Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma- tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.     

MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.     

Drosophila parthenogenesis: a tool to decipher centrosomal vs acentrosomal spindle assembly pathways

Development of unfertilized eggs in the parthenogenetic strain K23-O-im of Drosophila mercatorum requires the stochastic interactions of self-assembled centrosomes with the female chromatin. In a portion of the unfertilized eggs that do not assemble centrosomes, microtubules organize a bipolar anastral mitotic spindle around the chromatin like the one formed during the first female meiosis, suggesting that similar pathways may be operative. In the cytoplasm of eggs in which centrosomes do form, monastral and biastral spindles are found. Analysis by laser scanning confocal microscopy suggests that these spindles are derived from the stochastic interaction of astral microtubules directly with kinetochore regions or indirectly with kinetochore microtubules. Our findings are consistent with the idea that mitotic spindle assembly requires both acentrosomal and centrosomal pathways, strengthening the hypothesis that astral microtubules can dictate the organization of the spindle by capturing kinetochore microtubules.     

Peripheral, non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells

In centrosome-containing cells, microtubules utilized in spindle formation are thought to be nucleated at the centrosome. However, spindle formation can proceed following experimental destruction of centrosomes or in cells lacking centrosomes, suggesting that non-centrosome-associated microtubules may contribute to spindle formation, at least when centrosomes are absent. Direct observation of prometaphase cells expressing GFP-alpha-tubulin shows that peripheral, non-centrosome-associated microtubules are utilized in spindle formation, even in the presence of centrosomes. Clusters of peripheral microtubules moved into the centrosomal region, demonstrating that a centrosomal microtubule array can be composed of both centrosomally nucleated and peripheral microtubules. Peripheral bundles also moved laterally into the forming spindle between the spindle poles; 3D reconstructions of fixed cells reveal interactions between peripheral and centrosome-associated microtubules. The spindle pole component NuMA and gamma-tubulin were present at the foci of peripheral microtubule clusters, indicating that microtubules moved into the spindle with minus ends leading. Photobleach- and photoactivation-marking experiments of cells expressing GFP-tubulin or a photoactivatable variant of GFP-tubulin, respectively, demonstrate that microtubule motion into the forming spindle results from transport and sliding interactions, not treadmilling. Our results directly demonstrate that non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.     

Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.     

Molecular requirements for kinetochore-associated microtubule formation in mammalian cells

In centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly. In addition, microtubule formation at kinetochores has also been observed, most recently under physiological conditions in live cells. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-beta or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture.     

Centrosomal nucleolin is required for microtubule network organization

Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin co-localized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing 2 key centrosomal proteins, γ-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells.     

Survivin modulates microtubule dynamics and nucleation throughout the cell cycle

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.     

Influence of M-phase chromatin on the anisotropy of microtubule asters

In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long- range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long- range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.     

Regulation of microtubule nucleation mediated by γ-tubulin complexes

The microtubule cytoskeleton is critically important for spatio-temporal organization of eukaryotic cells. The nucleation of new microtubules is typically restricted to microtubule organizing centers (MTOCs) and requires γ-tubulin that assembles into multisubunit complexes of various sizes. γ-Tubulin ring complexes (TuRCs) are efficient microtubule nucleators and are associated with large number of targeting, activating and modulating proteins. γ-Tubulin-dependent nucleation of microtubules occurs both from canonical MTOCs, such as spindle pole bodies and centrosomes, and additional sites such as Golgi apparatus, nuclear envelope, plasma membrane-associated sites, chromatin and surface of pre-existing microtubules. Despite many advances in structure of γ-tubulin complexes and characterization of γTuRC interacting factors, regulatory mechanisms of microtubule nucleation are not fully understood. Here, we review recent work on the factors and regulatory mechanisms that are involved in centrosomal and non-centrosomal microtubule nucleation.     

Origin of kinetochore microtubules in Chinese hamster ovary cells

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 m thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 g/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. — In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubles were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.     

Dictyostelium EB1 is a genuine centrosomal component required for proper spindle formation

EB1 proteins are ubiquitous microtubule-associated proteins involved in microtubule search and capture, regulation of microtubule dynamics, cell polarity, and chromosome stability. We have cloned a complete cDNA of Dictyostelium EB1 (DdEB1), the largest known EB1 homolog (57 kDa). Immunofluorescence analysis and expression of a green fluorescent protein-DdEB1 fusion protein revealed that DdEB1 localizes along microtubules, at microtubule tips, centrosomes, and protruding pseudopods. During mitosis, it was found at the spindle, spindle poles, and kinetochores. DdEB1 is the first EB1-homolog that is also a genuine centrosomal component, because it was localized at isolated centrosomes that are free of microtubules. Furthermore, centrosomal DdEB1 distribution was unaffected by nocodazole treatment. DdEB1 colocalized with DdCP224, the XMAP215 homolog, at microtubule tips, the centrosome, and kinetochores. Furthermore, both proteins were part of the same cytosolic protein complex, suggesting that they may act together in their functions. DdEB1 deletion mutants expressed as green fluorescent protein or maltose-binding fusion proteins indicated that microtubule binding requires homo-oligomerization, which is mediated by a coiled-coil domain. A DdEB1 null mutant was viable but retarded in prometaphase progression due to a defect in spindle formation. Because spindle elongation was normal, DdEB1 seems to be required for the initiation of the outgrowth of spindle microtubules.     

Sperm chromatin acquires an activity that induces microtubule assembly during residence in the cytoplasm of metaphase oocytes of the mouse

Evidence from several cell types indicates that chromatin can induce microtubule assembly in its vicinity. To determine whether this activity is present in sperm chromatin, whose biochemical composition differs from somatic chromatin, mouse oocytes that were undergoing meiotic maturation were inseminated. Maturing oocytes are not activated by sperm penetration but remain arrested at metaphase. The sperm chromatin within the oocyte cytoplasm initially became dispersed and later, under the influence of oocyte cytoplasmic factors, recondensed into a small mass of individual chromosomes. When inseminated oocytes were processed for immunofluorescence using an anti--tubulin antibody, microtubules were never associated with dispersed sperm chromatin, although the chromosomes of the oocyte were arranged on a spindle. In contrast, microtubules were associated with the majority of sperm nuclei that had become recondensed, and were frequently arranged into a spindle-like structure. When oocytes had been penetrated by more than three sperm, most sperm nuclei remained at the dispersed chromatin stage and these were never associated with microtubules. Exposure of polyspermic oocytes to taxol, which promotes microtubule assembly, failed to induce microtubule assembly around dispersed sperm chromatin. Exposure of monospermic oocytes to nocodazole, which inhibits tubulin polymerization, prevented resolution of the recondensed sperm chromatin into individual chromosomes. These results suggest that sperm chromatin lacks an activity that can induce local microtubule assembly, and that it acquires this activity once modified by oocyte cytoplasmic factors.     

A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. We show that this "internal" matrix is a distinct structure from the microtubule spindle and from a lamin B-containing spindle envelope. By injection of 2000-kDa dextran, we show that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as "struts" stretching the spindle matrix. In addition, we demonstrate that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division.     

Astrin regulates meiotic spindle organization,spindle pole tethering and cell cycle progression in mouse oocytes

Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of Tof the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrestT. Thr24, Ser66 or Ser447 may be the potential phosphorylated sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at HTmetaphaseTH I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.     

Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly

It was recently reported that GTP-bound Ran induces microtubule and pseudo-spindle assembly in mitotic egg extracts in the absence of chromosomes and centrosomes, and that chromosomes induce the assembly of spindle microtubules in these extracts through generation of Ran-GTP. Here we examine the effects of Ran-GTP on microtubule nucleation and dynamics and show that Ran-GTP has independent effects on both the nucleation activity of centrosomes and the stability of centrosomal microtubules. We also show that inhibition of Ran-GTP production, even in the presence of duplicated centrosomes and kinetochores, prevents assembly of a bipolar spindle in M-phase extracts.     

Role of non-kinetochore microtubules in spindle elongation in mitotic PtK1 cells

PtK1 metaphase cells were treated with varying concentrations of nocodazole to reduce spindle microtubule number and spindle length. The range of concentrations employed reduced spindle length from approximately 47% to 82% of the original pole-pole distance. Electron microscopy of cells treated with the lowest concentration of nocodazole employed (0.01 microgram/ml) showed a small decrease in the number of non-kinetochore microtubules (nkMTs), particularly evident in the astral region, with no significant effect on kinetochore microtubule number. Metaphase cells treated with 1 microgram/ml nocodazole for 2 min demonstrated a reduction in spindle length and loss of most non-kinetochore microtubules with little effect on the number and arrangement of the kinetochore class of microtubules. Following nocodazole treatment, the cells were perfused with 0.5 M sucrose dissolved in tissue culture medium, a treatment which has previously been shown to induce spindle elongation in metaphase cells. In cells where nocodazole effected a large decrease in non-kinetochore microtubule number with a concomitant decrease in spindle length, sucrose treatment had a reduced effect in inducing spindle elongation. In cells treated with lower concentrations of nocodazole, where numerous non-kinetochore microtubules remained, sucrose had a greater effect in inducing spindle elongation. These data suggest that the non-kinetochore population of microtubules is responsible for the extent of sucrose-induced spindle elongation. An explanation of these data is provided which suggests that the role of non-kinetochore microtubules is to trap energy in the developing spindle, such that it can be used to separate spindle poles during anaphase B.     

Major sperm protein signaling promotes oocyte microtubule reorganization prior to fertilization in Caenorhabditis elegans

In most animals, female meiotic spindles assemble in the absence of centrosomes; instead, microtubule nucleation by chromatin, motor activity, and microtubule dynamics drive the self-organization of a bipolar meiotic spindle. Meiotic spindle assembly commences when microtubules gain access to chromatin after nuclear envelope breakdown (NEBD) during meiotic maturation. Although many studies have addressed the chromatin-based mechanism of female meiotic spindle assembly, it is less clear how signaling influences microtubule localization and dynamics prior to NEBD. Here we analyze microtubule behavior in Caenorhabditis elegans oocytes at early stages of the meiotic maturation process using confocal microscopy and live-cell imaging. In C. elegans, sperm trigger oocyte meiotic maturation and ovulation using the major sperm protein (MSP) as an extracellular signaling molecule. We show that MSP signaling reorganizes oocyte microtubules prior to NEBD and fertilization by affecting their localization and dynamics. We present evidence that MSP signaling reorganizes oocyte microtubules through a signaling network involving antagonistic G alpha(o/i) and G alpha(s) pathways and gap-junctional communication with somatic cells of the gonad. We propose that MSP-dependent microtubule reorganization promotes meiotic spindle assembly by facilitating the search and capture of microtubules by meiotic chromatin following NEBD.     

Reorganization of microtubule nucleation during muscle differentiation

Skeletal muscle differentiation involves a complete reorganization of the microtubule network. Nearly 20 years ago, Tassin et al. [1985: J Cell Biol 100:35-46] suggested a mechanism for this reorganization by showing a redistribution of the microtubule organizing center from the centrosome to the nuclear membrane. Little progress has been made since. It is still not clear whether centrosomal proteins are redistributed together, whether microtubules are nucleated at the nuclear membrane or transported there post-nucleation, and whether gamma-tubulin (gammatub) remains necessary for nucleation in myotubes. To investigate these questions, we have examined the redistribution of the centrosomal proteins pericentrin (PC), gammatub, and ninein in the C2 muscle cell line. Immunofluorescence of differentiated myotubes shows PC along the nuclear membrane whereas gammatub is only detected there after pre-fixation detergent extraction. After expression of a GFP-tagged gammatub, we observe a weak fluorescence along the nuclear membrane, confirming the presence of gammatub at a low concentration relative to PC. Microinjection of anti-gammatub antibodies into myotubes blocks microtubule growth from both nuclear membranes and centrosomal sites. The centrosomal microtubule-anchoring protein, ninein, is found at the nuclear membrane as well and its distribution appears independent of microtubule integrity. We conclude that centrosomal proteins are redistributed independently during muscle differentiation, to sites that nucleate microtubules both along the nuclear membranes and through the cytoplasm.     

Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes

Chromosome segregation in mammalian oocytes is driven by a microtubule spindle lacking centrosomes. Here, we analyze centrosome-independent spindle assembly by quantitative high-resolution confocal imaging in live maturing mouse oocytes. We show that spindle assembly proceeds by the self-organization of over 80 microtubule organizing centers (MTOCs) that form de novo from a cytoplasmic microtubule network in prophase and that functionally replace centrosomes. Initially distributed throughout the ooplasm, MTOCs congress at the center of the oocyte, where they contribute to a massive, Ran-dependent increase of the number of microtubules after nuclear envelope breakdown and to the individualization of clustered chromosomes. Through progressive MTOC clustering and activation of kinesin-5, the multipolar MTOC aggregate self-organizes into a bipolar intermediate, which then elongates and thereby establishes chromosome biorientation. Finally, a stable barrel-shaped acentrosomal metaphase spindle with oscillating chromosomes and astral-like microtubules forms that surprisingly exhibits key properties of a centrosomal spindle.