The centriolar cycle in polykaryons containing prematurely condensed chromosomes or telophase-like nuclei]

In fused interphase-mitotic cells, either interphase nuclei are induced to premature chromosome condensation (PCC) or mitotic chromosomes are induced to telophase-like nuclei (TLN) formation. This study concerns structural and functional changes in centrioles of fused cells in which PCC or TLN are induced. Embryonic pig kidney cells were fused using a modified PEG-DMSO-serum method. Cell cycle period of the nuclei was determined before cell fusion using double-labeling autoradiography. Polykaryons containing desirable type of PCC or interphase nuclear combination in TLN were selected on the basis of isotope labeling after being embedded in epon. Selected cells were cut into serial sections and studied under electron microscope. The data obtained showed that centrioles at every interphase period undergo mitotic activation when their nuclei are induced to PCC. They acquire fibrillar halo and form half-spindles. Daughter centrioles at G1, S and G2 periods are also capable of mitotic activation when separated from their mother centriole. Inert centrioles were found in some cells with G1-PCC. When mitotic nuclei are induced to TLN formation, their centrioles also become inactivated. They lose fibrillar halo and mitotic spindles break down. Some mitotic centrioles develop features characteristic of interphase period such as satellites and vacuoles. Induced nuclear and centriolar changes are simultaneous and may be controlled by the same factor. Mitotic factor of mitotic cell partner which induces PCC may also induce interphase centrioles to mitotic activation. Degradation of the mitotic factor leading to TLN formation may also cause the loss of the mitotic activity of centrioles and disorganization of mitotic spindles.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

Centrosome and spindle pole microtubules are main targets of a fluorescent taxoid inducing cell death

Microtubules offer a very large local concentration of binding sites for cytotoxic taxoids or for hypothetical endogenous regulators. Several compounds from diverse sources stabilize microtubules and arrest cell division similarly to the antitumour drug Taxol. We have investigated the subcellular location of the Taxol binding sites, employing a fluorescent taxoid (FLUTAX) that reversibly interacts with the Taxol binding sites of microtubules and induces cellular effects similar to Taxol. The specific binding of FLUTAX to a fraction of the available cellular binding sites effectively inhibits division of cultured human tumour cells at G(2)/M, and triggers apoptotic death. The loci of reversible binding, directly imaged in intact U937 cells treated with cytotoxic doses of fluorescent taxoid are the centrosomes, with a few associated microtubules in interphase cells, and the spindle pole microtubules in mitotic cells, instead of uniformly labelling the microtubule cytoskeleton. Cytoskeletal lesions induced and visualized with FLUTAX consist of microtubule bundles and abnormal mitoses, including monopolar spindles and highly fluorescent multipolar spindles. The multiple asters and monopolar spindles mark arrested U937 leukaemia and OVCAR-3 ovarian carcinoma cells on their path to apoptosis (as well as K562, HeLa, and MCF-7 cells). Depending on the FLUTAX treatment, OVCAR-3 cells died from abnormal mitosis or from a subsequent interphase with double chromatin content and lobulated nuclei (micronuclei), indicating impairment of centrosome separation. Fragmented centrosomes could be observed in FLUTAX-treated non-transformed 3T3.A31 cells, which developed micronuclei but were resistant to apoptosis. These results strongly suggest that centrosomal impairment by taxoid binding during interphase, in addition to the suppression of the kinetochore microtubule dynamics in the mitotic spindle, is a primary cause of cell cycle de-regulation and cell death.     

Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells

Actin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase cells began to disassemble. During this process, which began in the center of the cell, individual fibers shortened, and in a few fibers, adjacent bands of fluorescent myosin could be seen to move closer together. In most cells, stress fiber disassembly was complete by metaphase, resulting in a diffuse distribution of the fluorescent proteins throughout the cytoplasm with the greatest concentration present in the mitotic spindle. The first evidence of actin and myosin concentration in a cleavage ring occurred at late anaphase, just before furrowing could be detected. Initially, the intensity of fluorescence and the width of the fluorescent ring increased as the ring constricted. In cells with asymmetrically positioned mitotic spindles, both protein concentration and furrowing were first evident in the cortical regions closest to the equator of the mitotic spindle. As cytokinesis progressed in such asymmetrically dividing cells, fluorescent actin and myosin appeared at the opposite side of the cell just before furrowing activity could be seen there. At the end of cytokinesis, myosin and actin were concentrated beneath the membrane of the midbody and subsequently became organized in two rings at either end of the midbody.     

Association of BAF53 with mitotic chromosomes

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.     

Exogenous nucleation sites fail to induce detectable polymerization of actin in living cells

Most nonmuscle cells are known to maintain a relatively high concentration of unpolymerized actin. To determine how the polymerization of actin is regulated, exogenous nucleation sites, prepared by sonicating fluorescein phalloidin-labeled actin filaments, were microinjected into living Swiss 3T3 and NRK cells. The nucleation sites remained as a cluster for over an hour after microinjection, and caused no detectable change in the phase morphology of the cell. As determined by immunofluorescence specific for endogenous actin and by staining cells with rhodamine phalloidin, the microinjection induced neither an extensive polymerization of endogenous actin off the nucleation sites, nor changes in the distribution of actin filaments. In addition, the extent of actin polymerization, as estimated by integrating the fluorescence intensities of bound rhodamine phalloidin, did not appear to be affected. To determine whether the nucleation sites remained active after microinjection, cells were first injected with nucleation sites and, following a 20-min incubation, microinjected with monomeric rhodamine-labeled actin. The rhodamine-labeled actin became extensively associated with the nucleation sites, suggesting that at least some of the nucleation activity was maintained, and that the endogenous actin behaved in a different manner from the exogenous actin subunits. Similarly, when cells containing nucleation sites were extracted and incubated with rhodamine-labeled actin, the rhodamine-labeled actin became associated with the nucleation sites in a cytochalasin-sensitive manner. These observations suggest that capping and inhibition of nucleation cannot account for the regulation of actin polymerization in living cells. However, the sequestration of monomers probably plays a crucial role.     

Tubulin composition and microtubule nucleation of a griseofulvin-resistant Chinese hamster ovary cell mutant with abnormal spindles

A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered beta-tubulin subunit as well as wild-type beta-tubulin is temperature-sensitive (ts) for growth at 40.5 degrees C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) [19]. Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain beta-tubulin antibody revealed that both mutant and wild-type beta-tubulins were present in similar proportions in both bipolar spindles at 37 degrees C and multipolar spindles at 40.5 degrees C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant beta-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 degrees C.     

Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs

We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.     

Microtubule rearrangements during mitosis in multinucleate cells

The peroxidase-antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)-mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.     

Uniparental chromosome elimination at mitosis and interphase in wheat and pearl millet crosses involves micronucleus formation, progressive heterochromatinization, and DNA fragmentation

Complete uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanisms underlying selective elimination of the paternal chromosomes during the development of wheat (Triticum aestivum) x pearl millet (Pennisetum glaucum) hybrid embryos. All pearl millet chromosomes were eliminated in a random sequence between 6 and 23 d after pollination. Parental genomes were spatially separated within the hybrid nucleus, and pearl millet chromatin destined for elimination occupied peripheral interphase positions. Structural reorganization of the paternal chromosomes occurred, and mitotic behavior differed between the parental chromosomes. We provide evidence for a novel chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei is different, and heterochromatinization and DNA fragmentation of micronucleated pearl millet chromatin is the final step during haploidization.     

Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.     

The cortical actin cytoskeleton in a Dipteran embryo: Analysis of the spatial reorganization of F-actin aggregates during the early nuclear division cycles

Rhodamine phalloidin-staining was used to study the organization of the cortical actin cytoskeleton of the early Ceratitis capitata embryo. The dynamics of the actin aggregates and their changes in distribution during the formation of the syncytial blastoderm, were followed in detail. It was found that these aggregates formed a shell-like cluster around the interphase nuclei, and concentrated toward the poles of the mitotic apparatus when the nuclei divided. Laser scanning confocal microscopy revealed that aggregates not clustered at the poles of the mitotic apparatus were closely associated with fine fibers of a dense cytoplasmic network of actin filaments.     

Antibodies specific for mitotic human chromosomes

The induction of premature chromosome condensation in an interphase cell immediately following fusion with a mitotic cell suggests the presence of factors in the mitotic cell that are responsible for the transformation of an interphase nucleus into prematurely condensed chromosomes (PCC). Several lines of evidence suggest that these factors are proteins present in the cytoplasm of mitotic cells. The objective of this study was to raise antibodies to the factors responsible for PCC. Cytosol from synchronized mitotic HeLa cells was injected into rabbits in order to obtain antiserum. The IgG fraction from this antiserum reacted with 98% of mitotic HeLa cells when tested by indirect immunofluorescence. Most of the fluorescence was localized on the chromosomes. About 5% of the interphase nuclei also reacted with the antiserum, but 50% of these cells were in early G1. Antigenic reactivity was induced in the condensing interphase chromatin in 31% of the interphase nuclei found in mitotic-interphase fused cells. Rodent cells did not react with the antibody by indirect immunofluorescence. Mitotic HeLa cells were able to induce antigenic reactivity in 23 % of interphase Chinese hamster ovary (CHO) cell nuclei in fused binucleate cells, whereas the converse was not true of mitotic CHO cells. Enzyme digestion and incubation with denaturing agents suggested that antigenic reactivity depended on a DNA-non-histone protein complex.     

The Sac3 homologue shd1 is involved in mitotic progression in mammalian cells

Saccharomyces Sac3 required for actin assembly was shown to be involved in DNA replication. Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression. SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to alpha-tubulin, at M phase. RNA interference suppression of endogenous shd1 caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei. Conversely, increased expression of SHD1 by DNA transfection with shd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells. These results demonstrated that shd1 is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function.     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

Radiation-induced centrosome overduplication and multiple mitotic spindles in human tumor cells

The centrosome is a highly regulated organelle and its proper duplication is indispensable for the formation of bipolar mitotic spindles and balanced chromosome segregation. To elucidate a possible linkage between centrosome duplication and radiation-induced nuclear damage, we examined centrosome dynamics in U2-OS osteosarcoma cells following gamma-irradiation. Nearly all control cells contained one or two centrosomes, and at mitosis more than 97% of the cells displayed typical bipolar spindles. In contrast, over 20% of cells at 48 h after 10 Gy gamma-irradiation contained more than two centrosomes, and 60% of the mitotic cells showed aberrant spindles organized by multiple poles. Remarkably, the cells with multiple centrosomes frequently exhibited changes in size and/or morphology of the nucleus, including micronuclei formation. We conclude that abnormal centrosome duplication could be one of the key events involved in nuclear fragmentation and perhaps even cell death following irradiation.     

Sites of actin filament initiation and reorganization in cold-treated tobacco cells

Cytoskeletal proteins assemble into dynamic polymers that play many roles in nuclear and cell division, signal transduction, and determination of cell shape and polarity. The distribution and dynamics of microtubules (MTs) and actin filaments (AFs) are determined, among other factors, by the location of their nucleation sites. Whereas the sites of microtubule nucleation in plants are known to be located under the plasma membrane and on the nuclear envelope during interphase, there is a striking lack of information about nucleation sites of AFs. In the studies reported herein, low temperature (0 °C) was used to de‐polymerize AFs and MTs in tobacco BY‐2 (Nicotiana tabacum L.) cells at interphase. The extent of de‐polymerization of cytoskeletal filaments in interphase cells during cold treatment and the subcellular distribution of nucleation sites during subsequent recovery at 25 °C were monitored by means of fluorescence microscopy. The results show that AFs re‐polymerized rapidly from sites located in the cortical region and on the nuclear envelope, similarly to the initiation sites of MTs. In contrast to MTs, however, complete reconstitution of AFs was preceded by the formation of transient actin structures including actin dots, rods, and filaments with a dotted signal. Immunoblotting of soluble and sedimentable protein fractions showed no changes in the relative amounts of free and membrane‐bound actin or tubulin.     

Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells.

The interaction between the mitotic spindle and the cellular cortex is thought to play a critical role in stimulating cell cleavage. However, little is understood about the nature of such interactions, particularly in tissue culture cells. We have investigated the role of the spindle midzone in signaling cytokinesis by creating a barrier in cultured epithelial cells with a blunted needle, to block signals that may emanate from this region. When the barrier was created during metaphase or early anaphase, cleavage took place only on the sides of the cortex facing the mitotic spindle. Microtubules on the cleaving side showed organization typical of that in normal dividing cells. On the noncleaving side, most microtubules passed from one side of the equator into the other without any apparent organization, and actin filaments failed to organize in the equatorial region. When the barrier was created after the first minute of anaphase, cells showed successful cytokinesis, with normal organization of microtubules and actin filaments on both sides of the barrier. Our study suggests that transient signals from the midzone of early anaphase spindles are required for equatorial contraction in cultured cells and that such signaling may involve the organization of microtubules near the equator.