridine Orange↗
PubMed1995

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent.

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4-5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

Animals↗
PubMed1988

Ultrastructural autoradiographic localization of exogenous arachidonic acid in cultured endothelial and smooth muscle cells.

The uptake and intracellular localization of exogenous arachidonic acid (AA) were investigated in cultured endothelial (EC) and smooth muscle cells (SMC) isolated from bovine aorta. The [14C]AA uptake was assessed biochemically and by light and electron microscopic autoradiography. The highest values of silver grain surface density were associated with the mitochondria, lysosomes, and the Golgi apparatus of the EC. The grain linear density was greater on the nuclear envelope than on plasmalemma. On SMC, the grain density was highest on lipid droplets whereas the linear densities of the nuclear envelope and plasmalemma were similar. The share of each subcellular compartment in the AA distribution was estimated as the percentage of the individual silver grain count out of the total cell-associated radioactivity. The results showed that cytoplasm (including endoplasmic reticulum, ribosomes, and small vesicles) made the main contribution followed by the nucleus and at lower values by other organelles. These subcompartments may represent the intracellular sites from which AA could be mobilized for prostanoid synthesis by EC and SMC.

Animals↗
PubMed1988

Fine structural studies of early mitotic stages in untreated and nocodazole-treated HeLa cells.

When cells in mitosis are treated with nocodazole, a microtubule-disrupting drug, it can be shown in comparison to untreated cells that microtubules are responsible for the polarized formation of indentations, folds, tubes, and crypts of the nuclear envelope in prophase nuclei. No translocation of chromosomes within the nucleus takes place. Microtubules are not necessary for chromosome condensation, nuclear envelope breakdown, the formation of trilaminar kinetochores, and the orientation of sister-kinetochores within one chromosome in relation to each other. The orientation of kinetochores in relation to the mitotic poles, however, is mediated by microtubules. The data shown here support the working hypothesis about chromosome translocation in prophase nuclei which was presented in an earlier paper.

Benzimidazoles↗
PubMed1977

[Ultrastructure of rat cerebral cortex neurons at different levels of motor activity].

The reaction of neurons in rat motor cortex during the intensive motor activity and fatigue caused by swimming was investigated with the electron microscope. In both the cases the nuclear envelope in the neuron perikaryon was sharply enlarged resulting in the appearance of deep invaginations; the nucleolus displaced towards the nuclear envelope. Components of the endoplasmic reticulum and Golgi complex increase in size and number, ribosomes become more numerous, the cytoplasm getting denser. During fatigue the above changes appear more pronounced in addition to mitochondrial swelling, the matrix becomes less dense and cristae less numerous. These changes suggest the intensification of metabolitic activity of the cortical neurons during both the intensive motor activity and fatigue.

Animals↗