The Mechanics of Anaphase B in a Basidiomycete as Revealed by Laser Microbeam Microsurgery

Bayles, C. J., Aist, J. R., and Berns, M. W. 1993. The mechanics of anaphase B in a basidiomycete as revealed by laser microbeam microsurgery. Experimental Mycology 17, 191-199. Cytoplasmic forces were found to be actively pulling on the spindle pole bodies during anaphase B in the dikaryotic, basidiomycete fungus, Helicobasidium mompa. When the spindle of one nucleus was severed with a laser microbeam at mid anaphase B, its two spindle pole bodies separated at a much faster rate than did those of the intact spindle in the other nucleus of the same cell. Since astral microtubule populations apparently reach their maximum during anaphase B in this fungus, we suggest that these microtubules may be involved in the cytoplasmic pulling forces. The spindle appears to act primarily as a governor, regulating the rate at which the spindle pole bodies are separated.     

Spindle positioning in fibroblasts supports an astral microtubule length dependent force generation at the basal membrane.

V79 Chinese hamster fibroblasts that maintain an elongated shape in metaphase occur at a low frequency and often show the spindle asymmetrically positioned. We show here that this aberrant position is corrected in anaphase by an external force, pulling the spindle into place. The force was applied on astral microtubules because spindle motility was hampered when astral microtubules were poorly developed spontaneously, or destroyed by colcemid. Colcemid also abolished the observed downward positioning of centrosomes in anaphase. One pole of the spindle was usually dominant during correction, but occasionally both poles could become subject to pulling making the spindle move perpendicular to the long axis of the cell, which induced reshaping of the cell. The pulling force acted unevenly with short intervals of resting between the pulling. Spindle elongation also varied in rate but showed a different periodicity than translocation of the spindle, and therefore appeared independently regulated. The length of the spindle increased with the length of the cell, and the rate of spindle elongation and pole movement increased with distance moved, indicating that the forces mediated by astral microtubules increase with their length. Arp1/dynactin, not colocalising with tubulin, was more often continuous with microtubules in anaphase B than in metaphase, and was primarily located at the bottom of the cell. Further, shifts in the geometric gravity centre of the cell occurred in the same direction as migration of the spindle. To explain these results, we suggest that astral microtubles transiently anchored at the bottom of the cell are of particular importance for spindle translocation in fibroblasts.     

Anaphase B precedes anaphase A in the mouse egg

Segregation of chromosomes at the time of cell division is achieved by the microtubules and associated molecules of the spindle. Chromosomes attach to kinetochore microtubules (kMTs), which extend from the spindle pole region to kinetochores assembled upon centromeric DNA. In most animal cells studied, chromosome segregation occurs as a result of kMT shortening, which causes chromosomes to move toward the spindle poles (anaphase A). Anaphase A is typically followed by a spindle elongation that further separates the chromosomes (anaphase B). The experiments presented here provide the first detailed analysis of anaphase in a live vertebrate oocyte and show that chromosome segregation is initially driven by a significant spindle elongation (anaphase B), which is followed by a shortening of kMTs to fully segregate the chromosomes (anaphase A). Loss of tension across kMTs at anaphase onset produces a force imbalance, allowing the bipolar motor kinesin-5 to drive early anaphase B spindle elongation and chromosome segregation. Early anaphase B spindle elongation determines the extent of chromosome segregation and the size of the resulting cells. The vertebrate egg therefore employs a novel mode of anaphase wherein spindle elongation caused by loss of k-fiber tension is harnessed to kick-start chromosome segregation prior to anaphase A.     

Kinetochore fibre dynamics outside the context of the spindle during anaphase

Chromosomes move polewards as kinetochore fibres shorten during anaphase. Fibre dynamics and force production have been studied extensively, but little is known about these processes in the absence of the spindle matrix. Here we show that laser-microbeam-severed kinetochore fibres in the cytoplasm of grasshopper spermatocytes maintain a constant length while turning over in a polarized manner. Tubulin incorporates at or near the kinetochore and translocates towards severed ends without shortening the fibre. Consequently, the chromosome cannot move polewards unless the severed fibre reattaches to the pole through microtubules. A potential seclusion artefact has been ruled out, as fibres severed inside spindles behave identically despite being surrounded by the spindle matrix. Our data suggest that kinetochore microtubules constantly treadmill during anaphase in insect cells. Treadmilling is an intrinsic property of microtubules in the kinetochore fibre, independent of the context and attachment of the spindle. The machinery that depolymerizes minus ends of kinetochore microtubules is functional in a non-spindle context. Attachment to the pole, however, is required to cause net kinetochore fibre shortening to generate polewards forces during anaphase.     

Cyclin B degradation leads to NuMA release from dynein/dynactin and from spindle poles

The protein NuMA localizes to mitotic spindle poles where it contributes to the organization of microtubules. In this study, we demonstrate that NuMA loses its stable association with the spindle poles after anaphase onset. Using extracts from Xenopus laevis eggs, we show that NuMA is dephosphorylated in anaphase and released from dynein and dynactin. In the presence of a nondegradable form of cyclin B (Δ90), NuMA remains phosphorylated and associated with dynein and dynactin, and remains localized to stable spindle poles that fail to disassemble at the end of mitosis. Inhibition of NuMA or dynein allows completion of mitosis, despite inducing spindle pole abnormalities. We propose that NuMA functions early in mitosis during the formation of spindle poles, but is released from the spindle after anaphase, to allow spindle disassembly and remodelling of the microtubule network.     

Microtubule dynamics and chromosome motion visualized in living anaphase cells

Chromosome segregation in most animal cells is brought about through two events: the movement of the chromosomes to the poles (anaphase A) and the movement of the poles away from each other (anaphase B). Essential to an understanding of the mechanism of mitosis is information on the relative movements of components of the spindle and identification of sites of subunit loss from shortening microtubules. Through use of tubulin derivatized with X-rhodamine, photobleaching, and digital imaging microscopy of living cells, we directly determined the relative movements of poles, chromosomes, and a marked domain on kinetochore fibers during anaphase. During chromosome movement and pole-pole separation, the marked domain did not move significantly with respect to the near pole. Therefore, the kinetochore microtubules were shortened by the loss of subunits at the kinetochore, although a small amount of subunit loss elsewhere was not excluded. In anaphase A, chromosomes moved on kinetochore microtubules that remained stationary with respect to the near pole. In anaphase B, the kinetochore fiber microtubules accompanied the near pole in its movement away from the opposite pole. These results eliminate models of anaphase in which microtubules are thought to be traction elements that are drawn to and depolymerized at the pole. Our results are compatible with models of anaphase in which the kinetochore fiber microtubules remain anchored at the pole and in which microtubule dynamics are centered at the kinetochore.     

ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO : Direct Microtubule Counts

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK₁) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.     

The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.     

The Saccharomyces cerevisiae Kinesin-related Motor Kar3p Acts at Preanaphase Spindle Poles to Limit the Number and Length of Cytoplasmic Microtubules

The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle–specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential α-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.     

Spindle microtubule dynamics following ultraviolet-microbeam irradiations of mitotic diatoms

Lesions ("ARBs") generated in metaphase and anaphase central spindles of Hantzschia by an ultraviolet microbeam are devoid of microtubules previously present. In vivo, the poleward transverse edge of the lesion invariably loses birefringence poleward, until this segment has vanished; the loss is slow during metaphase and faster at anaphase. The other transverse edge, proximal to the overlap, remains stable until disassembly of the whole spindle. We conclude that the central spindle microtubules are not in flux during metaphase to telophase, and that depolymerization of these microtubules takes place only from the end distal to the pole, as during normal spindle disassembly. Microtubule polarity and the creation of free ends may determine which microtubules are disassembled during later mitosis and how disassembly proceeds.     

Mps1 kinase promotes sister-kinetochore bi-orientation by a tension-dependent mechanism

Segregation of sister chromatids to opposite spindle poles during anaphase is dependent on the prior capture of sister kinetochores by microtubules extending from opposite spindle poles (bi-orientation). If sister kinetochores attach to microtubules from the same pole (syntelic attachment), the kinetochore-spindle pole connections must be re-oriented to be converted to proper bi-orientation. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension. Mps1 is another evolutionarily conserved protein kinase, required for spindle-assembly checkpoint and, in some organisms, for duplication of microtubule-organizing centers. Separately from these functions, however, Mps1 has an important role in chromosome segregation. Here we show that, in budding yeast, Mps1 has a crucial role in establishing sister-kinetochore bi-orientation on the mitotic spindle. Failure in bi-orientation with inactive Mps1 is not due to a lack of kinetochore-spindle pole connections by microtubules, but due to a defect in properly orienting the connections. Mps1 promotes re-orientation of kinetochore-spindle pole connections and eliminates those that do not generate tension between sister kinetochores. We did not find evidence that Ipl1 regulates Mps1 or vice versa; therefore, they play similar, but possibly independent, roles in facilitating bi-orientation.     

Organelle motility within mitotic asters of the fungus Nectria haematococca.

The mitotic asters of the fungus, Nectria haematococca, pull on the spindle pole bodies during anaphase B and help to elongate the central spindle. Because these asters are invisible in vivo, studies of their functions during mitosis have been limited. Invisible asters in other organisms can be studied in vivo because of visible, membranous organelles that are held or transported within them. This is the first report of intra-astral motility of organelles in a fungus, and it lays the foundation for additional studies of aster function in vivo. Using phase-contrast, video-enhanced microscopy, we observed directed motility of mitochondria, small vesicles of various kinds, lipid bodies and, rarely, small vacuoles within the astral region during anaphase B. Both bidirectional motility--toward and away from the spindle pole body--and reversal of direction by an individual organelle were common. Organelles usually did not tend to accumulate either within the aster or near the spindle pole. They were drawn toward the spindle pole body from up to 5.0 microns away. Average velocities were 2.3 to 3.2 microns/s, depending on the organelle and its direction of movement. Transmission electron microscopy revealed apparent cross bridging between astral microtubules and mitochondria, vesicles, endoplasmic reticulum, microbodies, and vacuoles. The antimicrotubule drug, methylbenzimidazole-2-ylcarbamate (MBC), destroyed astral microtubules and virtually eliminated intra-astral motility in vivo, whereas the antiactin drug, cytochalasin E, did not greatly affect the frequency of intra-astral motility episodes. The results suggest a role for astral microtubules in intra-astral motility in this fungus.     

The role of self-association in Fin1 function on the mitotic spindle

Stabilization of spindle microtubules during anaphase is essential for proper chromosome segregation. Fin1 is a budding yeast protein that localizes to the poles and microtubules of the spindle during anaphase and contributes to spindle stability. The N-terminal half of Fin1 is phosphorylated at multiple sites by the cyclin-dependent kinase Clb5-Cdk1, and dephosphorylation in anaphase triggers its localization to the spindle. The C-terminal half of Fin1 contains coiled-coil motifs that are required for its self-association. Here we investigated the functional importance of the two regions of Fin1. Fin1 mutants lacking the C-terminal coiled-coil domains localized to spindle pole bodies but not along spindle microtubules. These mutants failed to self-associate and displayed reduced binding to microtubules in vitro but were functional in vivo and stabilized anaphase spindles when dephosphorylated. Deletion of the Fin1 C terminus suppressed the lethal phenotypes of the phospho-mutant Fin15A. Our findings suggest that the N-terminal region of Fin1 is sufficient for its regulated function as a spindle-stabilizing factor and that this function involves association with the spindle pole body. The ability of the C-terminal region to promote Fin1 self-association and microtubule binding may underlie the lethal effects of the deregulated Fin15A mutant.     

Bi-orienting chromosomes: acrobatics on the mitotic spindle

To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to bi-orientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.     

Spindle and kinetochore morphology of Dictyostelium discoideum

The metaphase spindle of haploid Dictyostelium discoideum (n = 7) is 2 mum long. It consists of some 20 microtubules which seem continuous between the spindle pole bodies and there are about 20 chromosomal microtubules at each end of the spindle. During anaphase the central spindle elongates and the chromosomal microtubules shorten. The spindle length and structure at this stage suggests that lengthening is caused by elongation as well as parallel sliding of the nonchromosomal microtubules. The nuclear envelope remains mostly intact during mitosis, and nuclear separation through medial constriction takes place when the spindle is 6 mum long. Cytokinesis occurs when the spindle is 10 mum long. At that time the kinetochores double in size. During interphase, the spindle pole body separates from the nucleus to a distance of 0.7 mum, and it returns at the onset of the next prophase when it becomes functionally double, thereby starting the formation of a central spindle. When comparing mitosis in the cellular slime molds Polysphondylium violaceum and D. discoideum, several similarities and some differences are apparent.     

Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.     

Mechanism for Anaphase B: Evaluation of “Slide-and-Cluster” versus “Slide-and-Flux-or-Elongate” Models

Elongation of the mitotic spindle during anaphase B contributes to chromosome segregation in many cells. Here, we quantitatively test the ability of two models for spindle length control to describe the dynamics of anaphase B spindle elongation using experimental data from Drosophila embryos. In the slide-and-flux-or-elongate (SAFE) model, kinesin-5 motors persistently slide apart antiparallel interpolar microtubules (ipMTs). During pre-anaphase B, this outward sliding of ipMTs is balanced by depolymerization of their minus ends at the poles, producing poleward flux, while the spindle maintains a constant length. Following cyclin B degradation, ipMT depolymerization ceases so the sliding ipMTs can push the poles apart. The competing slide-and-cluster (SAC) model proposes that MTs nucleated at the equator are slid outward by the cooperative actions of the bipolar kinesin-5 and a minus-end-directed motor, which then pulls the sliding MTs inward and clusters them at the poles. In assessing both models, we assume that kinesin-5 preferentially cross-links and slides apart antiparallel MTs while the MT plus ends exhibit dynamic instability. However, in the SAC model, minus-end-directed motors bind the minus ends of MTs as cargo and transport them poleward along adjacent, parallel MT tracks, whereas in the SAFE model, all MT minus ends that reach the pole are depolymerized by kinesin-13. Remarkably, the results show that within a narrow range of MT dynamic instability parameters, both models can reproduce the steady-state length and dynamics of pre-anaphase B spindles and the rate of anaphase B spindle elongation. However, only the SAFE model reproduces the change in MT dynamics observed experimentally at anaphase B onset. Thus, although both models explain many features of anaphase B in this system, our quantitative evaluation of experimental data regarding several different aspects of spindle dynamics suggests that the SAFE model provides a better fit.     

Polarity of spindle microtubules in Haemanthus endosperm

Structural polarities of mitotic spindle microtubules in the plant Haemanthus katherinae have been studied by lysing endosperm cells in solutions of neurotubulin under conditions that will decorate cellular microtubules with curved sheets of tubulin protofilaments. Microtubule polarity was observed at several positions in each cell by cutting serial thin sections perpendicular to the spindle axis. The majority of the microtubules present in a metaphase or anaphase half-spindle are oriented with their fast-growing or "plus" ends distal to the polar area. Near the polar ends of the spindle and up to about halfway between the kinetichores and the poles, the number of microtubules with opposite polarity is low: 8-20% in metaphase and 2-15% in anaphase cells. Direct examination of 10 kinetochore fibers shows that the majority of these microtubules, too, are oriented with their plus ends distal to the poles, as had been previously shown in animal cells. Sections from the region near the spindle equator reveal an increased fraction of microtubules with opposite polarity. Graphs of polarity vs. position along the spindle axis display a smooth transition from microtubules of one orientation near the first pole, through a region containing equal numbers of the two orientations, to a zone near the second pole where the opposite polarity predominates. We conclude that the spindle of endosperm cells is constructed from two sets of microtubules with opposite polarity that interdigitate near the spindle equator. The length of the zone of interdigitation shortens from metaphase through telophase, consistent with a model that states that during anaphase spindle elongation in Haemanthus, the interdigitating sets of microtubules are moved apart. We found no major changes in the distribution of microtubule polarity in the spindle interzone from anaphase to telophase when cells are engaged in phragmoplast formation. Therefore, the initiation and organization of new microtubules, thought to take place during phragmoplast assembly, must occur without significant alteration of the microtubule polarity distribution.     

Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors

Merotelic kinetochore orientation is a misattachment in which a single kinetochore binds microtubules from both spindle poles rather than just one and can produce anaphase lagging chromosomes, a major source of aneuploidy. Merotelic kinetochore orientation occurs frequently in early mitosis, does not block chromosome alignment at the metaphase plate, and is not detected by the spindle checkpoint. However, microtubules to the incorrect pole are usually significantly reduced or eliminated before anaphase. We discovered that the frequency of lagging chromosomes in anaphase is very sensitive to partial inhibition of Aurora kinase activity by ZM447439 at a dose, 3 microM, that has little effect on histone phosphorylation, metaphase chromosome alignment, and cytokinesis in PtK1 cells. Partial Aurora kinase inhibition increased the frequency of merotelic kinetochores in late metaphase, and the fraction of microtubules to the incorrect pole. Measurements of fluorescence dissipation after photoactivation showed that kinetochore-microtubule turnover in prometaphase is substantially suppressed by partial Aurora kinase inhibition. Our results support a preanaphase correction mechanism for merotelic attachments in which correct plus-end attachments are pulled away from high concentrations of Aurora B at the inner centromere, and incorrect merotelic attachments are destabilized by being pulled toward the inner centromere.     

Merotelic kinetochores in mammalian tissue cells

Merotelic kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian kinetochores typically have binding sites for about 20-25 kinetochore microtubules. In prometaphase, kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic kinetochores based on the ratio of kinetochore microtubules to the correct versus incorrect pole.