Analysis of the distribution of spindle microtubules in the diatom Fragilaria.

The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.     

Microtubule dynamics in the spindle. II. A thermodynamic and kinetic description

We have previously presented a model for the assembly and disassembly of mitotic spindle microtubules (MTs) (Pickett-Heaps et al., 1986). In this paper, we describe the thermodynamics of such spindle MT assembly and present equations to describe the polymerization kinetics of different classes of spindle MTs. These equations are used to predict, in terms of kinetics parameters, the magnitude of forces extant on spindle MTs and to define the critical force needed to halt MT assembly. We calculate several of these forces for a hypothetical model cell; our predicted value for the force generated along kinetochore fibers is in close agreement with measured values taken from living cells. The model and its implications are discussed with reference to other recent models of spindle and MT dynamics.     

Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.     

The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.     

Three-dimensional structure of the central mitotic spindle of Diatoma vulgare

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three- dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements.     

Effect on microtubule dynamics of XMAP230, a microtubule-associated protein present in Xenopus laevis eggs and dividing cells [published erratum appears in J Cell Biol 1995 Mar;128(5):following 988]

The reorganization from a radial [corrected] interphase microtubule (MT) network into a bipolar spindle at the onset of mitosis involves a dramatic change in MT dynamics. Microtubule-associated proteins (MAPs) and other factors are thought to regulate MT dynamics both in interphase and in mitosis. In this study we report the purification and functional in vitro characterization of a 230-KD MAP from Xenopus egg extract (XMAP230). This protein is present in eggs, oocytes, testis and a Xenopus tissue culture cell line. It is apparently absent from non- dividing cells in which an immunologically related 200-kD protein is found. XMAP230 is composed of two isoforms with slightly different molecular masses and pIs. It is localized to interphase MTs, dissociates from MTs at the onset of prophase and specifically binds to spindle MTs during metaphase and anaphase. The dissociation constant of XMAP230 is 500 nM, the stoichiometry of binding to MTs is between 1:8 and 1:4, and the in vivo concentration is approximately 200 nM. Both isoforms are phosphorylated and have reduced affinity for microtubules in mitotic extracts. Analysis of the effect of XMAP230 on MT dynamics by video microscopy shows that it increases the growth rate, decreases the shrinking rate of MTs and strongly suppresses catastrophes. These results suggest that in vivo, XMAP230 participates in the control of the MT elongation rate, stabilizes MTs and locally modulates MT dynamics during mitosis.     

Cross-sectional structure of the central mitotic spindle of Diatoma vulgare. Evidence for specific interactions between antiparallel microtubules

During the transition from prometaphase to metaphase, the cross- sectional area of the central spindle of Diatoma decreases by a factor of nearly two, both at the poles and at the region of overlapping microtubules (MTs) near the spindle equator. The density of spindle MT packing stays approximately constant throughout mitosis. Optical diffraction analysis of electron micrographs shows that the packing of the MTs at the poles at all stages of mitosis is similar to that expected for a two-dimensional liquid. Analysis of the region of overlap reveals more packing regularity: during prometaphase, a square packing emerges that displays sufficient organization by late metaphase to generate five orders of diffraction; during anaphase the packing in the overlap region shifts to hexagonal; at telophase, it returns to square. From the data provided by serial section reconstructions of the central spindle, it is possible to identify the polarity of almost every spindle MT, that is, to identify one pole with which the MT is associated. Near neighbor analyses of MTs in cross sections of the overlap region show that MTs prefer antiparallel near neighbors. These near neighbors are most often found at a spacing of approximately 40 nm center-to-center, while parallel near neighbors in the zone of overlap are spaced essentially at random. These results are evidence for a specific interaction between antiparallel MTs. In some sections definite bridges between MTs can be seen. Our findings show that certain necessary conditions for a sliding filament model of anaphase spindle elongation are met.     

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm

We have studied the effect of taxol on mitosis in Haemanthus endosperm. Immuno-Gold Stain (IGS), a new immunocytochemical method (17), was used to visualize microtubules (MTs) in the light microscope. Observations on MT arrangements were correlated with studies in vivo. Chromosome movements are affected in all stages of mitosis which progresses over at least 10(4) range of taxol concentrations. The three most characteristic effects on MTs are: (a) enhancement of the lateral associations between MTs, seen especially during the reorganization of the polar region of the spindle, (b) promotion of MT assembly, leading to the formation of additional MTs in the spindle and MT arrays in the cytoplasm, and (c) an increase in MT stability, demonstrated in their increased cold resistance. In this report, the emphasis is on the primary, immediate effects, occurring in the first 30 min of taxol action. Effects are detected after a few mins, are reversible, and are concentration/time dependent. The spindle and phragmoplast are remarkably modified due to the enhancement of lateral associations of MTs and the formation of abundant nonkinetochore and polar, asterlike MTs. The equatorial region of the interzone in anaphase may be entirely depleted of MTs, and the spindle may break perpendicular to the spindle axis. Mitosis is completed in these conditions, providing evidence for the motile autonomy of each half-spindle. Trailing chromosome arms in anaphase are often stretched and broken. Chromosome fragments are transported away from the polar regions, i.e., in the direction opposite to that expected (5, 6). This supplies the first direct evidence of pushing by elongating MTs in an anastral higher plant spindle. These observations draw attention to the relation between the lateral association of MT ends to assembly/disassembly and to the role of such an interaction in spindle function and organization.     

Mechanism,function and regulation of microtubule-dependent microtubule amplification in mitosis

Mitotic spindle mediates the segregation of chromosomes in the cell cycle and the proper function of the spindle is crucial to the high fidelity of chromosome segregation and to the stability of the genome. Nucleation of microtubules (MTs) from centrosomes and chromatin represents two well-characterized pathways essential for the assembly of a dynamic spindle in mitosis. Recently, we identified a third MT nucleation pathway, in which existing MTs in the spindle act as a template to promote the nucleation and polymerization of MTs, thereby efficiently amplifying MTs in the spindle. We will review here our current understanding on the molecular mechanism, the physiological function and the cell-cycle regulation of MT amplification.     

Compartmentalized Toxoplasma EB1 bundles spindle microtubules to secure accurate chromosome segregation

Toxoplasma gondii replicates asexually by a unique internal budding process characterized by interwoven closed mitosis and cytokinesis. Although it is known that the centrosome coordinates these processes, the spatiotemporal organization of mitosis remains poorly defined. Here we demonstrate that centrosome positioning around the nucleus may signal spindle assembly: spindle microtubules (MTs) are first assembled when the centrosome moves to the basal side and become extensively acetylated after the duplicated centrosomes reposition to the apical side. We also tracked the spindle MTs using the MT plus end–binding protein TgEB1. Endowed by a C-terminal NLS, TgEB1 resides in the nucleoplasm in interphase and associates with the spindle MTs during mitosis. TgEB1 also associates with the subpellicular MTs at the growing end of daughter buds toward the completion of karyokinesis. Depletion of TgEB1 results in escalated disintegration of kinetochore clustering. Furthermore, we show that TgEB1’s MT association in Toxoplasma and in a heterologous system (Xenopus) is based on the same principles. Finally, overexpression of a high-MT-affinity TgEB1 mutant promotes the formation of overstabilized MT bundles, resulting in avulsion of otherwise tightly clustered kinetochores. Overall we conclude that centrosome position controls spindle activity and that TgEB1 is critical for mitotic integrity.     

Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A-dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.     

A role for NuSAP in linking microtubules to mitotic chromosomes

The spindle apparatus is a microtubule (MT)-based machinery that attaches to and segregates the chromosomes during mitosis and meiosis. Self-organization of the spindle around chromatin involves the assembly of MTs, their attachment to the chromosomes, and their organization into a bipolar array. One regulator of spindle self-organization is RanGTP. RanGTP is generated at chromatin and activates a set of soluble, Ran-regulated spindle factors such as TPX2, NuMA, and NuSAP . How the spindle factors direct and attach MTs to the chromosomes are key open questions. Nucleolar and Spindle-Associated Protein (NuSAP) was recently identified as an essential MT-stabilizing and bundling protein that is enriched at the central part of the spindle . Here, we show by biochemical reconstitution that NuSAP efficiently adsorbs to isolated chromatin and DNA and that it can directly produce and retain high concentrations of MTs in the immediate vicinity of chromatin or DNA. Moreover, our data reveal that NuSAP-chromatin interaction is subject to Ran regulation and can be suppressed by Importin alpha (Impalpha) and Imp7. We propose that the presence of MT binding agents such as NuSAP, which can be directly immobilized on chromatin, are critical for targeting MT production to vertebrate chromosomes during spindle self-organization.     

Near-neighbor analysis of spindle microtubules in the alga Ochromonas

The near-neighbor spacing of microtubules (MTs) in the spindle of the alga Ochromonas is analyzed. The technique of near-neighbor analysis of MTs (as developed by McDonald et al. [9]) in the mid-region of the Ochromonas spindle (overlap) shows that MTs from one pole preferentially associate with MTs from the opposite pole at a center-to-center distance of 35 to 43 nm. However, in the half spindle between the chromosomes and the poles, kinetochore MTs (kMTs) do not preferentially associate with other MTs in the half spindle but instead are arranged essentially at random. Individual polar MTs (MTs attached to one pole), kMTs and free MTs (MTs unattached to the poles) were selected for near-neighbor analysis over their entire lengths. The spacing of MTs in the overlap is compatible with those models for mitosis which propose that separation of the poles is accomplished by sliding between closely spaced MTs of opposite polarity. In contrast to the overlap, the arrangement of MTs in the half spindle is not compatible with MT2MT sliding theories that propose that chromosome movement is accomplished by sliding between kMTs and polar MTs.     

Visualizing kinetochore architecture

Kinetochores are large macromolecular assemblies that link chromosomes to spindle microtubules (MTs) during mitosis. Here we review recent advances in the study of core MT-binding kinetochore complexes using electron microcopy methods in vitro and nanometer-accuracy fluorescence microscopy in vivo. We synthesize these findings in novel three-dimensional models of both the budding yeast and vertebrate kinetochore in different stages of mitosis. There is a growing consensus that kinetochores are highly dynamic, supra-molecular machines that undergo dramatic structural rearrangements in response to MT capture and spindle forces during mitosis.     

Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow

Astral microtubules (MTs) are known to be important for cleavage furrow induction and spindle positioning, and loss of astral MTs has been reported to increase cortical contractility. To investigate the effect of excess astral MT activity, we depleted the MT depolymerizer mitotic centromere-associated kinesin (MCAK) from HeLa cells to produce ultra-long, astral MTs during mitosis. MCAK depletion promoted dramatic spindle rocking in early anaphase, wherein the entire mitotic spindle oscillated along the spindle axis from one proto-daughter cell to the other, driven by oscillations of cortical nonmuscle myosin II. The effect was phenocopied by taxol treatment. Live imaging revealed that cortical actin partially vacates the polar cortex in favor of the equatorial cortex during anaphase. We propose that this renders the polar actin cortex vulnerable to rupture during normal contractile activity and that long astral MTs enlarge the blebs. Excessively large blebs displace mitotic spindle position by cytoplasmic flow, triggering the oscillations as the blebs resolve.     

Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle

A family of microtubule (MT)-binding proteins, Orbit/multiple asters/cytoplasmic linker protein-associated protein, has emerged as an important player during mitosis, but their functional mechanisms are poorly understood. In this study, we used meiotic egg extracts to gain insight into the role of the Xenopus laevis homologue Xorbit in spindle assembly and function. Xorbit immunodepletion or its inhibition by a dominant-negative fragment resulted in chromosome alignment defects and aberrant MT structures, including monopolar and small spindles. Xorbit-depleted extracts failed to nucleate MTs around chromatin-coated beads, indicating its essential requirement for spindle assembly in the absence of centrosomes and kinetochores. Xorbit's MT stabilizing effect was most apparent during anaphase, when spindle MTs depolymerized rapidly upon Xorbit inhibition. Biochemical interaction between a COOH-terminal Xorbit fragment and the kinetochore-associated kinesin centromeric protein E may contribute to Xorbit's role in chromosome congression. We propose that Xorbit tethers dynamic MT plus ends to kinetochores and chromatin, providing a stabilizing activity that is crucial for spindle assembly and chromosome segregation.     

Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase

The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)-dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.     

Dynamic partitioning of mitotic kinesin-5 cross-linkers between microtubule-bound and freely diffusing states

The dynamic behavior of homotetrameric kinesin-5 during mitosis is poorly understood. Kinesin-5 may function only by binding, cross-linking, and sliding adjacent spindle microtubules (MTs), or, alternatively, it may bind to a stable "spindle matrix" to generate mitotic movements. We created transgenic Drosophila melanogaster expressing fluorescent kinesin-5, KLP61F-GFP, in a klp61f mutant background, where it rescues mitosis and viability. KLP61F-GFP localizes to interpolar MT bundles, half spindles, and asters, and is enriched around spindle poles. In fluorescence recovery after photobleaching experiments, KLP61F-GFP displays dynamic mobility similar to tubulin, which is inconsistent with a substantial static pool of kinesin-5. The data conform to a reaction-diffusion model in which most KLP61F is bound to spindle MTs, with the remainder diffusing freely. KLP61F appears to transiently bind MTs, moving short distances along them before detaching. Thus, kinesin-5 motors can function by cross-linking and sliding adjacent spindle MTs without the need for a static spindle matrix.     

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.     

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)–containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.