Model of chromosome motility in Drosophila embryos: adaptation of a general mechanism for rapid mitosis

During mitosis, ensembles of dynamic MTs and motors exert forces that coordinate chromosome segregation. Typically, chromosomes align at the metaphase spindle equator where they oscillate along the pole-pole axis before disjoining and moving poleward during anaphase A, but spindles in different cell types display differences in MT dynamicity, in the amplitude of chromosome oscillations and in rates of chromatid-to-pole motion. Drosophila embryonic mitotic spindles, for example, display remarkably dynamic MTs, barely detectable metaphase chromosome oscillations, and a rapid rate of "flux-pacman-dependent" anaphase chromatid-to-pole motility. Here we develop a force-balance model that describes Drosophila embryo chromosome motility in terms of a balance of forces acting on kinetochores and kMTs that is generated by multiple polymer ratchets and mitotic motors coupled to tension-dependent kMT dynamics. The model shows that i), multiple MTs displaying high dynamic instability can drive steady and rapid chromosome motion; ii), chromosome motility during metaphase and anaphase A can be described by a single mechanism; iii), high kinetochore dynein activity is deployed to dampen metaphase oscillations, to augment the basic flux-pacman mechanism, and to drive rapid anaphase A; iv), modulation of the MT rescue frequency by the kinetochore-associated kinesin-13 depolymerase promotes metaphase chromosome oscillations; and v), this basic mechanism can be adapted to a broad range of spindles.     

Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.     

Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint

During mitosis, the motor molecule cytoplasmic dynein plays key direct and indirect roles in organizing microtubules (MTs) into a functional spindle. At this time, dynein is also recruited to kinetochores, but its role or roles at these organelles remain vague, partly because inhibiting dynein globally disrupts spindle assembly [1-4]. However, dynein can be selectively depleted from kinetochores by disruption of ZW10 [5], and recent studies with this approach conclude that kinetochore-associated dynein (KD) functions to silence the spindle-assembly checkpoint (SAC) [6]. Here we use dynein-antibody microinjection and the RNAi of ZW10 to explore the role of KD in chromosome behavior during mitosis in mammals. We find that depleting or inhibiting KD prevents the rapid poleward motion of attaching kinetochores but not kinetochore fiber (K fiber) formation. However, after kinetochores attach to the spindle, KD is required for stabilizing kinetochore MTs, which it probably does by generating tension on the kinetochore, and in its absence, chromosome congression is defective. Finally, depleting KD reduces the velocity of anaphase chromosome motion by approximately 40%, without affecting the rate of poleward MT flux. Thus, in addition to its role in silencing the SAC, KD is important for forming and stabilizing K fibers and in powering chromosome motion.     

Kinetochore dynein generates a poleward pulling force to facilitate congression and full chromosome alignment

For proper chromosome segregation, all kinetochores must achieve bipolar microtubule （MT） attachment and subsequently align at the spindle equator before anaphase onset. The MT minus end-directed motor dynein/dynactin binds kinetoehores in prometaphase and has long been implicated in chromosome congression. Unfortunately, inactivation of dynein usually disturbs spindle organization, thus hampering evaluation of its kinetochore roles. Here we specifically eliminated kinetochore dynein/dynactin by RNAi-mediated depletion of ZW10, a protein essential for kinetochore localization of the motor. Time-lapse microscopy indicated markedly-reduced congression efficiency, though congressing chromosomes displayed similar velocities as in control cells. Moreover, cells frequently failed to achieve full chromosome alignment, despite their normal spindles. Confocal microcopy revealed that the misaligned kinetochores were monooriented or unattached and mostly lying outside the spindle, suggesting a difficulty to capture MTs from the opposite pole. Kinetoehores on monoastral spindles were dispersed farther away from the pole and exhibited only mild oscillation. Furthermore, inactivating dynein by other means generated similar phenotypes. Therefore, kinetochore dynein produces on monooriented kinetochores a poleward pulling force, which may contribute to efficient bipolar attachment by facilitating their proper microtubule captures to promote congression as well as full chromosome alignment.     

Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.     

Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1

Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.     

Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors

Segregation of homologs at the first meiotic division (MI) is facilitated by crossovers and by a physical constraint imposed on sister kinetochores that facilitates monopolar attachment to the MI spindle. Recombination failure or premature separation of homologs results in univalent chromosomes at MI, and univalents constrained to form monopolar attachments should be inherently unstable and trigger the spindle assembly checkpoint (SAC). Although univalents trigger cell-cycle arrest in the male, this is not the case in mammalian oocytes. Because the spindle assembly portion of the SAC appears to function normally, two hypotheses have been proposed to explain the lack of response to univalents: (1) reduced stringency of the oocyte SAC to aberrant chromosome behavior, and (2) the ability of univalents to satisfy the SAC by forming bipolar attachments. The present study of Mlh1 mutant mice demonstrates that metaphase alignment is not a prerequisite for anaphase onset and provides strong evidence that MI spindle stabilization and anaphase onset require stable bipolar attachment of a critical mass--but not all--of chromosomes. We postulate that subtle differences in SAC-mediated control make the human oocyte inherently error prone and contribute to the age-related increase in aneuploidy.     

Multiple mechanisms of chromosome movement in vertebrate cells mediated through the Ndc80 complex and dynein/dynactin

Kinetochores bind microtubules laterally in a transient fashion and stably, by insertion of plus ends. These pathways may exist to carry out distinct tasks during different stages of mitosis and likely depend on distinct molecular mechanisms. On isolated chromosomes, we found microtubule nucleation/binding depended additively on both dynein/dynactin and on the Ndc80/Hec1 complex. Studying chromosome movement in living Xenopus cells within the simplified geometry of monopolar spindles, we quantified the relative contributions of dynein/dynactin and the Ndc80/Hec1 complex. Inhibition of dynein/dynactin alone had minor effects but did suppress transient, rapid, poleward movements. In contrast, inhibition of the Ndc80 complex blocked normal end-on attachments of microtubules to kinetochores resulting in persistent rapid poleward movements that required dynein/dynactin. In normal cells with bipolar spindles, dynein/dynactin activity on its own allowed attachment and rapid movement of chromosomes on prometaphase spindles but failed to support metaphase alignment and chromatid movement in anaphase. Thus, in prometaphase, dynein/dynactin likely mediates early transient, lateral interactions of kinetochores and microtubules. However, mature attachment via the Ndc80 complex is essential for metaphase alignment and anaphase A. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Meiosis I in Xenopus oocytes is not error-prone despite lacking spindle assembly checkpoint

The spindle assembly checkpoint, SAC, is a surveillance mechanism to control the onset of anaphase during cell division. SAC prevents anaphase initiation until all chromosome pairs have achieved bipolar attachment and aligned at the metaphase plate of the spindle. In doing so, SAC is thought to be the key mechanism to prevent chromosome nondisjunction in mitosis and meiosis. We have recently demonstrated that Xenopus oocyte meiosis lacks SAC control. This prompted the question of whether Xenopus oocyte meiosis is particularly error-prone. In this study, we have karyotyped a total of 313 Xenopus eggs following in vitro oocyte maturation. We found no hyperploid egg, out of 204 metaphase II eggs with countable chromosome spreads. Therefore, chromosome nondisjunction is very rare during Xenopus oocyte meiosis I, despite the lack of SAC.     

The dynactin complex maintains the integrity of metaphasic centrosomes to ensure transition to anaphase

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.     

Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein–generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated—it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)–mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle–regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.     

Lack of response to unaligned chromosomes in mammalian female gametes

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.     

A quantitative and semiautomated method for determining misaligned and lagging chromosomes during mitosis

Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore–microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Tools to sensitively, accurately, and quantitatively measure chromosome alignment at metaphase will facilitate understanding of the contribution of chromosome segregation errors to the development of aneuploidy. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semiautomated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.     

Lack of response to unaligned chromosomes in mammalian female gametes

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.     

Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation

The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.     

Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I

The spindle assembly checkpoint (SAC) ensures correct separation of sister chromatids in somatic cells and provokes a cell cycle arrest in metaphase if one chromatid is not correctly attached to the bipolar spindle. Prolonged metaphase arrest due to overexpression of Mad2 has been shown to be deleterious to the ensuing anaphase, leading to the generation of aneuploidies and tumorigenesis. Additionally, some SAC components are essential for correct timing of prometaphase. In meiosis, we and others have shown previously that the Mad2-dependent SAC is functional during the first meiotic division in mouse oocytes. Expression of a dominant-negative form of Mad2 interferes with the SAC in metaphase I, and a knock-down approach using RNA interference accelerates anaphase onset in meiosis I. To prove unambigiously the importance of SAC control for mammalian female meiosis I we analyzed oocyte maturation in Mad2 heterozygote mice, and in oocytes overexpressing a GFP-tagged version of Mad2. In this study we show for the first time that loss of one Mad2 allele, as well as overexpression of Mad2 lead to chromosome missegregation events in meiosis I, and therefore the generation of aneuploid metaphase II oocytes. Furthermore, SAC control is impaired in mad2+/- oocytes, also leading to the generation of aneuploidies in meiosis I.     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.     

Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells

Summary Treatment of metaphase PtK₁ cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK₁ metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 m in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 m in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.Abbreviations DIC differential interference contrast - kMT(s) kinetochore microtubule(s) - MT(s) microtubule(s) - nkMT(s) non-kinetochore microtubule(s)     

Evidence that the spindle assembly checkpoint does not regulate APC(Fzy) activity in Drosophila female meiosis

The spindle assembly checkpoint (SAC) plays an important role in mitotic cells to sense improper chromosome attachment to spindle microtubules and to inhibit APC(Fzy)-dependent destruction of cyclin B and Securin; consequent initiation of anaphase until correct attachments are made. In Drosophila , SAC genes have been found to play a role in ensuring proper chromosome segregation in meiosis, possibly reflecting a similar role for the SAC in APC(Fzy) inhibition during meiosis. We found that loss of function mutations in SAC genes, Mad2, zwilch, and mps1, do not lead to the predicted rise in APC(Fzy)-dependent degradation of cyclin B either globally throughout the egg or locally on the meiotic spindle. Further, the SAC is not responsible for the inability of APC(Fzy) to target cyclin B and promote anaphase in metaphase II arrested eggs from cort mutant females. Our findings support the argument that SAC proteins play checkpoint independent roles in Drosophila female meiosis and that other mechanisms must function to control APC activity.     

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.