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PubMed1982

Visualization of microtubules in interphase and mitotic plant cells of Haemanthus endosperm with the immuno-gold staining method.

A procedure is presented for the immunocytochemical visualization of microtubules in interphase and mitotic cells of Haemanthus endosperm. It includes preservation of microtubules (MTs) with glutaraldehyde and uses colloidal gold, coated with secondary antibodies, in a novel indirect-light microscopic technique: the immuno-gold staining method. This immunocytochemical stain allows us to follow the changes in distribution of MTs during mitosis with greater precision and specificity than allowed by other light microscopic techniques. Many aspects of MT arrangements, as reported from ultrastructural studies, are corroborated and extended. This demonstrates the reliability of the technique. In addition, a number of significant observations were made. These concern (i) the presence of a network of MTs in interphase cells, (ii) the transformation of this network into a spindle-like cage of MTs (the clear zone) surrounding the nucleus during prophase, (iii) the drastic rearrangement of MT distribution during prometaphase, (iv) new evidence for the formation of aster-like arrays of polar MTs during anaphase, and (v) the development of the phragmoplast.

Cell Division↗
PubMed1992

Localization of Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 in interphase and mitotic Burkitt lymphoma cells.

The subcellular distribution of the small Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 has been investigated by using a high-resolution in situ hybridization technique. The distribution patterns in Raji cells of fluorescent oligodeoxynucleotides complementary to each RNA were detected by confocal laser scanning microscopy. Both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. In contrast, use of the same technique indicated an exclusively nuclear location for cellular U2 RNA. In the cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs can bind, and was coincident with the rough endoplasmic reticulum. In cells undergoing mitosis the EBERs became localized around the chromosomes, whereas the protein kinase remained uniformly distributed in the cytoplasm. A cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control.

Base Sequence↗
PubMed1997

Protein kinase C-mediated interphase lamin B phosphorylation and solubilization.

Disassembly of the sperm nuclear envelope at fertilization is one of the earliest events in the development of the male pronucleus. We report that nuclear lamina disassembly in interphase sea urchin egg cytosol is a result of lamin B phosphorylation mediated by protein kinase C (PKC). Lamin B of permeabilized sea urchin sperm nuclei incubated in fertilized egg G1 phase cytosolic extract is phosphorylated within 1 min of incubation and solubilized prior to sperm chromatin decondensation. Phosphorylation is Ca2+-dependent. It is reversibly inhibited by the PKC-specific inhibitor chelerythrine, a PKC pseudosubstrate inhibitor peptide, and a PKC substrate peptide, but not by inhibitors of PKA, p34(cdc2) or calmodulin kinase II. Phosphorylation is inhibited by immunodepletion of cytosolic PKC and restored by addition of purified rat brain PKC. Sperm lamin B is a substrate for rat brain PKC in vitro, resulting in lamin B solubilization. Two-dimensional phosphopeptide maps of lamin B phosphorylated by the cytosolic kinase and by purified rat PKC are virtually identical. These data suggest that PKC is the major kinase required for interphase disassembly of the sperm lamina.

Animals↗
PubMed2003

Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity.

Cdc25A regulates cell cycle progression, has oncogenic and anti-apoptotic activity, and is over-expressed in many human tumors. Phosphorylation by Chk1 and Cds1/Chk2 down-regulates Cdc25A levels in response to genotoxic stresses. Nevertheless, it remains unclear whether Chk1 and Cds1/Chk2 are uniquely responsible for regulating Cdc25A stability during interphase or if other kinase activities contribute. Here we report that treatment of HeLa cells with the cyclin-dependent kinase inhibitor roscovitine caused a concentration- and time-dependent increase in Cdc25A protein levels. Transfection with dominant-negative Cdk mutants demonstrated that only a Cdk2 mutant increased Cdc25A protein levels; Cdk1 and Cdk3 mutants had no effect. The increased Cdc25A protein levels were the result of an increase in the half-life of the protein; no increase in Cdc25A mRNA levels was observed. These results demonstrate Cdk2 kinase activity contributes to the labile nature of Cdc25A during interphase and redefine the nature of the Cdc25A-Cdk2 autoamplification feedback loop.

CDC2-CDC28 Kinases↗
PubMed2003

Nek9, a novel FACT-associated protein, modulates interphase progression.

The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulators with important roles in replication and transcription. Although regarded as a transcription elongator for chromatin template, little is known about the mode of action and involvement in other molecular processes of the mammalian FACT. Here we report the identification of a novel interacting and functional partner of FACT, Nek9. Nek9 forms a stable, approximately 600-kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation-dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr(210)). When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr(210). Cell cycle analysis on the Nek9(dsRNAi) cells directly implicated Nek9 in maintaining proper G(1) and S progression, a role temporally correlated to the formation of a phospho-Nek9-FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression.

Animals↗
PubMed2004

Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.

14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement after activation of the DNA replication and DNA damage checkpoints. Here we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3 zeta affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary high pressure liquid chromatography tandem mass spectrometry analysis. Several known and novel 14-3-3-interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport as well as several other cellular processes. In some cases 14-3-3 binding was cell cycle-dependent, whereas in other cases the binding was shown to be cell cycle-independent. This study adds to the growing list of human 14-3-3-binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes.

14-3-3 Proteins↗
PubMed2000

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis.

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.

Animals↗
PubMed2003

The spindle pole body of the pathogenic yeast Exophiala dermatitidis: variation in morphology and positional relationship to the nucleolus and the bud in interphase cells.

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Exophiala dermatitidis was studied in detail. The SPB was located on the outer nuclear envelope and was 342 +/- 86 nm long in a haploid strain. It consisted of two disk elements that measured 151 +/- 43 nm in diameter and 103 +/- 17 nm in thickness, connected by a rod-shaped midpiece that measured 56 +/- 20 nm in length and 37 +/- 9 nm in diameter. There were considerable variations in size and morphology of interphase SPB. Some disk elements appeared spherical but others were more flattened, and there was variation in electron density. A few SPBs did not have the midpiece. The SPB of a diploid strain was 486 +/- 118 nm long, thus significantly bigger than that of the haploid strain. The SPB tended to be localized away from the nucleolus (110 +/- 48 degrees), but close to the bud (78 +/- 45 degrees). The present study highlights the necessity of observing a large number of micrographs in three-dimensions to describe accurately the ultrastructure of the SPB in yeast.

Animals↗
PubMed1997

Detection of minimal residual disease in an AML patient with trisomy 8 using interphase fish.

Patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) often exhibit clonal chromosomal abnormalities. Using a probe for the centromeric region of chromosome 8, fluorescence in situ hybridization (FISH) on interphase cells was used to detect trisomy 8 in an AML patient whose leukemia was characterised by the karyotype 47, XY, +8, del(9) (q21.1q32). We have demonstrated using FISH the presence of the trisomy at all stages of the patient's disease course (including remission, peripheral blood cell harvest and relapse), whereas conventional karyoptypic analysis was only able to detect the trisomy at diagnosis and clinical relapse. We have also shown using immunophenotyping, cell sorting and FISH, that the trisomic cells in this patient were restricted to the CD34+ subset of blood and bone marrow and could not be found in the CD 34-, T or B cell compartment. Overall we have shown FISH to be a rapid, quantitative method for the detection of cells with numerical chromosome abnormalities. FISH analysis of interphase cells provides valuable information on the status of the whole population, rather than just cycling cells, and can be applied successfully to monitor the level of leukemic cells.

Acute Disease↗
PubMed2002

Significant large-scale chromosome territory movement occurs as a result of mitosis, but not during interphase.

PURPOSE: To investigate large-scale relative movement (position change) of interphase chromosome territories (ICT), as indicated by the separation of chromosome derivatives following exposure to ionizing radiation. MATERIALS AND METHODS: A simple experiment was conducted to investigate large-scale movement of ICT, using whole chromosome 18 painting to measure the frequency of fluorescent ICT in irradiated lymphocytes, cultured over 9 days (seven cell cycles). After chromosome 18 painting, derivative chromosome territory separation was indicated by the observation of three fluorescent ICT in aberrant cells compared with the presence of two fluorescent ICT in normal cells. The frequencies of interphase nuclei containing three fluorescent chromosome territories for unirradiated resting lymphocytes and for lymphocytes acutely irradiated with 2.25 or 5.5 Gy 137Cs gamma-rays were measured for each culture time point of 0, 1, 2, 3, 4, 7 and 9 days. RESULTS: No significant difference was observed in the frequency of three ICT between the average of the controls and irradiated cells cultured for 0, 1 and 2 days. However, irradiated cells cultured for >or=3 days showed a significant increase in the frequency of three fluorescent ICT over those cultured for <or=2 days, as well as over all controls. The frequency of cells containing three territories for cultures that received a dose of 5.5 Gy was about twice that for cultures that received a dose of 2.25 Gy for 3-, 4-, 7- and 9-day cultures. CONCLUSION: The finding that significant large-scale movement was not observed within the first 48 h of cell culture, but was seen in cultures after 48 h, suggests that ICT do not undergo large-scale movement relative to each other until lymphocytes go through mitosis.

Chromosome Aberrations↗
PubMed1988

Thymocyte membrane changes and modifications of interphase death.

A reduction in the rate of interphase death of X-irradiated thymocytes is observed when they are previously treated with triton X-100 or trypsin at low concentrations. The data obtained demonstrate the importance of the structural integrity of the cell surface in the initiation of processes leading to interphase death of lymphoid cells.

Animals↗
PubMed1993

Biological dosimetry of absorbed radiation by C-banding of interphase chromosomes in peripheral blood lymphocytes.

Induction of premature chromosome condensation enables direct observation of radiation-induced cytogenetic damage in non-stimulated, interphase, human peripheral blood lymphocytes. This phenomenon can be explored in radiation protection for biological dosimetry in instances of accidental exposure to ionizing radiation. Quantification of an exposure by means of this approach has been limited so far mainly to the analysis of chromosome fragments. This limitation is due to the fact that conventional Giemsa staining of prematurely condensed chromosomes (PCCs) does not allow visualization of the centromeric regions and, as a result, the identification of dicentrics, centric rings and acentric fragments. In the present report a C-banding procedure, refined to avoid swelling and chromosome distortion of freshly prepared PCCs spreads, is used to identify such aberrations in non-stimulated human lymphocytes. The method allows immediate banding of the centromeric regions and enables scoring of aberrations within a time interval (3-4 h after blood sample withdrawal) that is only a fraction of that normally required when cells stimulated to proliferate are analysed at metaphase. The dose-response for dicentrics and centric rings measured in interphase lymphocytes was found to be similar to that obtained at metaphase. Measurement of dicentrics and centric rings in prematurely condensed chromosomes of human lymphocytes would provide valuable information on radiation dose estimates, especially in cases of extreme urgency.

Chromosome Banding↗
PubMed1999

Interphase chromosome domain re-organization following irradiation.

PURPOSE: To investigate chromosome domain movements during interphase in stationary cell nuclei. MATERIALS AND METHODS: Contact-inhibited primary human fibroblasts were irradiated with 4.0 Gy X-rays, BrdU was added, and air-dried cell preparations made at intervals up to 48 h. Chromosome 4 domain signals (1, 2 and >2) in BrdU negative nuclei (almost exclusively G0/G1) were counted using FISH. A similar experiment was performed using unstimulated human lymphocytes. RESULTS: A very significant rise in nuclei with >2 signals was found within 1 h after radiation. The frequencies observed were in very good agreement with those expected for simple and complex interchanges involving chromosome 4, scored at metaphase in these materials. CONCLUSIONS: The observations constitute evidence for significant domain movement and re-organization within a short time of radiation exposure in G0/G1 interphase nuclei, presumably induced by the formation of inter-domain exchanges. Such re-organization must be a very complicated and delicate topological problem for relaxed chromatin, and must have an important bearing on the interpretation of mechanistic premature chromosome condensation experiments performed whilst it is in operation.

Cell Line↗
PubMed1987

Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis.

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.

Actins↗
PubMed1990

Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis.

We have used autoantibodies to probe the function of three human centromere proteins in mitosis. These antibodies recognize three human polypeptides in immunoblots: CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD). Purified anticentromere antibodies (ACA-IgG) disrupt mitosis when introduced into tissue culture cells during interphase. We have identified two execution points for antibody inhibition. Antibodies injected into the nucleus greater than or equal to 3 h before mitosis prevent the chromosomes from undergoing normal prometaphase movements in the subsequent mitosis. Antibodies injected in the nucleus during late G2 cause cells to arrest in metaphase. Surprisingly, antibodies introduced subsequent to the beginning of prophase do not block mitosis. These results suggest that the CENP antigens are involved in two essential interphase events that are required for centromere action in mitosis. These may include centromere assembly coordinate with the replication of alpha-satellite DNA at the end of S phase and the structural maturation of the kinetochore that begins at prophase.

Anaphase↗
PubMed1990

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

The mouse mAb, mAb 327, that recognizes specifically both pp60v-src and pp60c-src in a wide variety of cells, has been used to determine precisely the various locations of pp60c-src in NIH c-src overexpresser cells, using the technique of immunofluorescence microscopy. In interphase cells, the protein exhibits two main distributions: one that appears uniform and in association with the cell surface and the other that is patchy and juxtanuclear and coincides with the centrosomes. The juxtanuclear aggregation of pp60c-src-containing patches depends on microtubules and does not seem to occur within the Golgi apparatus and the rough ER. At the G2-to-M-phase transition, a drastic change in the localization patterns of pp60c-src takes place. We also report experiments in which the NIH c-src overexpresser cells were exposed to Con A for various times to induce a redistribution of the cell surface Con A receptors. We show that, at each stage of the Con A-mediated endocytotic process, the Con A-receptor complexes redistribute into structures to which pp60c-src appears also to be associated: at first, into patches that form at the cell surface level and then, into a cap that stands at the cell center in a juxtanuclear position and that coincides with the Golgi apparatus. During this capping process, pp60c-src-containing vesicles continue to accumulate in a centriolar spot, as in interphase, Con A-untreated cells, from which Con A is excluded. The significance of the intracellular locations of pp60c-src to the possible functions of the protein is discussed. Also, the distribution patterns of the cellular protein in the NIH c-src overexpresser cells are compared with those of pp60v-src in RSV-transformed cells. The differences observed are discussed in relation with the differences in transforming capacities of the two proteins. Finally, the possible physiological significance of the association between pp60c-src and the structures generated after the binding of Con A to its surface receptors is addressed.

Animals↗
PubMed1991

Subcellular localization and sequence of sea urchin kinesin heavy chain: evidence for its association with membranes in the mitotic apparatus and interphase cytoplasm.

Kinesin was previously immunolocalized to mitotic apparatuses (MAs) of early sea urchin blastomeres (Scholey, J.M., M.E. Porter, P.M. Grissom, and J.R. McIntosh. 1985. Nature [Lond.]. 318:483-486). Here we report evidence that this MA-associated motor protein is a conventional membrane-bound kinesin, rather than a kinesin-like protein. Our evidence includes the observation that the deduced amino acid sequence of this sea urchin kinesin heavy chain is characteristic of a conventional kinesin. In addition, immunolocalizations using antibodies that distinguish kinesin from kinesin-like proteins confirm that conventional kinesin is concentrated in MAs. Finally, our immunocytochemical data further suggest that conventional kinesin is associated with membranes which accumulate in MAs and interphase asters of early sea urchin embryos, and with vesicles that are distributed in the perinuclear region of coelomocytes. Thus kinesin may function as a microtubule-based vesicle motor in some MAs, as well as in the interphase cytoplasm.

Adenosine Triphosphatases↗
PubMed1993

Molecular analysis of the INCENPs (inner centromere proteins): separate domains are required for association with microtubules during interphase and with the central spindle during anaphase.

It has recently been proposed that mitotic chromosomes transport certain cytoskeletal proteins to the metaphase plate so that these proteins are able to subsequently participate in the assembly of the anaphase spindle and the cleavage furrow. To understand how such proteins accomplish their dual chromosomal: cytoskeletal role, we have begun a molecular and functional analysis of the inner centromere proteins (INCENPs), founder members of the class of "chromosome passenger proteins". cDNA clones encoding the open reading frames of the two chicken INCENPs were recovered. The predicted proteins, class I INCENP (96,357 D) and class II INCENP (100,931 D) are novel, and differ from each other by the inclusion of a 38-codon insert within the class II coding region. Transient expression of the chicken INCENPs in mammalian cells confirms that the signals and structures required for the transfer of these proteins from chromosomes to cytoskeleton are evolutionarily conserved. Furthermore, these studies reveal that INCENP association with the cytoskeleton is complex. The amino-terminal 42-amino acid residues are required for transfer of the INCENPs from the chromosomes to the mitotic spindle at anaphase, but not for binding of INCENPs to cytoplasmic microtubules. In contrast, an internal 200 amino acid coiled-coil domain was required for association with microtubules, but dispensable for spindle association. These experiments suggest that proteins required for assembly of specialized cytoskeletal structures during mitosis from anaphase onwards might be sequestered in the nucleus throughout interphase to keep them from disrupting the interphase cytoskeleton, and to ensure their correct positioning during mitosis.

Amino Acid Sequence↗