Organization of mitotic apparatus poles in etoposide-treated CHO-K1 cells

Analyzed in this study is the organization of mitotic spindle poles in CHO-K1 cells dividing after treatment with etoposide (1 h, 25 μM). At various periods after the treatment, we studied the following: (1) the distribution of γ-tubulin in mitotic cells by immunofluorescent staining, (2) the level of post-translational modification of α-tubulin in spindle microtubules by immunoelectron microscopy, and (3) the ultrastructure of mitotic apparatus poles by standard electron microscopy. 48 h after the addition of etoposide, disturbances in the ultrastructure of mitotic spindle poles were observed in etoposide-treated CHO-K1 cells with both bipolar and with multipolar mitotic apparatuses. The increased number of centrioles was unevenly distributed between the mitotic spindle poles; some centrioles did not take an obvious part in the mitotic spindle organization and differed in their number of outgrowing microtubules. Most centrioles were without fibrillar halos. Immunoelectron microscopy showed the differences in the staining of the poles of a multipolar spindle within one cell with antibodies to tyrosinated α-tubulin, whereas the staining of cells with antibodies to acetylated α-tubulin did not reveal such differences. Immunofluorescence staining for γ-tubulin also indicated differing organizations of poles in the same spindle. Our data findings provided the first evidence that the pattern of immunostaining and ultrastructure of mitotic apparatus poles can differ in cells dividing at various time periods after the action of etoposide.     

Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles.     

Mitotic centromere-associated kinesin (MCAK) mediates paclitaxel resistance

Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III β-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.     

Polar organizers mark division axis prior to preprophase band formation in mitosis of the hepaticReboulia hemisphaerica (Bryophyta)

Summary Changes in the pattern of microtubules during the cell cycle of the hepaticReboulia hemisphaerica (Bryophyta) were studied by indirect immunofluorescence using conventional and confocal laser scanning microscopy (CLSM). The first indication that a cell is preparing for division is fusiform shaping of the nucleus accompanied by the appearance of well-defined polar organizers (POs) at the future spindle poles. Microtubules emanating from the POs ensheath the nucleus and eventually develop into the half-spindles of mitosis. Some of the microtubules from each PO pass tangential to the nucleus and interact in the region of the future mitotic equator. A preprophase band (PPB) forms in this region later in prophase and coexists with the prophase spindle. Thus, the plane of division appears to be determined by interaction of opposing arrays of microtubules emanating from POs. Prometaphase is marked by disappearance of the POs, loss of astral microtubules, and conversion of the fusiform spindle of prophase to a truncated, barrel-shaped spindle more typical of higher plants. Restoration of cortical microtubules in daughter cell occurs on the cell side distal to the new cell plate, but nucleation of microtubules is associated with the nuclear envelope and not with organized POs. At the next division POs appear at opposite poles of preprophase nuclei with no evidence of division and migration that is characteristic of cells with centriolar centrosomes. These data lend additional support for the view that mitosis in hepatics is transitional between green algae and higher plants.Abbreviations AMS axial microtubule system - CLSM confocal laser scanning microscopy - MTOC microtubule organizing center - PO polar organizer - PPB preprophase band of microtubules - QMS quadripolar microtubule system - TEM transmission electron microscopy     

Remodeling of nuclear architecture during the cell cycle in Drosophila embryos

Little is known about what determines the nuclear matrix or how its reorganization is regulated during mitosis. In this study we report on a monoclonal antibody, mAb2A, which identifies a novel nuclear structure in Drosophila embryos which forms a diffuse meshwork at interphase but which undergoes a striking reorganization into a spindle-like structure during pro- and metaphase. Double labelings with α-tubulin and mAb2A antibodies demonstrate that the microtubules of the mitotic apparatus co-localize with this mAb2A labeled structure during metaphase, suggesting it may serve a role in microtubule spindle assembly and/or function during nuclear division. That the mAb2A-labeled nuclear structure is essential for cell division and/or maintenance of nuclear integrity was directly demonstrated by microinjection of mAb2A into early syncytial embryos which resulted in a disintegration of nuclear morphology and perturbation of mitosis. © 1996 Wiley-Liss, Inc.     

Reorganization of mitotic apparatus in the etoposide-treated CHO-K1 cells precedes apoptotic death

In a culture of CHO-K1 cells, etoposide (1 h, 25 μM) has been shown to produce interphase arrest, after which the cells resume mitotic division and, after some time, are submitted to apoptotic death. Accumulation of apoptotic cells in the culture follows a gradual increase in the number of multipolar mitoses. Our findings provide the first evidence for differences in the pattern of immunofluorescent staining of multipolar mitotic spindle microtubules with antibodies to α-tubulin, acetylated α-tubulin, and tyrosinated α-tubulin in mitotic cells dividing in the period preceding apoptosis. Moreover, some parts of the multipolar mitotic spindle can differ by the presence of antigenic determinants accessible to anti-tyrosinated α-tubulin antibodies. These abnormalities of the mitotic apparatus are aggravated immediately before the increase in the number of cells submitted to apoptosis. Our data have also shown that some cells pass through at least two mitotic cycles prior to a sharp increase in the number of apoptotic cells in the cell culture.     

NAT10, a nucleolar protein, localizes to the midbody and regulates cytokinesis and acetylation of microtubules

The midbody is a structural organelle formed in late phase mitosis which is responsible for completion of cytokinesis. Although various kinds of proteins have been found to distribute or immigrate to this organelle, their functions have still not been completely worked out. In this study, we demonstrated that NAT10 (N-acetyltransferase 10, NAT10) is not only predominantly distributed in the nucleolus in interphase, but is also concentrated in the mitotic midbody during telophase. The domain in N-terminal residues 549-834 of NAT10 specifically mediated its subcellular localization. Treatment with genotoxic agents or irradiation increased concentration of NAT10 in both the nucleolus and midbody. Moreover, DNA damage induced increase of NAT10 in the midbody apparently accompanied by in situ elevation of the level of acetylated α-tubulin, suggesting that it plays a role in maintaining or enhancing stability of α-tubulin. The depletion of NAT10 induced defects in nucleolar assembly, cytokinesis and decreased acetylated α-tubulin, leading to G2/M cell cycle arrest or delay of mitotic exit. In addition, over-expression of NAT10 was found in a variety of soft tissue sarcomas, and correlated with tumor histological grading. These results indicate that NAT10 may play an important role in cell division through facilitating reformation of the nucleolus and midbody in the late phase of cell mitosis, and stabilization of microtubules.     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.     

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity—a mechanical paradigm that may be important throughout the spindle.     

Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly

The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 micrograms/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in "S" phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.     

Structural and regulatory roles of nonmotor spindle proteins

Chromosome alignment and segregation during cell division rely on a highly ordered bipolar microtubule array called the mitotic spindle. The organization of microtubules into bipolar spindles with focused poles during mitosis requires numerous microtubule-associated proteins including both motor and nonmotor proteins. Nonmotor microtubule-associated proteins display extraordinary diversity in how they contribute to mitotic spindle organization. These mechanisms include regulation of microtubule nucleation and organization, direct and indirect influences on motor function, and control of cell cycle progression. Furthermore, many nonmotor spindle proteins display altered expression in cancer cells emphasizing their important roles in cell proliferation.     

NuMA expression and function in mouse oocytes and early embryos

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage occytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the reformed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.     

Condensation-decondensation of the gamma-tubulin containing material in the absence of a structurally visible organelle during the cell cycle of Physarum plasmodia.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.     

Construction of centrosomes and spindle poles by molecular motor-driven assembly of protein particles

Centrosomes and other microtubule organizing centers are the largest non-membranous organelles in most cells. This morphologically diverse class of organelles shares a common ability to nucleate and organize microtubules in interphase and participates in the formation of mitotic spindles during cell division. This review summarizes recent evidence suggesting that assembly of centrosomes and mitotic spindle poles require transport of large protein particles along microtubules by the molecular motor cytoplasmic dynein.     

The kinesin ATK5 functions in early spindle assembly in Arabidopsis

During cell division, the mitotic spindle partitions chromosomes into daughter nuclei. In higher plants, the molecular mechanisms governing spindle assembly and function remain largely unexplored. Here, live cell imaging of mitosis in Arabidopsis thaliana plants lacking a kinesin-14 (ATK5) reveals defects during early spindle formation. Beginning during prophase and lasting until late prometaphase, spindles of atk5-1 plants become abnormally elongated, are frequently bent, and have splayed poles by prometaphase. The period of spindle elongation during prophase and prometaphase is prolonged in atk5-1 cells. Time-lapse imaging of yellow fluorescent protein:ATK5 reveals colocalization with perinuclear microtubules before nuclear envelope breakdown, after which it congresses inward from the poles to the midzone, where it becomes progressively enriched at regions of overlap between antiparallel microtubules. In vitro microtubule motility assays demonstrate that in the presence of ATK5, two microtubules encountering one another at an angle can interact and coalign, forming a linear bundle. These data indicate that ATK5 participates in the search and capture of antiparallel interpolar microtubules, where it aids in generating force to coalign microtubules, thereby affecting spindle length, width, and integrity.     

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.