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PubMed2003

Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI.

BACKGROUND: The recent development of a computer-assisted polarization microscopy system (Polscope) with which the meiotic spindle can be visualized in living oocytes on the basis of its birefringence permits analysis of the meiotic spindles of oocytes subjected to ICSI. Previous studies have shown that the meiotic spindle is not always aligned with the first polar body (PB) in metaphase II human oocytes prepared for ICSI. In the present study, the relationship between the degree of meiotic spindle deviation from the first PB location and ICSI outcome was analysed. METHODS: Oocytes were divided into four groups according to the angle of meiotic spindle deviation from the PB position. The angle of deviation was 0-5 degrees, 6-45 degrees, 46-90 degrees and >90 degrees for groups I to IV respectively. RESULTS: The rates of normal [2 pronuclei (PN)] and abnormal (1PN or >2PN) fertilization did not differ between groups I, II and III. However, the rate of normal fertilization was lower among oocytes in which the meiotic spindle deviation angle was >90 degrees; this led to an increased proportion of tripronucleated zygotes that failed to extrude the second PB. When embryos developed from normally fertilized oocytes were evaluated on day 3 after ICSI, no relationship was found between the angle of meiotic spindle deviation and embryo quality. The meiotic spindle was not detected in only 9% of oocytes, and these showed a higher incidence of fertilization and cleavage abnormalities than did oocytes in which the spindle was detected. When oocytes at metaphase I after cumulus oophorus and corona radiata removal were matured in vitro, the meiotic spindle was detected in 53.8% of those that reached metaphase II. In these in-vitro-matured oocytes the meiotic spindle was always aligned with the first PB, suggesting that misalignment seen in those oocytes matured in vivo resulted from PB displacement during manipulations for cumulus and corona removal. CONCLUSION: High degrees of misalignment between the meiotic spindle and the first PB predict an increased risk of fertilization abnormalities. However, when normal fertilization had occurred, the cleavage potential of embryos developing from such oocytes was not impaired. These findings facilitate the selection of oocytes for ICSI in situations when the creation of supernumerary embryos is to be avoided.

Cell Nucleus↗

PubMed1981

Some characteristics of sleep spindles derived from automatic analysis.

Sleep spindles in eight normal adults were analyzed automatically by a minicomputer. The accuracy of spindle detection was about 90%. Spindle wave frequency was measured by an approximation method for the spindle peaks; this method employed a quadratic equation on prior analog filtering. The accuracy of measurement was within 0.6 msec (SD 1). The average spindle wave frequency was about 13 Hz, but varied depending on the sleep stage and brain region. The frequency was highly variable and displayed no tendency. Concerning spindle duration, short spindles were followed by either short or long spindles, while long spindles rarely were followed by long ones. As sleep progressed, spindle duration tended to become shorter. There was a tendency for one long interval of spindle appearance not to be followed by another long interval of appearance. However, as sleep progressed, the interval of spindle appearance had a tendency to become prolonged.

Adult↗

PubMed1993

Self-expandable spindle for transcatheter vascular occlusion: in vivo experiments. Work in progress.

A mechanical vascular occluder was studied in eight pigs to test its efficacy in arteries of different diameters, stability over time, and safety in placement. The main component is a self-expandable spindle made of tubular stainless steel mesh. Simple steel spindles, double spindles, and Dacron-core spindles were placed in the common carotid artery, main renal artery, renal artery branches, and infrarenal aorta. Follow-up arteriograms were obtained immediately, 30-60 minutes, and (except for aortic spindles) 7 days after deployment. The left common carotid artery was occluded in all pigs, regardless of spindle type. In the main renal artery and its branches, simple steel spindles did not achieve complete occlusion. Double and Dacron-core spindles produced immediate occlusion without recanalization on the 1-week follow-up arteriogram, except for one spindle with an incomplete Dacron core and two spindles that did not completely fill the arterial lumen. Two Dacron-core spindles achieved immediate complete occlusion of the infrarenal aorta. No migration was seen with any spindle.

Animals↗

PubMed1991

Two kinds of resting discharge in cat muscle spindles.

1. The behavior of primary endings of cat soleus muscle spindles was studied during shortening steps carried out at different muscle lengths. 2. Spindles were of two kinds: one, silent spindles, whose afferents fell silent after the shortening, at least over part of the range of lengths tested. The second, spontaneous spindles, resumed firing at all lengths. 3. For silent spindles, the duration of the silent period, measured at lengths where they did recover a resting rate, depended directly on muscle length and became shorter at longer lengths. This is what would be expected if the slack introduced in the spindle by the shortening step was removed more rapidly at longer lengths by the higher passive tension. For spontaneous spindles, on the other hand, the duration of the silent period after the shortening was largely independent of muscle length and depended on the spindle's rate of firing immediately before the shortening. 4. At intermediate lengths the discharge of slack spontaneous spindles remained unaffected by an isometric muscle contraction. It was therefore not possible to produce a pause in the discharge, behavior normally taken as typical of spindles. The discharge could be interrupted by the contraction if this was combined with a large shortening movement. 5. It is proposed that when intrafusal fibers are slackened by a shortening step, the resting discharge in spontaneous spindles is generated by a maintained depolarization of the annulospiral ending resulting from extension of the terminal coils by forces from within the receptor. A shortening contraction compresses the spirals to interrupt the discharge. The sensory endings of silent spindles remain below threshold until the spirals have been opened out sufficiently by external stretch.

Afferent Pathways↗

PubMed1986

[The 4 second sleep spindle periodicity].

Polysomnographic EEGs were recorded from 12 adult volunteers between the ages of 20 and 50 years. They were given placebo, pentobarbital, carbromal, methaqualone, flunitrazepam, lormetazepam and triazolam. The visual evaluation was aimed at finding periodic sequences of centro-parietal sleep spindles at 13-15/s. Four consecutive spindles following each other regularly were defined as the shortest sequence. Placebo showed the following results: a considerable interindividual variance with 2 to 6 such sequences on the one hand and 25 on the other hand for each night. sequences with six periodically consecutive spindles dominated; the longest sequence contained 15 spindles. the inter-spindle distance was 4 seconds (m: 4.09; mean 4.14 s). All hypnotics caused an increase in periodic spindle sequences. Volunteers with a low tendency to have periodic spindle sequences remained at the lower end of the scale when taking hypnotics. Hypnotics increased the amount of longer spindle sequences, however, the average was still at sequences of 4-11 consecutive spindles. The inter-spindle distance became longer with all hypnotics; 4.34 s (carbromal) up to 4.58 s (flunitrazepam). Volunteers with a low tendency to have periodic spindle sequences showed shorter distances between spindles vice versa. Benzodiazepines do not reveal any characteristics due to their pharmacokinetic structure.

Adult↗

PubMed2025

Zea mays Meiotic Spindle Ultrastructure Reveals Kinetochore-Microtubule Interface and Embedded Membrane Components.

UNLABELLED: Introduction: Spindles are microtubules-based machines whose primary function is to accurately segregate chromosomes in both mitotic and meiotic cell division. The structure of spindles is critical for their function; errors in morphology or attachment to chromosomes lead to aneuploidy, potentially resulting in disease, infertility, and lethality. Electron microscopy studies have yielded fine-detail spindle ultrastructures in many plant and animal species, but no studies have investigated the spindle of Zea mays, a critical crop, and cytogenetic model system. METHODS: Here we use electron tomography (ET), reconstruction, and modeling to obtain three-dimensional, nanometer-resolution of the Z. mays meiotic spindle. Structures such as microtubules, kinetochores, vesicles, membrane channels, and nuclear envelope were modeled through a partial spindle reconstruction, and confirmed using immunostaining and live fluorescence microscopy. RESULTS: ET revealed that maize spindles contain 8-18 kinetochore microtubules (kMTs) per kinetochore, which are approximately 776 nm in diameter and 316 nm in depth. Small ∼37 nm vesicles were identified, as well as larger (∼5 µm long, 800 nm wide) membrane structures with channels that allow spindle microtubules to pass through. These membrane channels stain positively for the ER-marker protein disulfide isomerase. Imaging of prophase meiotic cells revealed a cross-hatch microtubule arrangement in the perinuclear ring on the external surface of the nuclear envelope, which also contained type II nuclear grooves with transnuclear microtubules passing from the nucleus to the cytoplasm. CONCLUSIONS: Z. mays meiotic spindles are similar to animal counterparts with a comparable number of kMTs and pre-spindle transnuclear microtubules but also plant-specific features such as Golgi-derived vesicles to assist cell plate formation, internal ER membrane channels, and a perinuclear microtubule ring that aids spindle assembly. Maize kinetochores have an electron-diffuse ball in cup morphology that is comparable in size to Drosophila kinetochores and larger than mammalian kinetochores. .

Zea mays↗

PubMed1999

Ultrastructural anatomy of physiologically identified jaw-muscle spindle afferent terminations onto retrogradely labeled jaw-elevator motoneurons in the rat.

Neuronal microcircuits involving jaw-muscle spindle afferents and jaw-elevator motoneurons were studied via retrograde and intracellular labeling in rats. Initially, trigeminal motoneurons were retrogradely labeled from horseradish peroxidase (HRP) injections into the temporalis and masseter muscles. The intracellular response of jaw-muscle spindle afferent neurons was then characterized during palpation, ramp and hold, and sinusoidal stretching of the jaw-closing muscles. Biotinamide was injected into these neurons, and the tissue was processed for the visualization of HRP and biotinamide. The ultrastructure of 243 intracellularly stained jaw-muscle spindle afferent boutons located within the trigeminal motor nucleus (Vmo) was examined. Eighty-five of these boutons synapsed with motoneurons retrogradely labeled with HRP, and 158 boutons synapsed with unlabeled structures within the Vmo. All spindle afferent boutons contained clear, spherical synaptic vesicles. Although the majority of boutons were S type, a few labeled jaw-muscle spindle afferent boutons possessed a long, narrow cleft, with a subsynaptic cistern comparable to previous descriptions of C-type boutons. Sixty-eight percent of spindle afferent boutons synapsed with large or medium-sized, retrogradely labeled motoneuron dendrites, and 32% synapsed with retrogradely labeled somata. In numerous instances, spindle afferent boutons synapsed with trigeminal motoneuron dendritic or somatic spines. Most of the synapses between spindle afferent boutons and trigeminal motoneuron dendrites were asymmetric, and the greatest percentage of axosomatic synapses between spindle afferents and trigeminal motoneurons were symmetric. Approximately 24% of spindle afferent boutons constituted the intermediate element of a axoaxodendritic or axoaxosomatic assemblage, implying that some jaw-muscle spindle afferent synapses with trigeminal motoneurons are presynaptically modulated.

Afferent Pathways↗

PubMed1987

Heterogeneity of spindle units in the cat tenuissimus muscle.

Three tandem spindles and their nerve supplies, reconstructed by light microscopy of serial transverse sections of the cat tenuissimus muscle, were compared to single spindle units. Each tandem spindle consisted of one large unit containing a dynamic bag1, a static bag2, and several static chain fibers (b1b2c unit) linked by the bag2 fiber to a small unit containing only a bag2 and chain fibers (b2c unit). Most features of primary afferents, secondary afferents, and motor neurons were qualitatively and quantitatively similar in both single and tandem b1b2c units. However, b1b2c units of tandem spindles had a lower density of skeletofusimotor innervation than did single b1b2c spindles. The b2c spindle units differed greatly from single or tandem b1b2c units. The b2c spindle units had fewer intrafusal fibers and incoming axons than either the tandem or single b1b2c units. The motor innervation of b2c units was typified by nonselective gamma axons that coinnervated both bag2 and chain fibers, in contrast to the regular occurrence of both selective and nonselective motor axons in b1b2c spindle units. The afferent located at the equator of b2c units differed in size, branching pattern, and intrafusal distribution of its ending from both the primary and secondary sensory axons of b1b2c units and, therefore, might represent a third category of spindle afferent. Thus, cat tenuissimus muscles contain three types of spindle units that differ in the number and organization of muscular and neural elements. These differences in structure and neural organization among tenuissimus spindle units may be a source for generation of different sensory signals in response to common mechanical or fusimotor stimuli.

Animals↗

PubMed2001

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↗

PubMed1986

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex.

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Potential roles of the nucleotide exchange factor ECT2 and Cdc42 GTPase in spindle assembly in Xenopus egg cell-free extracts.

The ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases. ECT2 contains motifs of cell cycle regulators at its N-terminal domain. We previously showed that ECT2 plays a critical role in cytokinesis. Here, we report a potential role of XECT2, the Xenopus homologue of the human ECT2, in spindle assembly in cell-free Xenopus egg extracts. Cloned XECT2 cDNA encodes a 100 kDa protein closely related to human ECT2. XECT2 is specifically phosphorylated in M phase extracts. Affinity-purified anti-XECT2 antibody strongly inhibited mitosis in Xenopus cell-free extracts. Instead of bipolar spindles, where chromosomes are aligned at the metaphase plane in control extracts, the addition of anti-XECT2 resulted in the appearance of abnormal spindles including monopolar and multipolar spindles as well as bipolar spindles with misaligned chromosomes. In these in vitro synthesized spindle structures, XECT2 was found to tightly associate with mitotic spindles. The N-terminal half of XECT2 lacking the catalytic domain also strongly inhibited spindle assembly in vitro, resulting in the formation of mitotic spindles with a low density. Among the representative Rho GTPases, a dominant-negative form of Cdc42 strongly inhibited spindle assembly in vitro. These results suggest that the Rho family GTPase Cdc42 and its exchange factor XECT2 are critical regulators of spindle assembly in Xenopus egg extracts.

Amino Acid Sequence↗

PubMed1985

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

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

Animals↗

PubMed2004

Roles of polo-like kinase 1 in the assembly of functional mitotic spindles.

BACKGROUND: The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. RESULTS: We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. CONCLUSIONS: Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

Length control of the metaphase spindle.

BACKGROUND: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. RESULTS: Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. CONCLUSIONS: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.

Animals↗

PubMed2002

hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division.

Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.

Animals↗

PubMed2001

Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo.

Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase. The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear. Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo. To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos. We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior-posterior embryonic axis. Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour. Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.

Animals↗

PubMed1995

Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

Anaphase↗

PubMed1995

Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes.

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.

Animals↗