Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation

During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.     

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

Chromosome micromanipulation

The attachment of individual chromosomes to the spindle has been studied by micromanipulation in functionally normal grasshopper spermatocytes. Prometaphase to anaphase I chromosomes can be repeatedly stretched with a microneedle without much increase in the distance between the kinetochores and the poles. Individual chromosomes can, however, be displaced laterally (prometaphase-anaphase) or toward the pole (anaphase) without loss of spindle attachment and without greatly disturbing other chromosomes. It is concluded that chromosomes are firmly and individually attached to the spindle by chromosomal spindle fibers which are capable of bearing any normal mitotic load, including the stretching of dikinetic (dicentric) chromosomes in anaphase. Prolonged or severe manipulation can produce a small — three or four micron — increase in the kinetochore-to-pole distance. Anaphase motion continues normally in spite of lateral or poleward displacements or of small increases in the kinetochore-to-pole distance. In late anaphase, chromosomes can be displaced to the opposite pole. An unusual, rapid motion back toward the original pole follows such displacements, but repeated displacements eventually result in non-disjunction. No evidence for firm interzonal connections between anaphase chromosomes was obtained. Prometaphase and metaphase bivalents can be detached from the spindle by manipulations other than bivalent stretching, but half-bivalents in anaphase are never detached by these manipulations.This investigation was supported in part by research grants GM-8480 and GM-13745 from the Division of General Medical Sciences, United States Public Health Service.     

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.     

Asymmetric positioning and organization of the meiotic spindle of mouse oocytes requires CDC42 function

The mature mammalian oocyte is highly polarized because asymmetrical spindle migration to the oocyte cortex ensures extrusion of small polar bodies in the two meiotic divisions, essential for generation of the large egg. Actin filaments, myosin motors, and formin-2, but not microtubules, are required for spindle migration. Here, we show that Cdc42, a key regulator of cytoskeleton and cell polarity in other systems , is essential for meiotic maturation and oocyte asymmetry. Disrupting CDC42 function by ectopic expression of its GTPase-defective mutants causes both halves of the first meiotic spindle to extend symmetrically toward opposing cortical regions and prevents an asymmetrical division. The elongated spindle has numerous astral-like microtubules, and aPKCzeta, normally associated with the spindle poles, is distributed along its length. Dynactin is displaced from kinetochores, consistently homologous chromosomes do not segregate, and polar body extrusion is prevented. Perturbing the function of aPKCzeta also causes elongation of the meiotic spindle but still permits spindle migration and polar body extrusion. Thus, at least two pathways appear to be downstream of CDC42: one affecting the actin cytoskeleton and required for migration of the meiotic spindle, and a second affecting the spindle microtubules in which aPKCzeta plays a role.     

Polar body emission requires a RhoA contractile ring and Cdc42-mediated membrane protrusion

Vertebrate oocyte maturation is an extreme form of asymmetric cell division, producing a mature egg alongside a diminutive polar body. Critical to this process is the attachment of one spindle pole to the oocyte cortex prior to anaphase. We report here that asymmetric spindle pole attachment and anaphase initiation are required for localized cortical activation of Cdc42, which in turn defines the surface of the impending polar body. The Cdc42 activity zone overlaps with dynamic F-actin and is circumscribed by a RhoA-based actomyosin contractile ring. During cytokinesis, constriction of the RhoA contractile ring is accompanied by Cdc42-mediated membrane outpocketing such that one spindle pole and one set of chromosomes are pulled into the Cdc42 enclosure. Unexpectedly, the guanine nucleotide exchange factor Ect2, which is necessary for contractile ring formation, does not colocalize with active RhoA. Polar body emission thus requires a classical RhoA contractile ring and Cdc42-mediated membrane protrusion.     

Kinesin-dependent transport results in polarized migration of the nucleus in oocytes and inward movement of yolk granules in meiotic embryos

During female meiosis, meiotic spindles are positioned at the oocyte cortex to allow expulsion of chromosomes into polar bodies. In C. elegans, kinesin-dependent translocation of the entire spindle to the cortex precedes dynein-dependent rotation of one spindle pole toward the cortex. To elucidate the role of kinesin-1 in spindle translocation, we examined the localization of kinesin subunits in meiotic embryos. Surprisingly, kinesin-1 was not associated with the spindle and instead was restricted to the cytoplasm in the middle of the embryo. Yolk granules moved on linear tracks, in a kinesin-dependent manner, away from the cortex, resulting in their concentration in the middle of the embryo where the kinesin was concentrated. These results suggest that cytoplasmic microtubules might be arranged with plus ends extending inward, away from the cortex. This microtubule arrangement would not be consistent with direct transport of the meiotic spindle toward the cortex by kinesin-1. In maturing oocytes, the nucleus underwent kinesin-dependent migration to the future site of spindle attachment at the anterior cortex. Thus the spindle translocation defect observed in kinesin-1 mutants may be a result of failed nuclear migration, which places the spindle too far from the cortex for the spindle translocation mechanism to function.     

The G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and Gialpha3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells

We addressed the role of the G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and G-proteins (Gialpha3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Gialpha3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Gialpha3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Gialpha. The influence of LGN and Gialpha3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry.     

Two PAR6 proteins become asymmetrically localized during establishment of polarity in mouse oocytes

Meiotic maturation in mammals is characterized by two asymmetric divisions, leading to the formation of two polar bodies and the female gamete. Whereas the mouse oocyte is a polarized cell, molecules implicated in the establishment of this polarity are still unknown. PAR proteins have been demonstrated to play an important role in cell polarity in many cell types, where they control spindle positioning and asymmetric distribution of determinants. Here we show that two PAR6-related proteins have distinct polarized distributions in mouse oocytes. mPARD6a is first localized on the spindle and then accumulates at the pole nearest the cortex during spindle migration. In the absence of microtubules, the chromosomes still migrate to the cortex, and mPARD6a was found associated with the chromosomes and was facing the cortex. mPARD6a is the first identified protein to associate with the spindle during spindle migration and to relocalize to the chromosomes in the absence of microtubule behavior, suggesting a role in spindle migration. The other protein, mPARD6b, was found on spindle microtubules until entry into meiosis II and relocalized to the cortex at the animal pole during metaphase II arrest. mPARD6b is the first identified protein to localize to the animal pole of the mouse oocyte and likely contributes to the polarization of the cortex.     

KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.     

Nuclear migration and spindle formation in the fourth cleavage of sea urchin eggs under the influence of inhibitors.

Mitosis of sea urchin eggs was inhibited when exposed to 3 micrograms/ml aphidicolin from the 2-cell stage onwards. Nevertheless the nuclei migrated to the vegetal pole at the time of the fourth unequal division in control eggs. Two or four equal or unequal asters developed. Asters in proximity to the vegetal pole were always considerably smaller than those close to the center of the two blastomeres. In contrast to colchicine, cytokinesis but not migration of the nuclei in the vegetal half was prevented by treatments with 5 microM cytochalasin B or D. Various mitotic figures were formed in the vegetal blastomeres of eggs treated with 0.4 mM colchicine or 3 microM griseofulvin after the third cleavage. In some eggs a centrally localized monaster with chromosomes in sphere-like arrangement was formed in others a monopolar mitotic figure pushed the chromosomes in bowl-like arrangements to the most vegetal cortex. In anaphase one set of chromatids migrated to the monopole leaving the scattered sister-chromatids behind. The mechanism of migration of the nuclei and of chromosome arrangement in the metaphase plate is discussed.     

Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p

Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Delta cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Delta cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation.     

Asymmetric division in mouse oocytes: with or without Mos

In both vertebrates and invertebrates, meiotic divisions in oocytes are typically asymmetric, resulting in the formation of a large oocyte and small polar bodies. The size difference between the daughter cells is usually a consequence of asymmetric positioning of the spindle before cytokinesis. Spindle movements are often related to interactions between the cell cortex and the spindle asters [1,2]. The spindles of mammalian oocytes are, however, typically devoid of astral microtubules, which normally connect the spindle to the cortex, suggesting that another mechanism is responsible for the unequal divisions in these oocytes. We observed the formation of the first polar body in wild-type oocytes and oocytes derived from c-Mos knockout mice [3]. In wild-type oocytes, the meiotic spindle formed in the centre of the cell and migrated to the cortex just before polar-body extrusion. The spindle did not elongate during anaphase. In mos-/- oocytes, the spindle formed centrally but did not migrate, although an asymmetric division still took place. In these oocytes, the spindle elongated during anaphase and the pole closest to the cortex moved while the other remained in place. Thus, a compensation mechanism exists in mouse oocytes and formation of the first polar body can be achieved in two ways: either after migration of the spindle to the cortex in wild-type oocytes, or after elongation, without migration, of the first meiotic spindle in mos-/- oocytes.     

Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules

In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, and KAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion of BIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein-Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.     

Unequal cleavage in the early Tubifex embryo

Unequal cleavage that produces two blastomeres of different size is a cleavage pattern that many animals in a variety of phyla, particularly in Spiralia, adopt during early development. This cleavage pattern is apparently instrumental for asymmetric segregation of developmental potential, but it is also indispensable for normal embryogenesis in many animals. Mechanically, unequal cleavage is achieved by either simple unequal cytokinesis or by forming a polar lobe at the egg's vegetal pole. In the present paper, the mechanisms for unequal cytokinesis involved in the first three cleavages in the oligochaete annelid Tubifex are reviewed. The three unequal cleavages are all brought about by an asymmetrically organized mitotic apparatus (MA). The MA of the first cleavage is monastral in that an aster is present at one pole of a bipolar spindle but not at the other. This monastral form, which arises as a result of the involvement of a single centrosome in the MA assembly, is both necessary and sufficient for unequal first cleavage. The egg cortex during the first mitosis is devoid of the ability to remodel spindle poles. In contrast to the non-cortical mechanisms for the first cleavage, asymmetry in the MA organization at the second and third cleavages depends solely on specialized properties of the cell cortex, to which one spindle pole is physically connected. A cortical attachment site for the second cleavage spindle is generated de novo at the cleavage membrane resulting from the first cleavage; it is an actin-based, cell contact-dependent structure. The cortical microtubule attachment site for the third cleavage, which functions independently of contact with other cells, is not generated at the cleavage membrane resulting from the second cleavage, but is located at the animal pole; it may originate from the second polar body formation and become functional at the 4-cell stage.     

Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67

Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.     

Live-cell analysis of mitotic spindle formation in taxol-treated cells

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.     

Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes

Mammalian meiotic divisions are asymmetrical and generate a large oocyte and two small polar bodies. This asymmetry results from the anchoring of the meiotic spindle to the oocyte cortex and subsequent cortical reorganization, but the mechanisms involved are poorly understood. We investigated the role of Rac in oocyte meiosis by using a fluorescent reporter for Rac-GTP. We find that Rac-GTP is polarized in the cortex overlying the meiotic spindle. Polarization of Rac activation occurs during spindle migration and is promoted by the proximity of chromatin to the cortex. Inhibition of Rac during oocyte maturation caused a permanent block at prometaphase I and spindle elongation. In metaphase II-arrested oocytes, Rac inhibition caused the spindle to detach from the cortex and prevented polar body emission after activation. These results demonstrate that Rac-GTP plays a major role in oocyte meiosis, via the regulation of spindle stability and anchoring to the cortex.     

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.