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PubMed2004

Proteome analysis of the human mitotic spindle.

The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. Malfunctioning of the spindle can lead to chromosome missegregation and hence result in aneuploidy, a hallmark of most human cancers. Despite major progress in deciphering the temporal and spatial regulation of the mitotic spindle, its composition and function are not fully understood. A more complete inventory of spindle components would therefore constitute an important advance. Here we describe the purification of human mitotic spindles and their analysis by MS/MS. We identified 151 proteins previously known to associate with the spindle apparatus, centrosomes, and/or kinetochores and 644 other proteins, including 154 uncharacterized components that did not show obvious homologies to known proteins and did not contain motifs indicative of a particular localization. Of these uncharacterized proteins, 17 were tagged and localized in transfected mitotic cells, resulting in the identification of six genuine spindle components (KIAA0008, CdcA8, KIAA1187, FLJ12649, FLJ90806, and C20Orf129). This study illustrates the strength of a proteomic approach for the analysis of isolated human spindles and identifies several novel spindle components for future functional studies.

Amino Acid Sequence↗

PubMed1985

Centriole number and the reproductive capacity of spindle poles.

The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.

Animals↗

PubMed1999

Chromosomal influence on meiotic spindle assembly: abnormal meiosis I in female Mlh1 mutant mice.

In mouse oocytes, the first meiotic spindle is formed through the action of multiple microtubule organizing centers rather than a pair of centrosomes. Although the chromosomes are thought to play a major role in organizing the meiotic spindle, it remains unclear how a stable bipolar spindle is established. We have studied the formation of the first meiotic spindle in murine oocytes from mice homozygous for a targeted disruption of the DNA mismatch repair gene, Mlh1. In the absence of the MLH1 protein meiotic recombination is dramatically reduced and, as a result, the vast majority of chromosomes are present as unpaired univalents at the first meiotic division. The orientation of these univalent chromosomes at prometaphase suggests that they are unable to establish stable bipolar spindle attachments, presumably due to the inability to differentiate functional kinetochore domains on individual sister chromatids. In the presence of this aberrant chromosome behavior a stable first meiotic spindle is not formed, the spindle poles continue to elongate, and the vast majority of cells never initiate anaphase. These results suggest that, in female meiotic systems in which spindle formation is based on the action of multiple microtubule organizing centers, the chromosomes not only promote microtubule polymerization and organization but their attachment to opposite spindle poles acts to stabilize the forming spindle poles.

Adaptor Proteins, Signal Transducing↗

PubMed1999

The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

Actins↗

PubMed2000

Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast.

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

CDC28 Protein Kinase, S cerevisiae↗

PubMed2003

Spindle assembly and cytokinesis in the absence of chromosomes during Drosophila male meiosis.

A large body of work indicates that chromosomes play a key role in the assembly of both a centrosomal and centrosome-containing spindles. In animal systems, the absence of chromosomes either prevents spindle formation or allows the assembly of a metaphase-like spindle that fails to evolve into an ana-telophase spindle. Here, we show that Drosophila secondary spermatocytes can assemble morphologically normal spindles in the absence of chromosomes. The Drosophila mutants fusolo and solofuso are severely defective in chromosome segregation and produce secondary spermatocytes that are devoid of chromosomes. The centrosomes of these anucleated cells form robust asters that give rise to bipolar spindles that undergo the same ana-telophase morphological transformations that characterize normal spindles. The cells containing chromosome-free spindles are also able to assemble regular cytokinetic structures and cleave normally. In addition, chromosome-free spindles normally accumulate the Aurora B kinase at their midzones. This suggests that the association of Aurora B with chromosomes is not a prerequisite for its accumulation at the central spindle, or for its function during cytokinesis.

Animals↗

PubMed2000

Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin.

In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus. The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck. Actin has been hypothesized to be involved in all these movements. To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin). We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells. In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function. Furthermore, nuclei segregated efficiently in latrunculin-treated cells. Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning. Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently. Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin.

Actins↗

PubMed1997

Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors.

Two Saccharomyces cerevisiae kinesin-related motors, Cin8p and Kip1p, perform an essential role in the separation of spindle poles during spindle assembly and a major role in spindle elongation. Cin8p and Kip1p are also required to prevent an inward spindle collapse prior to anaphase. A third kinesin-related motor, Kar3p, may act antagonistically to Cin8p and Kip1p since loss of Kar3p partially suppresses the spindle collapse in cin8 kip1 mutants. We have tested the relationship between Cin8p and Kar3p by overexpressing both motors using the inducible GAL1 promoter. Overexpression of KAR3 results in a shrinkage of spindle size and a temperature-dependent inhibition of the growth of wild-type cells. Excess Kar3p has a stronger inhibitory effect on the growth of cin8 kip1 mutants and can completely block anaphase spindle elongation in these cells. In contrast, overexpression of CIN8 leads to premature spindle elongation in all cells tested. This is the first direct demonstration of antagonistic motors acting on the intact spindle and suggests that spindle length is determined by the relative activity of Kar3p-like and Cin8p/Kip1p-like motors.

Anaphase↗

PubMed2003

A functional relationship between NuMA and kid is involved in both spindle organization and chromosome alignment in vertebrate cells.

We examined spindle morphology and chromosome alignment in vertebrate cells after simultaneous perturbation of the chromokinesin Kid and either NuMA, CENP-E, or HSET. Spindle morphology and chromosome alignment after simultaneous perturbation of Kid and either HSET or CENP-E were no different from when either HSET or CENP-E was perturbed alone. However, short bipolar spindles with organized poles formed after perturbation of both Kid and NuMA in stark contrast to splayed spindle poles observed after perturbation of NuMA alone. Spindles were disorganized if Kid, NuMA, and HSET were perturbed, indicating that HSET is sufficient for spindle organization in the absence of Kid and NuMA function. In addition, chromosomes failed to align efficiently at the spindle equator after simultaneous perturbation of Kid and NuMA despite appropriate kinetochore-microtubule interactions that generated chromosome movement at normal velocities. These data indicate that a functional relationship between the chromokinesin Kid and the spindle pole organizing protein NuMA influences spindle morphology, and we propose that this occurs because NuMA forms functional linkages between kinetochore and nonkinetochore microtubules at spindle poles. In addition, these data show that both Kid and NuMA contribute to chromosome alignment in mammalian cells.

Antigens, Nuclear↗

PubMed2001

The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint.

The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.

Aurora Kinases↗

PubMed1973

The muscle spindles in slow and twitch skeletal muscle of the lizard.

1. Responses from stretch receptors, identified as muscle spindles, were recorded in filaments of the nerve supplying a twitch muscle, semimembranosus, and a slow muscle, semitendinosus in the lizard Tiliqua.2. While recording afferent discharges in one filament of the motor nerve, several adjacent filaments were each in turn stimulated repetitively until one was encountered which on stimulation produced a powerful increase in spindle firing. Such an effect of the motor stimulus was interpreted as resulting from intrafusal contraction. Any interference with spindle firing patterns from extrafusal contraction produced by the motor stimulation was removed by differentially blocking the contraction with the drug curare.3. Discharge patterns of spindles in response to a slow stretch of the muscle were compared with the response to the same stretch, but during repetitive stimulation of the motor nerve filament which produced an intrafusal contraction.4. At the initial length, the firing rate of spindles in the twitch muscle was greatly increased by the motor tetanus. There was little further increase in the response during and following stretch of the muscle.5. While the spindles in the slow muscle were only moderately excited by the motor tetanus at the initial length of the muscle, a large increase was recorded during the dynamic component of the stretch. At the new length, the steady-state firing continued at a rate well above that for the initial length.6. The effect of the motor tetanus on the response to stretch of muscle spindles in the slow muscle could be mimicked by adding succinyl choline (5 mug/ml.) to the perfusion solution. Spindles in the twitch muscle did not show a sustained sensitivity to the drug.7. It is suggested that while the different effects of motor stimulation on the responses to stretch of spindles in slow and twitch muscle can be explained by propositions based on the sliding filament theory of contraction, the sustained elevation, at the new length, of firing frequencies of spindles in slow muscle might require an additional explanation.

Action Potentials↗

PubMed1980

Glycogen depletion of bag1 fibers elicited by stimulation of static gamma axons in cat peroneus brevis muscle spindles.

1. The distribution of static fusimotor axons to intrafusal muscle fibres in cat peroneus brevis spindles has been studied with the glycogen depletion method. 2. In each of six experiments three to seven static axons were stimulated. The muscle was subsequently quick-frozen and cut in serial transverse sections that were stained for glycogen. In each muscle, nearly all the spindles were examined for depletion. 3. Intrafusal muscle fibres displaying zones of complete glycogen depletion were observed in fifty-two whole spindles and seven half-spindles. Chain fibres were depleted in forty-eight (92%) of the whole spindles, bag2 fibres in thirty-five spindles (67%) and bag1 fibres in nineteen spindles (36%). 4. Seven other experiments were performed to test whether small amplitude sinusoidal stretching (30--100 Hz) of the muscle might produce glycogen depletion in bag1 fibres. Two hundred and seven spindles were examined. In nearly all of them the glycogen content of the intrafusal muscle fibres was normal. Two muscles had limited atrophic portions within which a few depleted spindles were found. 5. These experiments show that the intrafusal distribution of static gamma axons is not restricted to chain and bag2 fibres but that in a significant number of spindles the bag1 fibres are also supplied by static gamma axons.

Animals↗

PubMed1991

Responses of cat muscle spindles which lack a dynamic fusimotor supply.

1. The experiments reported here support the view that some spindles in the peroneus tertius muscle of the anaesthetized cat lack a nuclear bag1 intrafusal fibre. 2. The bag1 fibre is characterized by the fact that it is innervated exclusively by dynamic fusimotor axons. A method was devised to test each spindle in peroneus tertius for a dynamic fusimotor innervation. The ventral roots containing the muscle's motor supply were subdivided into five portions, approximately equal in terms of the tension they generated, and each piece was stimulated in turn, repetitively, at fusimotor strength, during ramp stretch of the muscle, to look for a large increase in dynamic response. 3. The method allowed confirmation that the majority of spindles in peroneus tertius had a dynamic fusimotor innervation. However, where the dynamic effect was weak and accompanied by a strong static fusimotor action and extrafusal unloading, it risked being overlooked. 4. The confirmatory test for the presence of a bag1 fibre was whether or not the spindle showed a large increase in dynamic response in the presence of the drug succinyl choline (SCh) injected arterially close to the muscle in which the spindle is located. SCh is known to induce a contracture in the bag1 fibre and therefore mimics tonic dynamic fusimotor stimulation. 5. In five experiments, of a total of forty-two spindles with afferents conducting within the group I range, five examples were encountered where there was no increase in dynamic response, either with ventral root stimulation or perfusion with SCh. It was concluded that these were spindles which lacked a bag1 fibre. 6. Passive stretch of such spindles revealed no feature in the response which allowed them to be distinguished from spindles in which the bag1 fibre was present. This conclusion posed the question, what contribution, if any, does the bag1 fibre make to the stretch response? 7. It was possible to show that under some conditions the bag1 fibre did contribute to the response to stretch. If the spindle was conditioned by repetitive nerve stimulation, at fusimotor strength, at a length longer than that at which a test stretch was applied, the response to the stretch was delayed, and it began at a lower rate than after conditioning stimulation at the test length. The delayed response was attributed to the presence of slack in intrafusal fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1975

Discharge of spindle afferents from jaw-closing muscles during chewing in alert monkeys.

The discharge of muscle spindle afferents from monkey spindle afferents from monkey jaw-closing muscles was studied during mastication of natural foods by extracellular recording from the fibers or cell bodies of the tract and mesencephalic nucleus of the fifth nerve. In all, 39 muscle afferents were studied. The spindle associated with 18 of the afferents was positively identified by the afferent's response to gentle, localized palpation of either the temporalis or masseter muscle. Discharge patterns were observed during mastication, and in the majority of cases the qualitative passive response characteristics of the spindle afferent were determined. During steady chewing spindle afferent discharge typically paused briefly during the initial rapid upward part of the chewing cycle. Firing generally began as the jaw slowed its upward movement, and firing rates during the slow grinding portion of the upward movement were within the range of 50-80 spikes/s. All spindles exhibited a brisk discharge during the opening movement, typically within the range of 100-150 spikes/s. One-third of the spindle afferents exhibited a brief, high-frequency burst of firing at the very beginning of the opening movement, presumably as a result of stretch applied to a spindle just previously subjects to fusimotor excitation. Although the results of the study make it clear that spindles in jaw-closing muscles are coactived along with the extrafusal muscle fibers, the fusimotor bias does not seem capable of sustaining discharge in the face of rapid shortening of the muscle. Furthermore, the fact that discharge rate during opening, when the jaw-closing motoneurons are quiescent, is much higher than at any part of the closing cycle, when the motoneurons are active, suggests that the muscle spindles cannot provide the primary excitatory drive to the motoneurons.

Acoustic Stimulation↗

PubMed2006

Inhibition of TRIP1/S8/hSug1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons.

Mitotic spindle poisons (e.g., Taxol and vinblastine), used as chemotherapy drugs, inhibit mitotic spindle function, activate the mitotic spindle checkpoint, arrest cells in mitosis, and then cause cell death by mechanisms that are poorly understood. By expression cloning, we identified a truncated version of human TRIP1 (also known as S8, hSug1), an AAA (ATPases associated with diverse cellular activities) family ATPase subunit of the 19S proteasome regulatory complex, as an enhancer of spindle poison-mediated apoptosis. Stable expression of the truncated TRIP1/S8/hSug1 in HeLa cells [OP-TRIP1(88-406)] resulted in a decrease of measurable cellular proteasome activity, indicating that OP-TRIP1(88-406) had a dominant-negative effect on proteasome function. OP-TRIP1(88-406) revealed an increased apoptotic response after treatment with spindle poisons or with proteasome inhibitors. The increased apoptosis coincided with a significant decrease in expression of BubR1, a kinase required for activation and maintenance of the mitotic spindle checkpoint in response to treatment with spindle poisons. Small interfering RNA (siRNA)-mediated knockdown of TRIP1/S8/hSug1 resulted in a reduction of general proteasome activity and an increase in mitotic index. The siRNA treatment also caused increased cell death after spindle poison treatment. These results indicate that inhibition of TRIP1/S8/hSug1 function by expression of a truncated version of the protein or by siRNA-mediated suppression enhances cell death in response to spindle poison treatment. Current proteasome inhibitor drugs in trial as anticancer agents target elements of the 20S catalytic subcomplex. Our results suggest that targeting the ATPase subunits in 19S regulatory complex in the proteasome may enhance the antitumor effects of spindle poisons.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1997

Cell-cell association directed mitotic spindle orientation in the early development of the marine shrimp Sicyonia ingentis.

During early cleavages of Sicyonia ingentis embryos, mitotic spindle orientations differ between blastomeres and change in a predictable manner with each successive mitosis. From 2nd through 7th cleavages, spindles orient at a 90 degrees angle with respect to the spindle of the parent blastomere. Thus, spindle orientation is parallel to the cleavage plane that formed the blastomere. To determine if specific spindle orientations were intrinsic properties of individual blastomeres, we altered blastomere associations and asked how mitotic spindle orientation was affected in successive cleavages using laser scanning confocal microscopy. Linear embryos were constructed by dissociating 4-cell embryos and recombining the blastomeres in a linear array. The ensuing cleavage (3rd embryonic cleavage) of these linear embryos was parallel to the long axis of the embryo, resulting in four parallel pairs of blastomeres which lay in a common plane that was parallel to the substratum. The 4th cleavage produced a linear embryo with the 16 blastomeres arranged in four parallel quartets. Then, in preparation for 5th cleavage, spindles oriented at a 45 degrees angle (not parallel as in normal development) with respect to the previous cleavage plane. When 8-cell linear embryos were separated into linear half-embryos, subsequent spindle orientations were not like those observed for intact 8-cell linear embryos, but rather regressed to the orientation seen in 4-cell linear embryos. We suggest that the reorientation of mitotic spindles during early cleavage of S. ingentis is neither an intrinsic property nor age dependent, but rather is cell contact related. Further, these results in conjunction with observations of non-manipulated embryos suggest that spindle poles (centrosomes) avoid cytoplasmic regions adjacent to where there is cell-cell contact during early development.

Animals↗

PubMed2005

Cortical capture of microtubules and spindle polarity in budding yeast - where's the catch?

In asymmetric divisions, the mitotic spindle must align according to the cell polarity axis. This is achieved through targeting astral microtubules emanating from each spindle pole to opposite cell cortex compartments. Saccharomyces cerevisiae is a powerful genetic model for dissection of this complex process. Intense research in this yeast has rendered detailed models for a program linking actin organization and spindle orientation along the mother-bud axis. This program requires the separate contributions of Kar9p, a protein guiding microtubules along polarized actin cables, and the polarity determinant Bud6p/Aip3 that marks sites for cortical capture at the bud tip and bud neck. In an added layer of complexity, cyclin-dependent kinase (Cdk) differentially regulates spindle pole function to dictate asymmetric spindle pole fate. Asymmetric contacts established by the spindle poles impart a further layer of extrinsic asymmetry restricting recruitment of Kar9p to the pole destined for the daughter cell. As a result, astral microtubules from a single pole are guided to the bud compartment after spindle assembly. Finally, Cdk might also translocate along astral microtubules in association with Kar9p to modulate microtubule-cortex interactions following spindle alignment. Insertion of the mitotic spindle into the bud neck is driven by the microtubule motor dynein. This process relies on the combined action of microtubule-plus-end-tracking proteins and kinesins that control the cell-cycle-dependent abundance of dynein at microtubule plus ends. Thus, this actin-independent pathway for spindle orientation might also be influenced by Cdk.

Actins↗

PubMed2005

Assembly pathway of the anastral Drosophila oocyte meiosis I spindle.

Oocyte meiotic spindles of many species are anastral and lack centrosomes to nucleate microtubules. Assembly of anastral spindles occurs by a pathway that differs from that of most mitotic spindles. Here we analyze assembly of the Drosophila oocyte meiosis I spindle and the role of the Nonclaret disjunctional (Ncd) motor in spindle assembly using wild-type and mutant Ncd fused to GFP. Unexpectedly, we observe motor-associated asters at germinal vesicle breakdown that migrate towards the condensed chromosomes, where they nucleate microtubules at the chromosomes. Newly nucleated microtubules are randomly oriented, then become organized around the bivalent chromosomes. We show that the meiotic spindle forms by lateral associations of microtubule-coated chromosomes into a bipolar spindle. Lateral interactions between microtubule-associated bivalent chromosomes may be mediated by microtubule crosslinking by the Ncd motor, based on analysis of fixed oocytes. We report here that spindle assembly occurs in an ncd mutant defective for microtubule motility, but lateral interactions between microtubule-coated chromosomes are unstable, indicating that Ncd movement along microtubules is needed to stabilize interactions between chromosomes. A more severe ncd mutant that probably lacks ATPase activity prevents formation of lateral interactions between chromosomes and causes defective microtubule elongation. Anastral Drosophila oocyte meiosis I spindle assembly thus involves motor-associated asters to nucleate microtubules and Ncd motor activity to form and stabilize interactions between microtubule-associated chromosomes during the assembly process. This is the first complete account of assembly of an anastral spindle and the specific steps that require Ncd motor activity, revealing new and unexpected features of the process.

Animals↗