Die Assembly-Hypothese der Chromosomenbewegung und die Veränderungen der Spindellänge während der Anaphase I in Spermatocyten von Pales ferruginea (Tipulidae,Diptera)

30 living first spermatocytes of the crane-fly Pales (Nephrotoma) ferruginea were photographed at intervals of 1 or 2 minutes throughout anaphase. In 11 cells the spindle length decreased in very early anaphase, in 4 cells it increased, and in the remaining 15 cells no significant changes occurred. There is a positive correlation between the decrease of spindle length during very early anaphase and spindle length at the beginning of anaphase (r = 0.40; P<0.05). During mid-anaphase the spindle length increased in all spermatocytes. The rate of length increase is again positively correlated with spindle length at the beginning of anaphase (r=0.47; P< 0.01). Nevertheless, the total length increase of spindles which have long axes at the beginning of anaphase, is not significantly higher than the length increase of those with short axes. This is so because the duration of spindle elongation is negatively correlated with spindle length at the beginning of anaphase (r=-0.39; P<0.05). In addition the duration of spindle elongation in mid-anaphase seems to be shorter the more the length of the spindle axes decreases during very early anaphase. The average velocity of the syntelically oriented chromosomes in early anaphase is positively correlated with the rate of the spindle elongation during mid-anaphase (r=0.66; P< 0.004). Both velocities are correlated with spindle length at the beginning of anaphase. An attempt was made to explain these phenomena on the basis of the assembly hypothesis of mitosis.     

The effects of colchicine and dinitrophenol on the in vivo rates of anaphase A and B in the diatom Surirella

The rates of chromosome-to-pole movement (anaphase A) and pole-pole separation (anaphase B) in vivo were measured in the pennate diatom Surirella, using differential interference contrast (DIC) light microscopy. In control cells, the rate of anaphase A is 1.6 +/- 0.6 micron/min, the rate of anaphase B is 2.3 +/- 0.3 micron/min, and the extent of anaphase B is 26.7 +/- 9.7% of metaphase spindle length. Colchicine was added to metaphase cells in order to inhibit any further addition of microtubule (MT) subunits onto the spindle. Colchicine, which does not break down the well-ordered Surirella central spindle, caused no significant change in the rate of anaphase A (1.3 +/- 0.3 micron/min) while it significantly decreased both the rate of anaphase B (1.2 +/- 0.4 micron/min) and the extent of anaphase B (14.8 +/- 8.3% of metaphase spindle length). Surirella cells were also treated with the metabolic inhibitor 2-4-dinitrophenol (DNP) in order to test the effects of energy depletion on anaphase. When DNP was added early in anaphase A, prior to the completion of sister chromosome separation, anaphase A was inhibited. When DNP was added after initiation of sister chromosome separation, anaphase A continued to completion, although at a lower rate than control cells (0.5 +/- 0.2 micron/min). Anaphase B was completely inhibited by DNP, but upon recovery from DNP resumed at a normal rate (2.2 +/- 0.5 micron/min) and progressed to a slightly larger than normal extent (44.0 +/- 13.0% of metaphase length).(ABSTRACT TRUNCATED AT 250 WORDS)     

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.     

Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells.

Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK₁ cells have been treated with 5 μg/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol-treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G₁ promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.     

Anaphase motions in dilute colchicine. Evidence of two phases in chromosome segregation

We have examined the rates of chromosome and pole motion during anaphase in HeLa cells using differential interference contrast and polarization optics. In early anaphase both chromosomes and poles move apart. When the chromosomes are separated by a distance about equal to the metaphase spindle length, both chromosomes and poles slow but continue to move at a reduced rate. Throughout anaphase, the chromosomes move faster than the poles, so the chromosome-to-pole distance decreases. Treatment of the cells with about 5 × 10^?8 M colchicine up to 45 min before observation tends to block normal formation of metaphase spindles, but more than half of the cells in metaphase go on through anaphase. In these cells, both chromosome and pole motions are essentially normal until the chromosomes are separated by a distance equal to the length of the metaphase spindle. After that time, chromosome motion is supressed and the poles move slowly toward one another. These data suggest that the mechanism of anaphase motion changes character when the chromosomes become spaced by the metaphase spindle length. We call anaphase before and after that time phase 1 and phase 2, respectively. The results are discussed in the light of a sliding tubule model for chromosome motion.     

Dynamics of Centromeres during Metaphase–Anaphase Transition in Fission Yeast: Dis1 Is Implicated in Force Balance in Metaphase Bipolar Spindle

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.     

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.     

Selective reduction of anaphase B in quinacrine-treated PtK1 cells

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non-kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10-19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 microM to determine energy requirements for chromosome motion. The rate and extent of chromosome-to-pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore-kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore-kinetochore separation by 20%, and addition of 12 microM quinacrine reduced the kinetochore-kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongation, quinacrine-treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373-379, 1985). Metaphase cells treated with 2-10 microM concentrations of quinacrine for 2-5 min reduced spindle lengths by 10-50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose-induced elongation was concomitantly diminished. These data suggest that quinacrine-sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose-induced metaphase spindle elongation.     

Ase1 domains dynamically slow anaphase spindle elongation and recruit Bim1 to the midzone

How cells regulate microtubule cross-linking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control the spindle elongation rate. Using the budding yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces higher elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.     

Effect of metabolic inhibitors on sucrose-induced metaphase spindle elongation and spindle recovery

Hyperosmotic sucrose treatment of metaphase PtK-1 cells has been shown to produce a reversible concentration-dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366-372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62-69, 1984 and 39:373-379, 1985). Addition of metabolic inhibitors to Ham's F-12 salts with deoxyglucose (D/F-12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose-induced spindle elongation; rates and extents are 60-75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F-12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose-treated cells released into D/F-12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP-containing D/F-12 medium and released into DNP-containing D/F-12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose-induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.     

Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase

The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B–dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.     

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.     

A regulatory model for spindle function during mitosis

Structural information on the mitotic spindle of Saccharomyces cerevisiae obtained from isolated whole mount preparations has shown that the spindle undergoes a two-fold increase in length whilst comprising only a single microtubule continuous between the two spindle pole bodies. Further data from immunofluorescence microscopy on the timing of anaphase B has suggested that microtubules do not directly produce the required force, but instead have a more passive role. Here a regulatory function for spindle microtubules during mitosis is explored and the existence of a non-microtubule force-generating system is postulated. Thus it is suggested that the continuous microtubules govern the velocity of anaphase B by providing a resistive force that is itself regulated by the number of microtubules and their rate of polymerization. On this basis a model for the forces acting on a spindle pole body during anaphase is proposed.     

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.     

Consequences of defective tubulin folding on heterodimer levels, mitosis and spindle morphology in Saccharomyces cerevisiae

In budding yeast, the essential roles of microtubules include segregating chromosomes and positioning the nucleus during mitosis. Defects in these functions can lead to aneuploidy and cell death. To ensure proper mitotic spindle and cytoplasmic microtubule formation, the cell must maintain appropriate stoichiometries of alpha- and beta-tubulin, the basic subunits of microtubules. The experiments described here investigate the minimal levels of tubulin heterodimers needed for mitotic function. We have found a triple-mutant strain, pac10Delta plp1Delta yap4Delta, which has only 20% of wild-type tubulin heterodimer levels due to synthesis and folding defects. The anaphase spindles in these cells are approximately 64% the length of wild-type spindles. The mutant cells are viable and accurately segregate chromosomes in mitosis, but they do have specific defects in mitosis such as abnormal nuclear positioning. The results establish that cells with 20% of wild-type levels of tubulin heterodimers can perform essential cellular functions with a short spindle, but require higher tubulin heterodimer concentrations to attain normal spindle length and prevent mitotic defects.     

Intramitotic controls in the fission yeast Schizosaccharomyces pombe: the effect of cell size on spindle length and the timing of mitotic events

We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns. Wild-type fission yeast cells divide at a length of 14 microns. Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell. Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells. This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1. In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40%. By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type. Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type. Thus, the events of mitosis can be extended but not abbreviated. These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.     

Dynein-mediated pulling forces drive rapid mitotic spindle elongation in Ustilago maydis

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein-mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin-5, which is located in the spindle mid-zone. When the spindle reaches approximately 2 microm in length, the elongation rate increases four-fold. This coincides with the appearance of long and less-dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser-mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less-dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off-loaded from the MT plus-end to the cortex.     

Temperature dependence of anaphase chromosome velocity and microtubule depolymerization

The time course of chromosome movement and decay of half-spindle birefringence retardation in anaphase have been precisely determined in the endosperm cell of a plant Tilia americana and in the egg of an animal Asterias forbesi. For each species, the anaphase retardation decay rate constant and chromosome velocity are similar exponential functions of temperature. Over the temperature range at which these cells can complete anaphase, chromosome velocity and retardation rate constant yield a positive linear relationship when plotted against each other. At the higher temperatures where the chromosomes move faster, the spindle retardation decays faster, even though the absolute spindle retardation is greater. Chromosome velocity thus parallels the anaphase spindle retardation decay rate, or rate of spindle microtubule depolymerization, rather than absolute spindle retardation, or the amount of microtubules in the spindle. These observations suggest that a common mechanism exists for mitosis in plant and animal cells. The rate of anaphase chromosome movement is associated with an apparent first-order process of spindle fiber disassembly. This process irreversibly prevents spindle fiber subunits from participating in the polymerization equilibrium and removes microtubular subunits from chromosomal spindle fibers.     

A quantitative study on anaphase movement of chromosomes in living grasshopper spermatocytes

Summary The present investigation has been undertaken to obtain data for the analysis of the chromosome movement at anaphase and the formation of a cleavage furrow. The study is based on simultaneous measurements of the spindle and cell diameters as well as of the chromosome separation in living spermatocyte divisions of the grasshoppers, Podisma sapporense and Acrydium japonicum.Evidence from the present investigation shows that the movement of chromosomes to the poles and the elongation of the spindle are separated in time; the spindle length remains unchanged through out anaphase. Spindle elongation is not associated with the separation of daughter chromosomes. The cell, and the spindle as well, elongate after the chromosomes have reached the poles. Cell elongation may follow the stretching of the spindle, and cause sufficient tension to distort the cell wall, resulting in the subsequent formation of a cleavage furrow.Contribution No. 327 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan. Aided by a grant from the Scientific Research Fund of the Ministry of Education.     

Chromosome segregation and spindle structure in crane fly spermatocytes following Colcemid treatment

Chromosome segregation in primary spermatocytes of the crane fly Nephrotoma suturalis was studied after exposure to Colcemid at doses that did not completely inhibit spindle formation. Colcemid was added either to the medium in which larvae were cultured or to Tricine buffer in which isolated testes were incubated. Patterns of chromosome segregation were analyzed in fixed, Feulgen-stained smears of testes from Colcemid-treated larvae and in living cell preparations. Anomalies observed during the first meiotic division at higher than normal frequencies in Colcemid-treated spermatocytes included anaphase lagging of autosomes, chromosomal strands, tripolar and tetrapolar divisions, and unequal distribution of chromosomes to secondary cells. Following those doses of Colcemid that induced the above anomalies, the length of the birefringent spindle in primary spermatocytes was shorter than normal. This effect on spindle length also was apparent in Giemsastained preparations of fixed cells, in which the two centrosomes at the spindle poles were differentiated from the rest of the cytoplasm. The results indicate a correlation between the inhibition of spindle formation and the induction of anomalous patterns of chromosome segregation.