The relative roles of centrosomal and kinetochore-driven microtubules in Drosophila spindle formation

Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. Here we review the studies on spindle formation in Drosophila centrosome-containing cells. Mutants with impaired centrosome function assemble functional anastral spindles in somatic tissues and survive to adulthood. In contrast, mutants defective in chromosome-driven MT formation form highly aberrant mitotic spindles and die at larval stages. The requirements for spindle assembly in Drosophila male meiotic cells are diametrically opposed to those of somatic cells. Spermatocytes assemble morphologically normal spindles in the complete absence of chromosome-induced MTs, but are unable to organize a functional spindle in the absence of centrosomal MTs. Male meiotic spindles are much larger than mitotic spindles as they contain most of the tubulin needed for sperm tail formation. We suggest that the centrosome-based mechanism of spindle assembly in spermatocytes reflects their need for rapid and efficient polymerization of a particularly large amount of tubulin.     

Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.     

Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.     

Inhibition of Aurora kinases perturbs chromosome alignment and spindle checkpoint signaling in rat spermatocytes

In somatic cells, integrity of cell division is safeguarded by the spindle checkpoint, a signaling cascade that delays the separation of sister chromatids in the presence of misaligned chromosomes. Aurora kinases play important roles in this process by promoting centrosome maturation, chromosome bi-orientation, spindle checkpoint signaling, and cytokinesis. To investigate the functions of Aurora kinases in male meiosis, we applied a small molecule Aurora inhibitor, ZM447439, to seminiferous tubules in vitro. Primary and secondary spermatocytes exposed to ZM447439 exhibit defects in the spindle morphology and fail to align their chromosomes at the metaphase plate. Moreover, the treated spermatocytes undergo a forced exit from the meiotic M-phase without cytokinesis. These results suggest that the activities of Aurora kinases are required for normal spindle assembly as well as for establishment and maintenance of proper microtubule-kinetochore attachments and spindle checkpoint signaling in male mammalian meiosis.     

Contribution of Noncentrosomal Microtubules to Spindle Assembly in Drosophila Spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.     

Chromatin-induced spindle assembly plays an important role in metaphase congression of silkworm holocentric chromosomes

The kinetochore plays important roles in cell cycle progression. Interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell and to segregate the sister chromatids into daughter cells in mitosis. The chromosome passenger complex (CPC), composed of the Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin, plays multiple roles in these chromosomal events. In the genome of the silkworm, Bombyx mori, which has holocentric chromosomes, the CPC components and their molecular interactions were highly conserved. In contrast to monocentric species, however, the silkworm CPC co-localized with the chromatin-driven spindles on the upper side of prometaphase chromosomes without forming bipolar mitotic spindles. Depletion of the CPC by RNAi arrested the cell cycle progression at prometaphase and disrupted the microtubule network of the chromatin-driven spindles. Interestingly, depletion of mitotic centromere-associated kinesin (MCAK) recovered formation of the microtubule network but did not overcome the cell cycle arrest at prometaphase. These results suggest that the CPC modulates the chromatin-induced spindle assembly and metaphase congression of silkworm holocentric chromosomes.     

A pathway containing the Ipl1/aurora protein kinase and the spindle midzone protein Ase1 regulates yeast spindle assembly

It is critical to elucidate the pathways that mediate spindle assembly and therefore ensure accurate chromosome segregation during cell division. Our studies of a unique allele of the budding yeast Ipl1/Aurora protein kinase revealed that it is required for centrosome-mediated spindle assembly in the absence of the BimC motor protein Cin8. In addition, we found that the Ase1 spindle midzone-associated protein is required for bipolar spindle assembly. The cin8 ipl1 and cin8 ase1 double mutant cells exhibit similar defects, and Ase1 overexpression completely restores spindle assembly in cin8 ipl1 strains. Consistent with the possibility that Ipl1 regulates Ase1, an ase1 mutant lacking the Ipl1 consensus phosphorylation sites cannot assemble spindles in the absence of Cin8. In addition, Ase1 phosphorylation and localization were altered in an ipl1 mutant. We therefore propose that Ipl1/Aurora and Ase1 constitute a previously unidentified spindle assembly pathway that becomes essential in the absence of Cin8.     

Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes

The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.     

Centrosome inheritance in the male germ line of Drosophila requires hu-li tai-shao function

Cytokinesis partitions a centrosome to each daughter cell at cell division that will duplicate and assemble a bipolar spindle in the subsequent M phase. Cytokinesis is incomplete in proliferating germ cells in Drosophila and cytoplasmic channels connect sibling germ cells. Although centrosomes are essential to male fertility, the molecular mechanism that retains centrosomes in parental germ cells is not known. Cortical cytoplasmic structures known as fusomes extend through ring canals and connect cells within the cyst. Fusome assembly in males requires function of hu-li tai-shao (hts), an adducin like protein found in fusomes and in the cortical membrane cytoskeleton of somatic cells. This work used immunological and cytological methods to place hts mutants in an allelic series. Male fertile hts mutants express hts protein and generate apparently normal or fragmented fusomes. A male sterile allele does not express hts protein or show fusome structures. Gonial cells in all hts mutants showed 2 centrosomes and mitotic spindles were bipolar. Yet, primary spermatocytes, with and without fusome structures, frequently contained too many or too few centrosomes. Although spindle structures were not found in spermatocytes without centrosomes, meiotic spermatocytes with centrosomes generated bipolar, monopolar, and multipolar spindles. Collectively, these results indicate that hts function is necessary for centrosome inheritance in spermatocytes as well as for male fertility.     

Misregulation of the kinesin-like protein Subito induces meiotic spindle formation in the absence of chromosomes and centrosomes

Bipolar spindles assemble in the absence of centrosomes in the oocytes of many species. In Drosophila melanogaster oocytes, the chromosomes have been proposed to initiate spindle assembly by nucleating or capturing microtubules, although the mechanism is not understood. An important contributor to this process is Subito, which is a kinesin-6 protein that is required for bundling interpolar microtubules located within the central spindle at metaphase I. We have characterized the domains of Subito that regulate its activity and its specificity for antiparallel microtubules. This analysis has revealed that the C-terminal domain may interact independently with microtubules while the motor domain is required for maintaining the interaction with the antiparallel microtubules. Surprisingly, deletion of the N-terminal domain resulted in a Subito protein capable of promoting the assembly of bipolar spindles that do not include centrosomes or chromosomes. Bipolar acentrosomal spindle formation during meiosis in oocytes may be driven by the bundling of antiparallel microtubules. Furthermore, these experiments have revealed evidence of a nuclear- or chromosome-based signal that acts at a distance to activate Subito. Instead of the chromosomes directly capturing microtubules, signals released upon nuclear envelope breakdown may activate proteins like Subito, which in turn bundles together microtubules.     

The impact of chromosomes and centrosomes on spindle assembly as observed in living cells

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video- enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.     

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.     

Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin''s spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo''s negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.     

MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.     

Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos

During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles. Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants. Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles. By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles. At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively). However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A.     

Loss of Drosophila borealin causes polyploidy, delayed apoptosis and abnormal tissue development

The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. Here, we report the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), a pronounced attenuation at prometaphase and multipolar spindles. Our evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium that involve Wingless signalling, which ultimately perturb the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy.     

Localized Aurora B activity spatially controls non-kinetochore microtubules during spindle assembly

Efficient spindle assembly involves the generation of spatial cues around chromosomes that locally stabilize microtubule (MT) plus-ends. In addition to the small GTPase Ran, there is evidence that Aurora B kinase might also generate a spatial cue around chromosomes but direct proof for this is still lacking. Here, we find that the Aurora B substrate MCAK localizes to MT plus-ends throughout the mitotic spindle, but its accumulation is strongly reduced on MT plus-ends near chromatin, suggesting that a signal emanating from chromosomes negatively regulates MCAK plus-end binding. Indeed, we show that Aurora B is the kinase responsible for producing this chromosome-derived signal. These results are the first to visualize spatially restricted Aurora B kinase activity around chromosomes on an endogenous substrate and explain how Aurora B could spatially control the dynamics of non-kinetochore MTs during spindle assembly.     

The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes

In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle.