The role of substrate curvature in actin-based pushing forces

The extension of the plasma membrane during cell crawling or spreading is known to require actin polymerization; however, the question of how pushing forces derive from actin polymerization remains open. A leading theory (herein referred to as elastic propulsion) illustrates how elastic stresses in networks growing on curved surfaces can result in forces that push particles. To date all examples of reconstituted motility have used curved surfaces, raising the possibility that such squeezing forces are essential for actin-based pushing. By contrast, other theories, such as molecular ratchets, neither require nor consider surface curvature to explain pushing forces. Here, we critically test the requirement of substrate curvature by reconstituting actin-based motility on polystyrene disks. We find that disks move through extracts in a manner that indicates pushing forces on their flat surfaces and that disks typically move faster than the spheres they are manufactured from. For a subset of actin tails that form on the perimeter of disks, we find no correlation between local surface curvature and tail position. Collectively the data indicate that curvature-dependent mechanisms are not required for actin-based pushing.     

Actin-driven chromosomal motility leads to symmetry breaking in mammalian meiotic oocytes

Movement of meiosis I (MI) chromosomes from the oocyte centre to a subcortical location is the first step in the establishment of cortical polarity. This is required for two consecutive rounds of asymmetric meiotic cell divisions, which generate a mature egg and two polar bodies. Here we use live-cell imaging and genetic and pharmacological manipulations to determine the force-generating mechanism underlying this chromosome movement. Chromosomes were observed to move toward the cortex in a pulsatile manner along a meandering path. This movement is not propelled by myosin-II-driven cortical flow but is associated with a cloud of dynamic actin filaments trailing behind the chromosomes/spindle. Formation of these filaments depends on the actin nucleation activity of Fmn2, a formin-family protein that concentrates around chromosomes through its amino-terminal region. Symmetry breaking of the actin cloud relative to chromosomes, and net chromosome translocation toward the cortex require actin turnover.     

Aurora kinase A controls meiosis I progression in mouse oocytes

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6- treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Over-expression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.     

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.     

Emerging roles for centromeres in meiosis I chromosome segregation

Centromeres are an essential and conserved feature of eukaryotic chromosomes, yet recent research indicates that we are just beginning to understand the numerous roles that centromeres have in chromosome segregation. During meiosis I, in particular, centromeres seem to function in many processes in addition to their canonical role in assembling kinetochores, the sites of microtubule attachment. Here we summarize recent advances that place centromeres at the centre of meiosis I, and discuss how these studies affect a variety of basic research fields and thus hold promise for increasing our understanding of human reproductive defects and disease states.     

CDC25A phosphatase controls meiosis I progression in mouse oocytes

CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a −/− mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.     

Mad2 and spindle assembly checkpoint function during meiosis I in mammalian oocytes

During mammalian mitosis, a proofreading network called the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation. An inhibitory SAC signal is deputed to inhibits mitotic cell-cycle progression in response to misaligned chromosomes until such imperfections are rectified thereby ensuring equitable chromosome partitioning to daughter cells. Amongst the cast of SAC proteins, mitotic arrest deficient 2 (Mad2) plays a leading role in transducing the SAC signal. The aneuploidy and cancer predispositions of individuals who harbour genetic mutations in SAC genes emphasise the in vivo significance of this surveillance mechanism. In humans, congenital aneuploidies such as Down's syndrome demonstrate an exponential increase with advancing female age. Although largely the result of female meiosis I errors, the molecular entities that succumb with age in oocytes remain elusive. Declining oocyte SAC function could plausibly contribute to such errors. Until recently however, convincing evidence for a functional SAC in mammalian oocytes during meiosis I was unforthcoming. Here I review the evidence regarding the SAC in female mammalian meiosis I and how our understanding of this system has evolved in recent years. This review will focus on Mad2 as this is the SAC protein that has been most comprehensively investigated.     

A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes

Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin beta. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.     

Inverted meiosis and meiotic drive in mealybugs

In the males of lecanoid coccids, or mealybugs, an entire, paternally derived, haploid chromosome set becomes heterochromatic after the seventh embryonic mitotic cycle. In females, both haploid sets are euchromatic throughout the life cycle. In mealybugs, as in all homopteran species, chromosomes are holocentric. Holocentric chromosomes are characterized by the lack of a localized centromere and consequently of a localized kinetic activity. In monocentric species, sister chromatid cohesion and monopolar attachment play a pivotal role in regulating chromosome behavior during the two meiotic divisions. Both these processes rely upon the presence of a single, localized centromere and as such cannot be properly executed by holocentric chromosomes. Here we furnish further evidence that meiosis is inverted in both sexes of mealybugs and we suggest how this might represent an adaptation to chromosome holocentrism. Moreover, we reveal that at the second meiotic division in males a monopolar spindle is formed, to which only euchromatic chromosomes become attached. By this mechanism the paternally derived, heterochromatic, haploid chromosome set strictly segregates from the euchromatic one, and it is then excluded from the genetic continuum as a result of meiotic drive.Communicated by E.A. Nigg     

Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylated around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.     

Regulation of asymmetrical cytokinesis by cAMP during meiosis I in mouse oocytes

Mammalian oocytes undergo an asymmetrical first meiotic division, extruding half of their chromosomes in a small polar body to preserve maternal resources for embryonic development. To divide asymmetrically, mammalian oocytes relocate chromosomes from the center of the cell to the cortex, but little is known about the underlying mechanisms. Here, we show that upon the elevation of intracellular cAMP level, mouse oocytes produced two daughter cells with similar sizes. This symmetrical cell division could be rescued by the inhibition of PKA, a cAMP-dependent protein kinase. Live cell imaging revealed that a symmetrically localized cleavage furrow resulted in symmetrical cell division. Detailed analyses demonstrated that symmetrically localized cleavage furrows were caused by the inappropriate central positioning of chromosome clusters at anaphase onset, indicating that chromosome cluster migration was impaired. Notably, high intracellular cAMP reduced myosin II activity, and the microinjection of phospho-myosin II antibody into the oocytes impeded chromosome migration and promoted symmetrical cell division. Our results support the hypothesis that cAMP plays a role in regulating asymmetrical cell division by modulating myosin II activity during mouse oocyte meiosis I, providing a novel insight into the regulation of female gamete formation in mammals.     

Calmodulin in starfish oocytes. I. Calmodulin antagonists inhibit meiosis reinitiation

A heat-stable factor has been found in starfish (Patiria miniata and Marthasterias glacialis) oocytes that activates two calmodulin-dependent enzymes: bovine brain phosphodiesterase (10-fold increase) and sea urchin egg NAD-kinase (10- to 50-fold increase). The dose-response curves for activation of these enzymes were found to be parallel for the starfish egg extract and pure mammalian brain calmodulin. The active factor was purified by chromatography on DE 52 cellulose to which it remained bound and was eluted by 0.225 M ammonium sulfate. Active fractions were pooled, dialyzed, and run on a polyacrylamide gel. The starfish active factor comigrated with pure bovine brain calmodulin. A radioimmunoassay was performed on the purified factor; it cross-reacted with antibodies against pure calmodulin. That calmodulin may play a role in hormonally induced maturation of starfish oocytes is suggested by the fact that two calmodulin antagonists (trifluoperazine and vinblastine), which are also inhibitors of NAD-kinase, were found to block 1-methyladenine-induced oocyte maturation. The inhibition could be reversed by increasing the hormone concentration. Oocytes were sensitive to trifluoperazine only during the hormone-dependent period.     

Erp1/Emi2 is essential for the meiosis I to meiosis II transition in Xenopus oocytes

Erp1 (also called Emi2), an inhibitor of the APC/C ubiquitin ligase, is a key component of cytostatic factor (CSF) responsible for Meta-II arrest in vertebrate eggs. Reportedly, however, Erp1 is expressed even during meiosis I in Xenopus oocytes. If so, it is a puzzle why normally maturing oocytes cannot arrest at Meta-I. Here, we show that actually Erp1 synthesis begins only around the end of meiosis I in Xenopus oocytes, and that specific inhibition of Erp1 synthesis by morpholino oligos prevents entry into meiosis II. Furthermore, we demonstrate that premature, ectopic expression of Erp1 at physiological Meta-II levels can arrest maturing oocytes at Meta-I. Thus, our results show the essential role for Erp1 in the meiosis I/meiosis II transition in Xenopus oocytes and can explain why normally maturing oocytes cannot arrest at Meta-I.     

Sequential immunocytogenetics, molecular cytogenetics and transmission electron microscopy of microspread meiosis I oocytes from a human fetal carrier of an unbalanced translocation

. The oocytes of a 17 week human fetus carrying an unbalanced 46,XX,add(18)(p13) translocation were studied with a sequential combination of microspreading, immunocytogenetics, fluorescence in situ hybridization (FISH) and transmission electron microscopy. This combination of technologies allowed the collection of data of unique accuracy and resolution. The translocated chromosome was found to be involved in five different synaptic configurations. A consistent feature of these configurations was the involvement of a second small bivalent, presumably chromosome 21 or 22, the normal synapsis of which was often disrupted. We conclude that chromosome 21 or 22 was the source of the translocated material, which was found to be either homologously triply synapsed, heterologously synapsed or asynapsed. Received: 31 August 1996; in revised form: 1 April 1997 / Accepted: 24 May 1997     

Essential CDK1-inhibitory role for separase during meiosis I in vertebrate oocytes

Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms, but in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) on liberation from the inhibitory protein, securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar-body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity.     

Homologous recombination and transposition generate chromosome I neopolymorphism during meiosis in Saccharomyces cerevisiae

We have studied the meiotic segregation of a chromosome length polymorphism (CLP) in the yeast Saccharomyces cerevisiae. The neopolymorphism frequently observed within the smallest chromosomes (I, VI, III and IX) is not completely understood. We focused on the analysis of the structure of chromosome I in 88 segregants from a cross between YNN295 and FL100trp. Strain FL100trp is known to carry a reciprocal translocation between the left arm of chromosome III and the right arm of chromosome I. PCR and Southern hybridization analyses were performed and a method for the rapid detection of chromosome I rearrangements was developed. Seven chromosome I types were identified among the 88 segregants. We detected 22 recombination events between homologous chromosomes I and seven ectopic recombination events between FL100trp chromosome III and YNN295 chromosome I. These recombination events occurred in 20 of the 22 tetrads studied (91%). Nine tetrads (41%) showed two recombination events. This showed that homologous recombination involving polymorphic homologues or heterologous chromosomes is the main source of neopolymorphism. Only one of the seven chromosome I variants resulted from a transposition event rather than a recombination event. We demonstrated that a Ty1 element had transposed within the translocated region of chromosome I, generating mutations in the 3′ LTR, at the border between U5 and PBS. Received: 7 May 1999 / Accepted: 14 February 2000     

Studies in chiral symmetry breaking crystallization I: The effects of stirring and evaporation rates

Chiral symmetry breaking can be realized in stirred crystallization of Na-ClO₃. We present experimental and theoretical studies of the random distribution of crystal enantiomeric excess (cee) for various stirring and solvent evaporation rates. For a fixed solvent evaporation rate, as the stirring RPM is increased, the probability distribution of cee initially broadens and subsequently develops a sharp peak close to cee = 1. On further increase of stirring rate, the probability distribution once again broadens. This broad probability distribution becomes narrow, with a sharp peak near cee = 1, if the solvent evaporation rate is decreased. Thus we show some ways in which the probability distribution of cee can be controlled in stirred crystallization. In particular, our study shows that the stirring rate and the solvent evaporation rate can be adjusted to maximize crystal enantiomeric excess. © 1995 Wiley-Liss, Inc.     

Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes.

Oocytes from LTXBO mice exhibit a delayed entry into anaphase I and frequently enter interphase after the first meiotic division. This unique oocyte model was used to test the hypothesis that protein kinase C (PKC) may regulate the meiosis I-to-meiosis II transition. PKC activity was detected in LTXBO oocytes at prophase I and increased with meiotic maturation, with the highest (P < 0.05) activity observed at late metaphase I (MI). Treatment of late MI-stage oocytes with the PKC inhibitor, bisindolylmaleimide I (BIM), transiently reduced (P < 0.05) M-phase-promoting factor (MPF) activity and promoted (P < 0.05) progression to metaphase II (MII), while mitogen-activated protein kinase (MAPK) activity remained elevated during the MI-to-MII transition. Confocal microscopy analysis of LTXBO oocytes during this transition showed PKC-delta associated with the meiotic spindle and then with the chromosomes at MII. Inhibition of PKC activity also prevented untimely entry into interphase, but only when PKC activity was reduced in oocytes before the progression to MII and thus indicates that the transition into interphase is directly associated with the delayed triggering of anaphase I. Moreover, the defect(s) that initiate activation occur upstream of MAPK, as suppression of PKC activity failed to prevent activation by Mos(tm1Ev)/ Mos(tm1Ev) LTXBO oocytes expressing no detectable MAPK activity. In summary, PKC participates in the regulatory mechanisms that delay entry into anaphase I in LTXBO oocytes, and the disruption promotes untimely entry into interphase. Thus, loss of regulatory control over PKC activity during oocyte maturation disrupts the critical MI-to-MII transition, leading to a precocious exit from meiosis.