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Self-organization of interphase microtubule arrays in fission yeast.

Microtubule organization is key to eukaryotic cell structure and function. In most animal cells, interphase microtubules organize around the centrosome, the major microtubule organizing centre (MTOC). Interphase microtubules can also become organized independently of a centrosome, but how acentrosomal microtubules arrays form and whether they are functionally equivalent to centrosomal arrays remains poorly understood. Here, we show that the interphase microtubule arrays of fission yeast cells can persist independently of nuclear-associated MTOCs, including the spindle pole body (SPB)--the centrosomal equivalent. By artificially enucleating cells, we show that arrays can form de novo (self-organize) without nuclear-associated MTOCs, but require the microtubule nucleator mod20-mbo1-mto1 (refs 3-5), the bundling factor ase1 (refs 6,7), and the kinesin klp2 (refs 8,9). Microtubule arrays in enucleated and nucleated cells are morphologically indistinguishable and similarly locate to the cellular axis and centre. By simultaneously tracking nuclear-independent and SPB-associated microtubule arrays within individual nucleated cells, we show that both define the cell centre with comparable precision. We propose that in fission yeast, nuclear-independent, self-organized, acentrosomal microtubule arrays are structurally and functionally equivalent to centrosomal arrays.

Cell Nucleus↗

Development of an interphase fluorescent in situ hybridization (FISH) test to detect t(8;21) in AML patients.

The translocation (8;21) is a chromosome abnormality associated with acute myeloid leukemia (AML). As a consequence of the translocation the AML1 (CBFA2) gene in the 21q22 region is fused to the ETO(CDR,MTG8) gene in the 8q22 region, resulting in one transcriptionally active gene on the 8q- derivative chromosome. In this report we demonstrate the use of a highly specific dual-colour FISH method for the detection of t(8;21) on interphase cells. Genomic probes able to detect the chimeric AML1/ETO gene on the 8q- derivative chromosome were assayed on both normal and leukemic bone marrow and peripheral blood samples. Cut-off values were established by independent analysis of 15 bone marrow specimens negative for the translocation. The cut-off value of positive nuclei was determined to be 2% and the cut-off value for both positive nuclei and nuclei of uncertain classification, 4%. Persistence of cells above these cut-off values was interpreted as persistence of the mutated clone. A total of 36 samples at different disease stages were tested. Interphase cytogenetics detected the translocation at the onset and relapse in the BM or the PB of 14 AML patients with t(8;21). The technique appears to be an alternative tool to both conventional cytogenetics and reverse transcription polymerase chain reaction (RT-PCR) for the monitoring of disease during patients' follow-up. By enabling the analysis of individual cells, interphase FISH is ideal for clonality studies both for clinical and experimental applications.

Acute Disease↗

Detection of BCR/ABL rearrangements in adult acute lymphoblastic leukemia using a highly sensitive interphase fluorescence in situ hybridization method (D-FISH).

INTRODUCTION: One hundred-and-six adult cases of acute lymphoblastic leukemia were prospectively investigated using a highly sensitive interphase fluorescence in situ hybridization assay which utilizes DNA probes that detect a double BCR/ABL fusion signal (D-FISH) in cells carrying the t(9;22) to evaluate the reliability and specificity of this method for the detection of the Ph translocation. The results were compared with those obtained in the same cases by conventional cytogenetics and by reverse-transcription polymerase chain reaction. MATERIALS AND METHODS: The study was performed using DNA probes that span the common breakpoints of the t(9;22) translocation and that detect a double BCR/ABL fusion in cells carrying this karyotypic anomaly, one on the abnormal chromosome 9 and one on the Ph chromosome. RESULTS: Interphase D-FISH detected a high number of rearranged cases (22/106) compared to conventional cytogenetics (15/106) and RT-PCR (21/106). CONCLUSION: Interphase D-FISH emerges as a reliable, fast and relatively inexpensive tool for the detection of BCR/ABL rearrangements in adult ALL patients at diagnosis. It has a sensitivity clearly higher than conventional karyotyping and it may prove also superior to that of RT-PCR in cases with unusual BCR/ABL breakpoints. Our results suggest that D-FISH might be considered as the initial test for the diagnosis of Ph+ adult ALL.

Adolescent↗

Interphase cytogenetics and competitive RT-PCR for residual disease monitoring in patients with chronic myeloid leukaemia during interferon-alpha therapy.

There is a need for fast and sensitive methods to evaluate the response of patients with chronic myeloid leukaemia (CML) to interferon-alpha (IFN-alpha) therapy to complement cytogenetic analysis of Philadelphia (Ph) chromosome-positive metaphases. We have used interphase FISH (fluorescence in situ hybridization) and competitive RT-PCR (reverse transcriptase-polymerase chain reaction) techniques for detection of BCR-ABL-positive cells to measure suppression of leukaemic clone in a series of 51 follow-up samples from 24 CML patients undergoing IFN-alpha treatment. Interphase FISH analysis of the malignant clone in bone marrow using BCR and ABL probes was found to be highly correlated to conventional G-banding metaphase examination (r = 0.98). RT-PCR quantification of BCR-ABL mRNA transcripts in blood also showed a high degree of concordance with the proportion of Ph-positive metaphases (r = 0.93). In addition, the degree of cytogenetic response did not influence the equivalence between karyotype analysis and molecular methods. We concluded that interphase FISH and competitive RT-PCR provide reliable information on residual tumour burden and response to IFN-alpha in CML patients. These molecular methods may significantly improve the efficiency of residual disease monitoring during IFN-alpha therapy of CML.

Antineoplastic Agents↗

Interphase fluorescence in situ hybridization and spectral karyotyping reveals hidden genetic aberrations in children with acute lymphoblastic leukaemia and a normal banded karyotype.

Twenty-two cases of childhood acute lymphoblastic leukaemia (ALL) with normal G- or Q-banded karyotypes were studied by interphase fluorescence in situ hybridization (FISH) and spectral karyotyping. Probes detecting MLL, BCR/ABL and TEL/AML1 rearrangements were used for the interphase studies, along with centromere-specific probes from chromosomes 17 and X. In 10 patients (45%), previously undetected aberrations were demonstrable. Specific gene rearrangements and structural changes were found in six cases and numerical changes in five. Five of these aberrations have previously been reported to have an impact on prognosis. Three cases were massively hyperdiploid and, in one, the prognostically important BCR/ABL fusion was detected. In addition, a near-haploid karyotype with 27 chromosomes was found in one patient and TEL/AML1 rearrangements were detected in two cases. This study indicates that about half of childhood ALL cases with apparently normal karyotypes harbour genetic aberrations that may be detected using interphase FISH and spectral karyotyping.

Adolescent↗

Application of the multi-colour FISH to interphase nuclei and metaphase spreads for simultaneous examination of monosomy 7 and trisomies 8 and 11 in acute myelocytic leukaemia (AML).

We have used the multi-colour (three) fluorescence in situ hybridization (FISH) technique based on the ratio labelling for the detection of monosomy 7 and trisomies 8 and 11 in 13 cases of acute myeloid leukaemia (AML). Two out of the 13 AML cases showed monosomy 7 and two out of the remaining cases exhibited trisomy 8 in interphase nuclei. Three of these results were confirmed by metaphase-FISH study. Trisomy 11 was not found either by the interphase FISH study or in the metaphase FISH study. These results demonstrate the potential power of multi-colour FISH using ratio labelling to produce more fluorescein colour in interphase nuclei for the detection of aneuploidies in leukaemia.

Adolescent↗

Cut1/separase C-terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formation.

BACKGROUND: The separase-securin complex is required for anaphase. Separase activated by securin destruction cleaves the cohesin subunit Scc1/Rad21 enriched in kinetochores. Fission yeast Cut1/separase resides in interphase cytoplasm and mobilizes to the spindle and the spindle pole bodies (SPBs) in mitosis, while Cut2/securin remains in the nucleus from interphase to metaphase, and temporarily locates at the short spindle. RESULTS: We here report a novel SPB-led dynamic nuclear movement in fission yeast, when the Cut1 C-terminal fragment is over-expressed. The tip of the pointed nucleus contained both SPB and centromeric DNA, and rapidly moved along the bundled cytoplasmic microtubules. The same pointed nucleus was produced when the human separase C-fragment was over-expressed. The pointed nuclear formation did not require the protease site of separase, but required the conserved C-terminus and a microtubule- and kinetochore-binding protein Mtc1/Alp14, a homologue of frog XMAP215 and budding yeast Stu2. The movement-inducing C-fragment should be cytoplasmic, as the pointed nucleus was abolished when the fragment contained the NLS (nuclear localization signal). CONCLUSIONS: Overproduced separase C-fragment abolishes correct SPB-positioning in interphase. Resulting pointed nuclear formation (alternatively called 'pigtail movement') requires cytoplasmic microtubules and Mtc1/Alp14.

Catalytic Domain↗

Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase.

We have investigated the organization of transgenes delivered by particle bombardment into the wheat genome, combining conventional molecular analysis with fluorescence in situ hybridization (FISH) and three-dimensional confocal microscopy. We selected a representative population of transformed wheat lines and carried out molecular and expression analysis. FISH on metaphase chromosomes showed that transgene integration sites were often separated by considerable lengths of genomic DNA (>1 Mbp), or could even be on opposite chromosome arms. Plants showing multiple integration sites on a single chromosome were selected for three-dimensional confocal analysis of interphase nuclei in root and embryo tissue sections. Confocal microscopy revealed that these sites lay in close physical proximity in the interphase nuclei. Our results clearly show that multiple transgenes physically separated by large intervening regions of endogenous DNA at metaphase can be brought together at interphase. This may reflect the original physical organization of the endogenous DNA at the moment of transformation, with DNA strand breaks introduced into several co-localized DNA loops by the intruding gold particles. Alternatively, the transgenes may be brought together after transformation, either by an ectopic homologous pairing mechanism, or by recruitment to a common transcription site.

Chromosome Mapping↗

[Interphase cytogenetics with DNA-probes for chromosome 8 to detect circulating tumor cells in breast cancer patients].

The detection of micrometastases in the bone marrow or peripheral blood of cancer patients is increasingly used for a more sensitive tumor staging and prognostication. The potential value of the currently used techniques for the detection of epithelial antigens by RT-PCR or immunohistochemistry in respect of specificity is currently controversially discussed. In the present study we demonstrate a new approach which enables the direct visualization of the tumor specific alteration of chromosome 8 in circulating tumor cells. We have therefore studied breast cancer patients with various tumor stages and tried to determine the frequency of circulating tumor cells in the peripheral blood by using interphase cytogenetics for chromosome 7 and 8. Imprints of primary breast cancers and cytospins with circulating tumor cells of corresponding patients were studied in a blinded fashion. The blood samples were generated by immunomagnetic enrichment of circulating tumor cells from peripheral blood by ferrofluid and centrifugation onto cover slips. These cytospins were then hybridized with centromer probes 7 and 8. After analyzing 27 patients with benign as well as malignant breast tumors we can demonstrate that the chromosomal pattern between malignant tumor and corresponding circulating tumor cells is identical. Furthermore, the detection of circulating tumor cells directly correlates with the primary tumor stage. We did not find any cells with chromosome 8 alterations in the patients with benign disease. Surprisingly, even in early breast cancers (T1N0) interphase cytogenetics identified circulating tumor cells in 2 out of 4 patients. In conclusion, interphase cytogenetics represent a non-invasive, sensitive and specific assay for the direct visualization of circulating tumor cells in the peripheral blood. The prognostic value of these findings remains to be further evaluated in larger prospective studies.

Breast Neoplasms↗

c-Myc induces chromosomal rearrangements through telomere and chromosome remodeling in the interphase nucleus.

In previous work, we showed that telomeres of normal cells are organized within the 3D space of the interphase nucleus in a nonoverlapping and cell cycle-dependent manner. This order is distorted in tumor cell nuclei where telomeres are found in close association forming aggregates of various numbers and sizes. Here we show that c-Myc overexpression induces telomeric aggregations in the interphase nucleus. Directly proportional to the duration of c-Myc deregulation, we observe three or five cycles of telomeric aggregate formation in interphase nuclei. These cycles reflect the onset and propagation of breakage-bridge-fusion cycles that are initiated by end-to-end telomeric fusions of chromosomes. Subsequent to initial chromosomal breakages, new fusions follow and the breakage-bridge-fusion cycles continue. During this time, nonreciprocal translocations are generated. c-Myc-dependent remodeling of the organization of telomeres thus precedes the onset of genomic instability and subsequently leads to chromosomal rearrangements. Our findings reveal that c-Myc possesses the ability to structurally modify chromosomes through telomeric fusions, thereby reorganizing the genetic information.

Animals↗

Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1.

H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr137 and Thr154, the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serine- or threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.

Cell Line, Transformed↗

Evaluation of interphase fluorescence in situ hybridization on direct hematological bone marrow smears.

Traditional cytogenetic analysis from bone marrow aspirates is time consuming and frequently suboptimal due to poor viability of cells in culture. Fluorescence in situ hybridization (FISH) with appropriate DNA probes is a potential alternative to routine cytogenetics. Our study examined the reliability of uptake of specific alpha satellite centromere probes from chromosome 18 (D18Z1) and X (DXZ1) and the Yq heterochromatin (pHY3.4) directly from routine hematological bone marrow smears. Altogether 34 separate hybridizations, performed on slides from 20 patients, were scored for fluorescence signals. Cells in interphase were examined with each probe using unstained slides. In addition Giemsa stained slides were destained and then used for interphase FISH. Between 412 and 631 cells were scored for the expected number of signals; 2 for the 18 centromere, 2 for the X centromere in females, one signal for the Yqh in males. The results showed the expected number of signals in 87-97% of cells with the 18 and X probes and 95-97% of cells with the Y probe. Interphase FISH is a reliable, reproducible technique for use on direct bone marrow smears.

Aneuploidy↗

Mouse lymphoma cells that undergo interphase death show markedly increased sensitivity to radiation-induced DNA double-strand breakage as compared with cells that undergo mitotic death.

The relationship between radiation-induced DNA double-strand breakage (dsb) and reproductive death (clonogenicity) for two mouse lymphoma cell lines was compared with that for the fibroblast-like hamster cell line V79. One of the lymphoma lines (STRij-4-2.2), which undergoes rapid disintegration following cytotoxic insult, showed extreme sensitivity to gamma-ray or DNA-associated 125I decay-induced DNA dsb (7 +/- 1 125I decays per clonogenic lethal event). Surprisingly, the other lymphoma line (WEHI-22.1), which does not undergo rapid disintegration, was also much more sensitive to DNA dsb than were V79 cells (17 +/- 1 versus 61 +/- 2 125I decays per clonogenic lethal event). Ultrastructure, DNA degradation, and flow cytometric cell cycle data suggested that both lymphoma cell lines may undergo interphase death, but that the induction of this process in WEHI-22.1 may depend upon blockage in the G2 phase. It is concluded that there are marked differences between the radiation responses of lymphoma and fibroblast lines, that there may be different forms of radiation-induced interphase death, and that the low number of DNA dsb required to produce a clonogenic lethal event in cells undergoing interphase death could explain the radiosensitivity of organs such as ovary, testis and thymus.

Animals↗

Mitosis-inducing factors are present in a latent form during interphase in the Xenopus embryo.

During the conversion to the mitotic state, higher eukaryotic cells activate a cascade of reactions which result in the disintegration of the nuclear envelope, the condensation of the DNA into chromosomes, and the reorganization of the cytoskeleton. In Xenopus, the induction of the mitotic state appears to be under the control of a cytoplasmic factor(s) known as mitosis-promoting factor or MPF. We have developed a rapid and highly sensitive version of an in vitro assay for MPF. The assay uses reconstituted nuclei in interphase cytoplasm from activated Xenopus eggs. The MPF-induced conversion from interphase to mitosis is conveniently monitored by the visual observation of the loss of the nuclear envelope from the substrate nuclei. At near saturating concentrations of MPF, nuclear breakdown requires 20-30 min. Preincubation experiments have revealed that the action of MPF requires only a few minutes and that the disassembly process itself takes up the remainder of the incubation period. Using this cell-free system, we have investigated the observation that protein synthesis is required for the progression through each successive mitotic cycle in the developing Xenopus embryo. A simple explanation for this finding would be that MPF is degraded after each mitosis and then resynthesized before the next mitotic cycle. However, using in vitro reactivation experiments, we have found that MPF is present in a latent, inactive form during interphase. These results suggest that the block in the cell cycle induced by inhibitors of protein synthesis is due to the lack of production of an activator of MPF.

Animals↗

Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis.

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase-arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.

Calcium↗

Oxygen tension and the rates of mitosis and interphase in roots.

The object of this work was to determine the influence of a wide range of oxygen tensions upon the relative rates of respiration, mitosis, and interphase in pea root tips, compared with the normal rates of these processes in air. From the rates of disappearance of mitotic figures in excised tips kept in various oxygen tensions, the relative rates of mitosis were found to decrease gradually from 122 per cent in 100 per cent oxygen to 24 per cent in 0.0007 per cent oxygen. From the mitotic indices of intact seedlings, the relative rates of interphase were found to decrease sharply from 82 per cent in 10 per cent oxygen to 6 per cent in 5 per cent oxygen. The data on relative rates of respiration, mitosis, and interphase in root tips were compared, and it was shown that the three processes are perfectly distinct in their quantitative relationships to low oxygen tensions.

Cell Division↗

Chromatin dynamics in interphase nuclei and its implications for nuclear structure.

Translational dynamics of chromatin in interphase nuclei of living Swiss 3T3 and HeLa cells was studied using fluorescence microscopy and fluorescence recovery after photobleaching. Chromatin was fluorescently labeled using dihydroethidium, a membrane-permeant derivative of ethidium bromide. After labeling, a laser was used to bleach small (approximately 0.4 microm radius) spots in the heterochromatin and euchromatin of cells of both types. These spots were observed to persist for >1 h, implying that interphase chromatin is immobile over distance scales >/=0.4 microm. Over very short times (<1 s), a partial fluorescence recovery within the spots was observed. This partial recovery is attributed to independent dye motion, based on comparison with results obtained using ethidium homodimer-1, which binds essentially irreversibly to nucleic acids. The immobility observed here is consistent with chromosome confinement to domains in interphase nuclei. This immobility may reflect motion-impeding steric interactions that arise in the highly concentrated nuclear milieu or outright attachment of the chromatin to underlying nuclear substructures, such as nucleoli, the nuclear lamina, or the nuclear matrix.

3T3 Cells↗

Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis.

The mechanisms of localization and retention of membrane proteins in the inner nuclear membrane and the fate of this membrane system during mitosis were studied in living cells using the inner nuclear membrane protein, lamin B receptor, fused to green fluorescent protein (LBR-GFP). Photobleaching techniques revealed the majority of LBR-GFP to be completely immobilized in the nuclear envelope (NE) of interphase cells, suggesting a tight binding to heterochromatin and/or lamins. A subpopulation of LBR-GFP within ER membranes, by contrast, was entirely mobile and diffused rapidly and freely (D = 0. 41 +/- 0.1 microm2/s). High resolution confocal time-lapse imaging in mitotic cells revealed LBR-GFP redistributing into the interconnected ER membrane system in prometaphase, exhibiting the same high mobility and diffusion constant as observed in interphase ER membranes. LBR-GFP rapidly diffused across the cell within the membrane network defined by the ER, suggesting the integrity of the ER was maintained in mitosis, with little or no fragmentation and vesiculation. At the end of mitosis, nuclear membrane reformation coincided with immobilization of LBR-GFP in ER elements at contact sites with chromatin. LBR-GFP-containing ER membranes then wrapped around chromatin over the course of 2-3 min, quickly and efficiently compartmentalizing nuclear material. Expansion of the NE followed over the course of 30-80 min. Thus, selective changes in lateral mobility of LBR-GFP within the ER/NE membrane system form the basis for its localization to the inner nuclear membrane during interphase. Such changes, rather than vesiculation mechanisms, also underlie the redistribution of this molecule during NE disassembly and reformation in mitosis.

Animals↗