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The Small GTPase Cdc42 Promotes Membrane Protrusion during Polar Body Emission via ARP2-Nucleated Actin Polymerization.

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The Small GTPase Cdc42 Promotes Membrane Protrusion during Polar Body Emission via ARP2-Nucleated Actin Polymerization.

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Abstract

Polar body emission is a specialized cell division throughout the animal kingdom, serving to reduce chromosome ploidy while preserving the egg cytoplasm. Critical to polar body emission are the asymmetric positioning of the meiotic spindle prior to anaphase, with one pole attached to the oocyte cortex, and the simultaneous membrane protrusion during subsequent cytokinesis. We have shown that, during Xenopus oocyte maturation, the small GTPase Cdc42 promotes membrane protrusion while a classical RhoA contractile ring forms and constricts at the base of the protrusion. We report here that treating oocytes with low concentrations of nocodazole diminished the size of metaphase I spindles and prevented polar body emission, and yet an active Cdc42 cap of correspondingly diminished size still developed, on time, atop of the spindle pole. Conversely, treating oocytes with low concentrations of taxol resulted in a spindle with multiple poles attached to the cortex, but still each of these poles were associated with activated cortical Cdc42 at the appropriate time. Therefore, the asymmetric positioning of the meiotic spindle with one pole anchored to the cortex is a prerequisite for Cdc42 activation. Furthermore, we demonstrated that the Cdc42-regulated F-actin nucleator ARP2/3 complex were similarly localized at the cortex of the protruding polar body membrane, suggesting that Cdc42 promotes membrane protrusion through an F-actin meshwork mechanism. Finally, we demonstrated that Cdc42 and RhoA formed similarly complementary activity zones during egg activation and that inhibition of Cdc42 prevented second polar body emission. Therefore, Cdc42 activation likely promotes membrane protrusion during polar body emission in widespread systems.

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... Imaging was performed using a 603 oil objective on a Zeiss Axiovert microscope with a Bio-Rad 1024 laser scanning confocal imaging system. Images were rendered to three dimensions (3D) and analyzed using Volocity imaging software (PerkinElmer) [17]. ...
... Mature, metaphase II-arrested oocytes were stimulated to emit the second polar body by pricking them in the animal hemisphere with a fine needle (needle tip diameter, approximately 10 lm) in OR2 medium (82.50 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM Na 2 HPO 4 , 5 mM N-2hydroxyethylpiperazine-N 0 9-2-ethanesulfonic acid [HEPES], pH 7.8) [17]. Sister chromatid separation (anaphase II) occurred approximately 12 min after pricking and completion of second polar body emission takes an additional 15 min [17]. ...
... Mature, metaphase II-arrested oocytes were stimulated to emit the second polar body by pricking them in the animal hemisphere with a fine needle (needle tip diameter, approximately 10 lm) in OR2 medium (82.50 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM Na 2 HPO 4 , 5 mM N-2hydroxyethylpiperazine-N 0 9-2-ethanesulfonic acid [HEPES], pH 7.8) [17]. Sister chromatid separation (anaphase II) occurred approximately 12 min after pricking and completion of second polar body emission takes an additional 15 min [17]. Oocytes are either scanned for the duration of second polar body emission, or examined at least 30 min after prick-activation. ...
Article
The chromosome passenger complex (CPC) consists of Aurora-B kinase and several other subunits. One of these, incenp, binds Aurora-B and regulates its kinase activity. During Xenopus oocyte maturation, incenp accumulates through translation, contributing to aurora-b activation. A previous study has demonstrated that inhibition of incenp translation during oocyte maturation diminishes aurora-b activation but does not interfere with oocyte maturation, characterized by normal maturation-specific cyclin-b phosphorylation, degradation, and resynthesis. Here we have extended these findings, showing that inhibition of incenp translation during oocyte maturation did not interfere with meiosis I or II, as indicated by the normal emission of the first polar body and metaphase II arrest, followed by the successful emission of the second polar body upon parthenogenetic egg activation. Most importantly, however, when transferred to host frogs and subsequently ovulated, the incenp-deficient eggs were fertilized but failed to undergo mitotic cleavage. Thus, translation of incenp during oocyte maturation appears to be part of oocyte cytoplasmic maturation, preparing the egg for the rapid mitosis following fertilization.
... What are these microscopic MTOCs [Huchon et al., 1981;Maro et al., 1985;Gard, 1992;Schuh and Ellenberg, 2007] to which the microtubule minus-ends are anchored? A recent study [Zhang et al., 2011] suggests that mini Golgi stacks may participate in spindle assembly in mouse oocytes. The pattern of GM130 (a Golgi marker) localization [Zhang et al., 2011] is somewhat similar to that of the c-tubulin-and pericentrin-containing mini MTOCs during mouse oocyte maturation [Schuh and Ellenberg, 2007]. ...
... A recent study [Zhang et al., 2011] suggests that mini Golgi stacks may participate in spindle assembly in mouse oocytes. The pattern of GM130 (a Golgi marker) localization [Zhang et al., 2011] is somewhat similar to that of the c-tubulin-and pericentrin-containing mini MTOCs during mouse oocyte maturation [Schuh and Ellenberg, 2007]. Do MTOCs in mouse oocytes contain mini-Golgi stacks? ...
... Such direct studies, namely whether disruption of the spindle causes metaphase I arrest (with bivalent chromosomes), are lacking. Treating frog oocytes with high concentrations of nocodazole inhibits polar body emission [Leblanc et al., 2011]. However, unlike in mouse oocytes, washing out the drug does not permit reformation of the spindle (H Shao and XJ Liu, unpublished results), consistent with an earlier study indicating that frog oocytes treated with nocodazole do not repolymerize microtubules following drug removal [Jessus et al., 1987]. ...
Article
Generation of a haploid female germ cell, the egg, consists of two rounds of asymmetric cell division (meiosis I and meiosis II), yielding two diminutive and nonviable polar bodies and a large haploid egg. Animal eggs are also unique in the lack of centrioles and therefore form meiotic spindles without the pre-existence of the two dominant microtubule organizing centers (centrosomes) found in mitosis. Meiotic spindle assembly is further complicated by the unique requirement of sister chromatid mono-oriented in meiosis I. Nonetheless, the eggs appear to adopt many of the same proteins and mechanisms described in mitosis, with necessary modifications to accommodate their special needs. Unraveling these special modifications will not only help understanding animal reproduction, but should also enhance our understanding of cell division in general. © 2012 Wiley Periodicals, Inc.
... In addition to Ran, the Rho family GTPase Cdc42 has been implicated in multiple events during oocyte meiosis including spindle positioning and the cortical polarity maintenance in both mouse and frog oocytes [Ma et al., 2006;Na and Zernicka-Goetz, 2006;Bielak-Zmijewska et al., 2008;Leblanc et al., 2011]. In Xenopus oocytes, active Cdc42 was found to localize to the polar cortical domain in a spindle-dependent manner [Ma et al., 2006;Zhang et al., 2008]. ...
... In Xenopus oocytes, active Cdc42 was found to localize to the polar cortical domain in a spindle-dependent manner [Ma et al., 2006;Zhang et al., 2008]. Dominant negative Cdc42 inhibited cortical actin assembly inside the contractile ring and prevented polar body extrusion, possibly through blockage of membrane protrusion mediated by Arp2/3-dependent actin dynamics [Leblanc et al., 2011]. In mouse oocytes, Cdc42 perturbation led to disorganized and abnormally long MI spindles, with an associated defect in spindle migration [Na and Zernicka-Goetz, 2006]. ...
... It was found that Cdc42 forms an activity zone overlapping the cortical cap, and Cdc42 activity is crucial for a dynamic F-actin cap [Ma et al., 2006;Zhang et al., 2008]. A more recent study demonstrated that the Arp2/3 complex is localized at the cortex of the protruding cap, suggesting that Cdc42 may promote cortical cap protrusion through Arp2/3-nucleated actin assembly [Leblanc et al., 2011]. As discussed earlier, the Arp2/3 nucleated actin assembly has been indicated to be crucial for the dynamic maintenance of asymmetric meiotic spindle position in mouse MII oocyte [Yi et al., 2011]. ...
Article
Mammalian oocyte maturation involves two successive rounds of extremely asymmetric cell divisions (known as polar body extrusion) to generate a functional haploid egg. Successful polar body extrusion relies on establishment of an asymmetric spindle position and cortical polarity. Decades of studies using mouse oocytes as a model have revealed critical roles for a dynamic actin cytoskeleton in this process. Here, we review the contribution of actin to the critical events during oocyte meiotic cell divisions with an emphasis on recent advances in understanding the underlying molecular and physical mechanisms. © 2012 Wiley Periodicals, Inc.
... This activity is essential for PBE [Ma et al., 2006]. It is thought that Leblanc et al., 2011] Cdc42 activity restricts activation of RhoA to the contractile ring, and that RhoA in turn exerts feedback to control local Cdc42 activation [Ma et al., 2006;Zhang et al., 2008]. These two distinct regions of GTPase activity overlap with two different populations of actin filaments: Cdc42 activity with dynamic F-actin and RhoA with stable F-actin . ...
... Despite the difference in Cdc42 localization in oocytes of different species, it is interesting to note that Cdc42 activity is not known to play any role in mitotic cytokinesis and appears to be exclusively required for cytokinesis during female meiosis [Leblanc et al., 2011]. This could be due to the fact that PBE uniquely involves membrane protrusion as well as ring constriction. ...
... The exact roles of Rho are difficult to demonstrate due to its earlier necessity for spindle-cortex attachment, at least in Xenopus [Ma et al., 2006]. Nevertheless, endogenous Rho and a probe for active Rho enrich in the meiotic contractile ring in mouse and Xenopus [Elbaz et al., 2010;Leblanc et al., 2011]. ...
Article
Polar body cytokinesis is the physical separation of a small polar body from a larger oocyte or ovum. This maternal meiotic division shares many similarities with mitotic and spermatogenic cytokinesis, but there are several distinctions, which will be discussed in this review. We synthesize results from many different model species, including those popular for their genetics and several that are more obscure in modern cell biology. The site of polar body division is determined before anaphase, by the eccentric, cortically associated meiotic spindle. Depending on the species, either the actin or microtubule cytoskeleton is required for spindle anchoring. Chromatin is necessary and sufficient to elicit differentiation of the associated cortex, via Ran-based signaling. The midzone of the anaphase spindle serves as a hub for regulatory complexes that elicit Rho activation, and ultimately actomyosin contractile ring assembly and contraction. Polar body cytokinesis uniquely requires another Rho family GTPase, Cdc42, for dynamic reorganization of the polar cortex. This is perhaps due to the considerable asymmetry of this division, wherein the polar body and the oocyte/ovum have distinct fates and very different sizes. Thus, maternal meiotic cytokinesis appears to occur via simultaneous polar relaxation and equatorial contraction, since the polar body is extruded from the spherical oocyte through the nascent contractile ring. As such, polar body cytokinesis is an interesting and important variation on the theme of cell division. © 2012 Wiley Periodicals, Inc.
... Making mini-cells after parthenogenetic activation (pricking with a fine glass needle, mimicking fertilization) of metaphase II eggs (Leblanc et al., 2011) was much more challenging due to the significant surface contraction, which often prevented the mini-cell shows two dyads. (C) A representative karyotype image of oocytes injected with xSecurin-DM mRNA at 3 h after GVBD. ...
... Bars: (insets) 10 µm. (Leblanc et al., 2011). To effect complete microtubule destruction, we added 3.3 µM nocodazole (1 mg/ml) 1 h after GVBD (when a bipolar meiosis I spindle is seen perpendicular to the animal pole cortex; Fig. 2 A). ...
... These results indicated that nocodazole-treated frog oocytes successfully completed bivalent-to-dyad transition, suggesting that these oocytes were arrested at metaphase II. However, the lack of spindle reformation upon nocodazole removal had precluded the direct confirmation that these were truly metaphase II eggs capable of responding to parthenogenetic activation to emit the second polar body (Leblanc et al., 2011). To circumvent this, we used colcemid, a UV-labile microtubuledisrupting drug (Sluder, 1979). ...
Article
Full-text available
The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC.
... The oocytes were activated twice, with a 2-h interval between the two activations. Interestingly, some studies revealed that the oocytes were able to complete the emissions of the first and the second polar bodies within 3 h of the germinal vesicle breakdown (GVBD) stage [35,36]. Moreover, double and triple activation has been applied to improve the developmental competence in human [37,38] and mouse [37] oocytes. ...
... Several artificial activation methods mimic the transient increases in Ca 2+ oscillations in camel oocytes, including ionomycin [27,41] and the electrical pulse [41]. The rise of cytosolic Ca 2+ following the activation is a potent factor for meiosis and polar body extrusion [35,42]. Direct current (DC) pulses can increase the pores in the cell membrane and increase the membrane permeability, which allows a transmembrane influx of Ca 2+ and stimulates the release of calcium from intracellular stores; additionally, electrical activation induces a more rapid reduction in histone H1 kinase activity [43]. ...
Article
The aim of the current study was to improve the selection method of camel oocytes after in vitro maturation by reducing exclusion criteria that were based only on the presence of the first polar body. A combined nuclear and morphometric assessment of camel oocytes after in vitro maturation was included to perform a judgment. The nuclear status of the oocytes, including the presence of the first polar body, meiosis I stage, and lack of nuclear materials, was investigated. The morphometric criteria that comprised the dimensions of each oocyte were as follows: diameter of the whole oocyte, including the zona pellucida (ZPO), zona pellucida thickness (ZPT), ooplasm diameter (OD), the perivitelline space (PVS) area, and PVS diameter. Among the oocytes with different nuclear status, there were no differences in ZPO and ZPT. However, oocytes with no nuclear material showed a significant reduction in OD (110.19 ± 1.4 μm) and a significant increase in PVS area (2139 ± 324.6 μm²) and PVS diameter (13.9 ± 1.96 μm) when compared with oocytes in the meiosis I stage (117.41 ± 2.85 μm, 1287.4 ± 123.4 μm², and 8.56 ± 0.65 μm, respectively). To simplify the selection, the major difference between meiosis I and degenerated oocytes was the diameter of the PVS, which was greater than the ZPT in degenerated oocytes. Therefore, three groups were morphologically differentiated into oocytes with polar bodies (PB1), meiosis I (MI) oocytes, and degenerated oocytes. MI oocytes were able to extrude the polar body after activation but were not able to develop into blastocysts. In contrast, MI oocytes were able to develop into blastocysts after a biphasic activation protocol in which the oocytes were electrically activated and treated with ionomycin after 2 h. In conclusion, the results obtained by the morphometric assessment allowed us to develop a simple and objective classification system for in vitro matured dromedary camel oocytes, which will lead to accurate oocyte selection for the support of subsequent embryonic development.
... Cdc42 is required for coupling asymmetric spindle positioning with cytokinesis (Ma et al., 2006) and regulates the Arp2/3 complex-mediated membrane protrusion and polar body extrusion in Xenopus oocyte maturation (Zhang et al., 2008;Leblanc et al., 2011). By using GTPase-defective Cdc42 mutants, it has been reported that Cdc42 might be required for asymmetric spindle migration, homologous chromosome segregation, and polar body extrusion in mouse oocytes (Na and Zernicka-Goetz, 2006). ...
... Of interest, Cdc42regulated polarity mediates a special process of "protrusion" budding in yeast, resembling the protrusion in polar body extrusion during Xenopus oocyte maturation (Zhang et al., 2008). During polar body extrusion, the protrusion is mediated by actin dynamics regulated by the Arp2/3 complex in Xenopus oocytes (Leblanc et al., 2011), and the Arp2/3 protein complex has been widely reported as an actin nucleator downstream of Cdc42. In our study, Cdc42 deletion leads to impaired cortex localization of Arp2 ( Figure 4E, yellow arrowhead). ...
Article
Full-text available
Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I (MI) spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here, we investigated the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We found that Cdc42 deletion caused female infertility in mice. Cdc42 deletion had little effect on meiotic spindle organization and migration to the cortex, but inhibited polar body emission, although homologous chromosome segregation occurred. The failure of cytokinesis was due to the loss of polarized Arp2/3 accumulation and actin cap formation, and thus the defected contract ring. Additionally, we correlated active Cdc42 dynamics with its function during polar body emission and revealed a relationship between Cdc42 and polarity as well as polar body emission in mouse oocytes.
... As shown in table 1 , formin-2 (Fmn2) is a specific protein that is co-localized with the spindle during meiosis and is involved in the formation of a dynamic actin meshwork [9] . After an asymmetric positioning of the meiotic spindle and by Cdc42 activation, the membrane protrusion activity probably operates through an actin meshwork mechanism [10] ( table 1 ). It seems that in some animals' oocytes, such as the mouse's, the actin-nucleating protein of Fmn2, an important protein in cytokinesis, is required for spindle migration [9] . ...
... This function of Cdc42 appears to be unique to female meiosis, so inhibiting Cdc42 was shown to be sufficient to prevent the extrusion of the second polar body. All these processes are suggesting an essential function for this protein [10] . Src family kinase proteins may be involved in maintaining the precise temporal restraints of asymmetric division, regulating the size of polar bodies by controlling filamentous actin turnover during the formation and ingression of the cleavage furrow [11] ( table 1 ). ...
Article
Full-text available
Background: Polar bodies degenerate on the first day of embryonic life, and there is no known or specific role for them in the human embryo yet. However, it is not logical to have useless cells. Our previous reports indicated a role for the primo vascular system (PVS) in human beings. There is a possible link between polar bodies and the PVS in embryonic life. Methods: The following databases were searched for peer-reviewed articles in English: Cochrane Collaboration, PubMed, MEDLINE, CINAHL, AMED, Age line, and Social Services Abstracts. Our search included the fol-lowing key words individually or in combination: alternative medicine(s), integrated medicine, holistic care, complementary alternative medicine, embryology, traditional Chinese medicine, traditional medicine, PVS, Bong Han duct in relation to polar bodies. Results: There are no re-ports relating polar bodies to our keywords. Conclusion: The PVS has been suggested to be responsible for embryonic development. This system contains some pluripotent cells and pro-teins, some of which are not even present in the blood circulation in chicken embryos. The sys-tem becomes only visible after staining with trypan blue. Accordingly, this report tries to present the PVS as the ruler of embryonic cell division and development, which regulates all complicated events during that period of life. To the best of our knowledge, this is the first report suggesting that embryonic development is controlled via the PVS, which originates from polar bodies.
... Manually defolliculated oocytes were injected with mRNA encoding various probes, as described in our previous publications (Leblanc et al., 2011;Shao et al., 2012;Zhang et al., 2008). For sfGFP-ER-3 and GFP-IP 3 R1 plasmids, which lack viral (such as SP6 and T7) promoters for in vitro transcription, purified plasmids were injected into the oocyte germinal vesicle (~3nL of 1mg/mL DNA per oocyte). ...
... Oocytes were imaged in poly-lysine-coated glass bottom microwell dishes (MatTek Corporation, P35G-1.5-10-C) with a 60x oil objective on a Zeiss Axiovert with a BioRad 1024 laser scanning confocal imaging system (Leblanc et al., 2011), or a Quorum Spinning Disk confocal system. High resolution images (Fig. 3D, AiryScan) were obtained using Zeiss LSM880 AxioObserver Z1 with AiryScan FAST. ...
Article
The extent to which calcium signaling participates in specific events of animal cell meiosis or mitosis is a subject of enduring controversy. We have previously demonstrated that buffering intracellular calcium with 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, a fast calcium chelator), but not Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA, a slow calcium chelator), rapidly depolymerizes spindle microtubules in Xenopus oocytes, suggesting that spindle assembly and/or stability requires calcium nanodomains – calcium transients at extremely restricted spatial-temporal scales. In this study, we have investigated the function of inositol-1,4,5-trisphosphate receptor (IP 3 R), an endoplasmic reticulum (ER) calcium channel, in spindle assembly using Trim21-mediated depletion of IP 3 R. Oocytes depleted of IP 3 R underwent germinal vesicle breakdown but failed to emit the first polar body and failed to assemble proper meiotic spindles. Further, we developed a cell-free spindle assembly assay in which cytoplasm was aspirated from single oocytes. Spindles assembled in this cell-free system were encased in ER membranes, with IP 3 R enriched at the poles, while disruption of either ER organization or calcium signaling resulted in rapid spindle disassembly. As in intact oocytes, formation of spindles in cell-free oocyte extracts also required IP 3 R. We conclude that intracellular calcium signaling involving IP 3 R-mediated calcium release is required for meiotic spindle assembly in Xenopus oocytes.
... Mature (metaphase II) eggs were imaged to determine the basal fluorescence signals of the two probes, focusing on the animal pole including the first polar body (Fig. 1B, 00:00, arrow). The eggs were then pricked (mimicking fertilization (Leblanc et al., 2011)) followed immediately by time lapse confocal imaging. Robust Lck-GCaMP3 (eGFP) signal was observed at the animal pole cortex 2 minutes after pricking (Fig. 1B,00:02,upper panel,and the graph), consistent with the travelling speed of the calcium wave (Miller et al., 1993) initiated at the site of pricking (1/4 of pole-to-pole distance). ...
... The spindle collapse caused by DB-BAPTA injection or UV activation of diazo-2 exhibited a speed identical to nocodazole treatment (Shao et al., 2013), suggesting similar mechanism: inhibition of microtubule polymerization. Consistent with this interpretation, injection of DB-BAPTA at the junction of metaphase-to-anaphase transition inhibited cortical Cdc42 activation and membrane protrusion (Fig. 6A), both of which are dependent on spindle microtubules (Leblanc et al., 2011) and spindle positioning, with one pole anchored to the cortex (Zhang et al., 2008). In contrast, the complete collapse of microtubule spindle, caused by DB-BAPTA, did not inhibit RhoA activation or formation of the contractile ring (Fig. 6B), suggesting that chromosomes and/or the closely associated MTOC in these acentrosomal oocytes (Gard, 1992;Schuh and Ellenberg, 2007;Shao et al., 2012), were sufficient to induce the contractile ring. ...
Article
Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca(2+) in second polar body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation and, yet, manipulating intracellular calcium levels interferes with first polar body emission, in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we used live cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators caused immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA, at similar or higher intracellular concentrations, had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation, at the spindle or elsewhere. Finally, endogenous calmodulin was found to colocalize with spindle microtubules throughout all stages of meiosis. Our results, most importantly the different sensitivity of the spindle to BAPTA and EGTA, suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium signaling.
... 7 complex to interconnect it, 35 and in this pathway, although N-WASP is a weak stimulator of the Arp2/3 complex, its activity can be enhanced by upstream factors such as CDC42 and PI(4,5)p2 36 and that CDC42 can stimulate membrane protrusion in oocytes through the ARP2/3 complex.. 37 This is in contrast to the downregulation of the Arp2/3 complex by CDC42, which inhibits oocyte membrane protrusion. 38 In mouse oocytes, the chromatin signal Ran GTPase drives the formation of polarized actin caps by promoting the accumulation of polarized N-WASP signalling across the spindle cortex, 39 and the silent expression of CDC42 resulting from its diminished signalling also allows for the disruption of actin cap formation. 40 As such, we hypothesize that the signalling cascade of oocyte actin polarization: ...
... 31 In mouse oocytes, Rho-GTPase contributes to membrane protrusion during polar body formation and ring contraction. 38 During meiosis in mouse oocytes, the Ran GTPase-CDC42 GTP-N WASP-Arp2/3 signalling pathway is conserved during late meiosis, and CDC42 is an essential component of the pathway that is required for first polar body emission in vivo. In contrast, the inhibition of CDC42 signalling can lead to the release of N-WASP into the cytoplasm, allowing secondary complete failure of second polar body extrusion in oocytes. ...
Article
Full-text available
CDC42 is a member of the Rho‐GTPase family and is involved in a variety of cellular functions including regulation of cell cycle progression, constitution of the actin backbone and membrane transport. In particular, CDC42 plays a key role in the establishment of polarity in female vertebrate oocytes, and essential to this major regulatory role is its local occupation of specific regions of the cell to ensure that the contractile ring is assembled at the right time and place to ensure proper gametogenesis. The multifactor controlled ‘inactivation‐activation’ process of CDC42 also allows it to play an important role in the multilevel signalling network, and the synergistic regulation of multiple genes ensures maximum precision during gametogenesis. The purpose of this paper is to review the role of CDC42 in the control of gametogenesis and to explore its related mechanisms, with the aim of further understanding the great research potential of CDC42 in female vertebrate germ cells and its future clinical translation.
... Thus, cortical outpocketing during anaphase I and II is thought to be induced by the cortical accumulation of active Cdc42 in Xenopus and mouse oocytes. However, it is not entirely clear what triggers the accumulation of active Cdc42 at the cortex driving outpocketing [18], and indeed how one outpocket rather than two are formed during meiosis II. Here in the ascidian we found that during meiosis II the midbody formed during PB1 emission becomes visible as a small "polar corps" sitting on the protrusive outpocket and that this polar corps predicts the precise site of PB2 outpocketing; we thus speculate that the polar corps may be involved in attracting one spindle pole into the protrusive outpocket during Ana II. ...
... Thus, in both Xenopus and mouse oocytes, the activation of the anaphase promoting complex (APC), which leads to the destruction of cyclin B (inactivating MPF) and securin (activating separase), is permissive for the cortical recruitment of active Cdc42 driving outpocketing during anaphase. Finally, although the fall in MPF activity is permissive for outpocketing, it is not clear how active Cdc42 is recruited to the actin cap during anaphase, although spindle pole proximity to the cortex is thought to be required [18]. Whether chromatin is also involved in driving Cdc42 recruitment to the actin cap during outpocketing (when spindle microtubules are depolymerized) is difficult to assess in mouse oocytes, because microtubule depolymerization activates the spindle assembly checkpoint (SAC) thus preventing the fall in MPF activity [41]. ...
Article
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Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 µm in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the "polar corps". As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.
... Rare budding from polynuclear giant cells was still found even 7-10 weeks post-DOX treatment ( Figure 1F); however, on the whole, the culture phenotype returned to that of the untreated cells ( Figure 1H). We decided to check the cells for the transcription of the Rho GTPases that are responsible for cell motility involved in the amoeboid transition, cell membrane protrusion for spore budding, and for invasion, CDC42 and RAC1 [55,56], as well as for the main stemness transcription factor POU5F1/OCT4A in the prolonged time course post-DOX. The results of Selfie digital PCR expressed per gene copy and per cell (multiplied by the average ploidy number, shown in Table 1, determined in the same culture) for each sampling term are presented in Figure 2E, per gene copy and gene dosage. ...
Article
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Mitotic slippage (MS), the incomplete mitosis that results in a doubled genome in interphase, is a typical response of TP53-mutant tumors resistant to genotoxic therapy. These polyploidized cells display premature senescence and sort the damaged DNA into the cytoplasm. In this study, we explored MS in the MDA-MB-231 cell line treated with doxorubicin (DOX). We found selective release into the cytoplasm of telomere fragments enriched in telomerase reverse transcriptase (hTERT), telomere capping protein TRF2, and DNA double-strand breaks marked by γH2AX, in association with ubiquitin-binding protein SQSTM1/p62. This occurs along with the alternative lengthening of telomeres (ALT) and DNA repair by homologous recombination (HR) in the nuclear promyelocytic leukemia (PML) bodies. The cells in repeated MS cycles activate meiotic genes and display holocentric chromosomes characteristic for inverted meiosis (IM). These giant cells acquire an amoeboid phenotype and finally bud the depolyploidized progeny, restarting the mitotic cycling. We suggest the reversible conversion of the telomerase-driven telomere maintenance into ALT coupled with IM at the sub-telomere breakage sites introduced by meiotic nuclease SPO11. All three MS mechanisms converging at telomeres recapitulate the amoeba-like agamic life-cycle, decreasing the mutagenic load and enabling the recovery of recombined, reduced progeny for return into the mitotic cycle.
... Downstream targets of RanGTP involved in this process have not yet been identified but this finding indicates a link between chromosomes and cortical differentiation. The differentiated cortex is the site of recruitment of the small GTPase Cdc42 [77], which promotes cortical relaxation and polar body `outpocketing' through assembly of a highly dynamic F-actin network [73,78,79]. Polar body cytokinesis relies on a ring of active RhoA that circumscribes the region of active Cdc42 and directs assembly of an actomyosin contractile ring that extends into a tube before abscission [77,80]. ...
Article
The ability to reproduce relies in most eukaryotes on specialized cells called gametes. Gametes are formed by the process of meiosis in which, after a single round of replication, two successive cell divisions reduce the ploidy of the genome. Fusion of gametes at fertilization reconstitutes diploidy. In most animal species, chromosome segregation during female meiosis occurs on spindles assembled in the absence of the major microtubule-organizing center, the centrosome. In mammals, oocyte meiosis is error prone and underlies most birth aneuploidies. Here, we review recent work on acentrosomal spindle formation and chromosome alignment/separation during oocyte meiosis in different animal models.
... This provides a convenient landmark to position the oocyte for live cell imaging. 17 We used 4D imaging 18 to study the process of meiosis by collecting time series of confocal Z-stacks. After collection, stacks were rendered into 3D representations and, in the figures below, are presented either in the transverse direction (side view) or en face (top view). ...
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Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine(122) is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.
... There are no more than 15 terms per cluster and no more than 250 terms in total. The nodes are coloured by cluster identification, where nodes that share the same cluster identification are generally close to each other body I extrusion occur in several mammalian species [30,31]. Meiosis requires highly specialised chromosomal connections. ...
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Background: The ewe lamb nutritional and physiological state interfere with the ovarian environment and fertility. The lack or excess of circulating nutrients reaching the ovary can change its gene expression. A protein deficiency in the blood caused by an Haemonchus contortus abomasal infection is detrimental to the organism's development during puberty. The peripubertal period is a time of intensive growth that requires a high level of nutrients. An essential feature controlling pubertal arousal and female reproductive potential is ovarian follicle growth activation. Protein supplementation improves the sheep's immune response to helminthic infections. We aimed to determine if supplementing protein in infected ewe lambs' diet would impact the ovarian environment leading to earlier ovarian follicle activation than in infected not supplemented animals. Methods: We fed 18 Santa Ines ewe lambs (Ovis aries) - bred by the same ram - with either 12% protein (Control groups) or 19% protein (Supplemented groups) in their diets. After 35 days of the diet, they were each artificially infected or not with 10,000 Haemonchus contortus L3 larvae. Following 77 days of the diet and 42 days of infection, we surgically collected their left ovaries and examined their genes expression through RNA sequencing. Results: We found that protein supplementation in infected animals led to an up-regulation of genes (FDR p-values < 0.05) and biological processes (p-value cut-off = 0.01) linked to meiotic activation in pre-ovulatory follicles and primordial follicle activation, among others. The supplemented not infected animals also up-regulated genes and processes linked to meiosis and others, such as circadian behaviour. The not supplemented animals had these same processes down-regulated while up-regulated processes related to tissue morphogenesis, inflammation and immune response. Conclusion: Diet's protein supplementation of peripubertal infected animals allowed them to express genes related to a more mature ovarian follicle stage than their half-sisters that were not supplemented. These results could be modelling potential effects of the interaction between environmental factors, nutrition and infection on reproductive health. When ovarian activation is achieved in a timely fashion, the ewe may generate more lambs during its reproductive life, increasing sheep breeders' productivity.
... It should be also noted here that asymmetric reduction division of maturing oocytes, wherein polar bodies are released during anaphase through an actin ring [191,192] is similar to sporogenesis and the asymmetric reduction divisions observed in some PGCC, suggesting their mutual natureexamples from four DNA-damage stressed tumour types are presented in Suppl. Fig. 2. In the case of parthenogenesis, a polar body is further reunited with the female pronucleus and is also very similar to the processes observed in the amoeboid. ...
Article
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The fundamental understanding of how Cancer initiates, persists and then progresses is evolving. High-resolution technologies, including single-cell mutation and gene expression measurements, are now attainable, providing an ever-increasing insight into the molecular details. However, this higher resolution has shown that somatic mutation theory itself cannot explain the extraordinary resistance of cancer to extinction. There is a need for a more Systems-based framework of understanding cancer complexity, which in particular explains the regulation of gene expression during cell-fate decisions. Cancer displays a series of paradoxes. Here we attempt to approach them from the view-point of adaptive exploration of gene regulatory networks at the edge of order and chaos, where cell-fate is changed by oscillations between alternative regulators of cellular senescence and reprogramming operating through self-organisation. On this background, the role of polyploidy in accessing the phylogenetically pre-programmed “oncofetal attractor” state, related to unicellularity, and the de-selection of unsuitable variants at the brink of cell survival is highlighted. The concepts of the embryological and atavistic theory of cancer, cancer cell “life-cycle”, and cancer aneuploidy paradox are dissected under this lense. Finally, we challenge researchers to consider that cancer “defects'' are mostly the adaptation tools of survival programs that have arisen during evolution and are intrinsic of cancer. Recognition of these features should help in the development of more successful anti-cancer treatments.
... Additionally, elegant RNAi and antibody injection experiments, resulting in the targeted degradation of JMY mRNA or blockade of protein function, respectively, confirm its essential role in asymmetric division, producing MII oocytes resembling a 2-cell embryo. Leblanc et al. (2011) from the Ottawa Hospital Research Institute, also present data that illuminate the process of polar body formation. They concentrate on Cdc42, a GTPase which is first detected as a circular cap directly overlaying the spindle pole following initiation of anaphase I. LeBlanc confirms that Cdc42 promotes the membrane protrusion essential for polar body formation, a function of Cdc42 that appears to be unique to female meiosis and has not been noted in other forms of cell division. ...
... Homologous chromosomes are segregated and one set is discarded into a membrane extrusion that eventually forms a small extra cell called 'polar body (PB)'. The membrane extrusion occurs after anaphase onset through Cdc42-dependent F-actin polymerisation (Zhang et al, 2008;Leblanc et al, 2011) at the site of spindle attachment to the cortex. A RhoA-controlled, contractile F-actin/myosin II ring then encompasses the membrane extrusion (Zhang et al, 2008) and drives segregation of the PB similar to somatic cell division (Green et al, 2011;Liu, 2012;Maddox et al, 2012). ...
Article
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Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis.
... Expression of constitutively active Rac had a strong, inhibitory effect on cytokinesis in sea urchin zygotes (Figure 2), in agreement with studies in cultured cells (Yoshizaki et al., 2004). However, both Rac and Arp2/3 are required for meiotic divisions in mouse oocytes, through the regulation of cytoplasmic streaming and spindle migration (Halet and Carroll, 2007;Leblanc et al., 2011;Sun et al., 2011a;Chaigne et al., 2013Chaigne et al., , 2016Yi et al., 2013;Wang et al., 2014). Polar body formation during meiotic maturation is thought to occur through a combination of a SCW, a localized protrusion of the cortex induced by the meiotic spindle, and the canonical cytokinetic signaling apparatus (Maddox et al., 2012;Satoh et al., 2013;Bischof et al., 2017;Klughammer et al., 2018). ...
Article
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In motile cells, the activities of the different Rho family GTPases are spatially segregated within the cell, and during cytokinesis there is evidence that this may also be the case. But while Rho’s role as the central organizer for contractile ring assembly is well established, the role of Rac and the branched actin networks it promotes is less well understood. To characterize the contributions of these proteins during cytokinesis, we manipulated Rac and Arp2/3 activity during mitosis and meiosis in sea urchin embryos and sea star oocytes. While neither Rac nor Arp2/3 were essential for early embryonic divisions, loss of either Rac or Arp2/3 activity resulted in polar body defects. Expression of activated Rac resulted in cytokinesis failure as early as the first division, and in oocytes, activated Rac suppressed both the Rho wave that traverses the oocyte prior to polar body extrusion as well as polar body formation itself. However, the inhibitory effect of Rac on cytokinesis, polar body formation and the Rho wave could be suppressed by effector-binding mutations or direct inhibition of Arp2/3. Together, these results suggest that Rac- and Arp2/3 mediated actin networks may directly antagonize Rho signaling, thus providing a potential mechanism to explain why Arp2/3-nucleated branched actin networks must be suppressed at the cell equator for successful cytokinesis.
... Within a range of 20 mm from the chromatin position, this RAN-GTP gradient is able to promote the formation of the actin cap (Deng et al., 2007) by a mechanism only partially elucidated but that involves the regulation by and co-localization of cdc42 (Dehapiot et al., 2013), a cell polarization mediator. In turn, Cdc42 activates the actin-related protein 2/3 (ARP2/3) complex (Leblanc et al., 2011; Sun et al., 2011), an actin nucleator, and its activator neural Wiskott – Aldrich syndrome protein (NWASP; Rohatgi et al., 1999 ). Our demonstration of an actin cap in human MII oocytes, together with the absence of information on how such cytoskeletal polarization is established and regulated, clearly opens exciting new opportunities for future research The intensity of actin signal was measured along a segment drawn through an equatorial plane of the oocyte, coaxially with the spindle. ...
Article
In mature mammalian oocytes, cortical f-actin distribution is polarized, as evidenced by a prominent cap subtended by the metaphase II (MII) spindle. Formation of a polarized actin cap is a consequence of a complex actomyosin-driven contractile process that directs polar body extrusion. Human mature oocytes also display a network of sub-oolemmal actin, but so far there has been no suggestion of an actin-rich domain in the vicinity of the spindle.By high-resolution confocal microscopy, we generated semi-quantitative data of the actin cytoskeleton in human mature and immature oocytes, with the aim to better understand the characteristics and remodelling of this cytoskeletal component in the female gamete. In mature MII oocytes, the cortical domain near the spindle showed a more intense actin signal in comparison to the opposite cortical domain (177.2±59.0 vs. 126.8±61.0, P<0.0001; data expressed in arbitrary units). The extent of cortical f-actin polarity was comparable between in vivo and in vitro matured oocytes. However, both the degree of polarity and relative abundance of signal were diminished with increasing maternal age. Mean intensity of cytoplasmic actin was significantly higher in oocytes matured in vitro derived from in vitro maturation (IVM) cycle, in comparison to oocytes matured in vivo or in vitro obtained from controlled ovarian stimulation cycles (35.0±8.0, 21.1±12.4 and 25.9±8.6, respectively; P=0.025). In germinal vesicle (GV)-stage oocytes obtained from both IVM and controlled ovarian stimulation cycles, cortical actin did not appear polarized, irrespective of whether the GV was located centrally or asymmetrically.These data indicate that, during maturation, cortical actin acquires a polarized distribution involving an accumulation in the domain adjacent the spindle. They also propose new questions concerning the existence of cytoplasmic actin in mature oocytes. Finally, they are suggestive of an influence of maternal age on the actin cytoskeleton.
... An intriguing observation of this study is that capping protein did not necessarily localize in actin-rich regions of oocytes. The cortical actin cap, which forms near to the approaching spindle, contains a dense actin network (Yi and Li, 2012;Yi et al., 2013a), and its formation is dependent on the Arp2/3 complex, Rho family GTPases and various nucleation-promoting factors (Dehapiot et al., 2013;Deng et al., 2007;Leblanc et al., 2011;Wang et al., 2013). Capping protein is an essential factor in an Arp2/3-mediated actin polymerization and depolymerization model called the 'dendritic treadmilling model' (Blanchoin et al., 2000a;Blanchoin et al., 2000b;Pollard and Borisy, 2003); therefore, this difference between the localization of capping protein and that of the actin-rich cortical cap is unexpected. ...
Article
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Actin polymerization is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion, and cytokinesis. The heterodimeric actin-capping protein (CP) is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. However, the roles of CP in mammalian oocyte maturation are poorly understood. We investigated the roles of CP in mouse oocytes and found that CP is essential for correct asymmetric spindle migration and polar body extrusion. CP mainly localized in the cytoplasm during maturation. By knockdown or ectopically overexpression of CP revealed that CP is critical for efficient spindle migration and maintenance of the cytoplasmic actin mesh density. Expression of the CP inhibiting protein CARMIL impaired spindle migration and polar body extrusion during oocyte maturation and decreased the cytoplasmic actin mesh density. Taken together, these findings show that CP is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation, presumably by controlling the cytoplasmic actin mesh density.
... polarization of the worm zygote, which can also be triggered by a microtubule-based mechanism (Motegi et al., 2011;Zonies et al., 2010). In Drosophila and mammalian oocytes, the actin cytoskeleton is polarized and plays an important role with Cdc42 in breaking symmetry (Leblanc et al., 2011;Leibfried et al., 2013;Ma et al., 2006;Wang et al., 2013;Yi et al., 2013;Yi et al., 2011;Zhang et al., 2008). In epithelial cells, which of these two mechanisms, oligomeric clustering of determinants versus cytoskeletal transport of determinants, is responsible for directing polarity remains a fundamental unsolved problem. ...
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Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and Hippo signalling component. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. We use the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins - Merlin, Moesin and Expanded - whose simultaneous loss disrupts apical localization of Crb. Accordingly, deletion of the Crb FERM-binding motif also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.
... Creation of these "softer" domains at the cell poles and in the polar lobe might serve the dual function of constraining myosin II as well as conferring a cortex that is easier to deform. On a much smaller scale, the polar bodies extruded during meiosis may represent a similar scenario, given that Arp2/3 is similarly enriched in the polar bodies present in Chlamys and Crassostrea (Fig. 6, S2 and S3), as well as mouse oocytes (Leblanc et al., 2011). Thus, polar bodies and polar lobes may represent similar structures whereby Arp2/3 enrichment creates a zone of lowered DMSO or 100 μM CK666 to inhibit Arp2/3 after completing second meiosis. ...
Article
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In many spiralians, asymmetry in the first two cleavages is achieved through the formation of a polar lobe (PL), which transiently constricts to sequester vegetal cytoplasm into the CD and D blastomeres. While microtubules and actin filaments are required for polar lobe formation, little else is known regarding the structural and functional similarities with the contractile ring, or how the PL constriction is able to form perpendicular to the cleavage plane. Examination of scallop embryos revealed that while activated myosin II could be detected in both the cleavage furrow and early PL constriction, astral or central spindle microtubules were not observed associated with the PL neck until the constriction was nearly complete. Further, inhibition of Aurora B had no effect on polar lobe initiation, but blocked both contractile ring ingression and PL constriction beyond phase II. The cortex destined for PL sequestration was marked by enrichment of the Arp2/3 complex, which was first detected during meiosis and remained enriched at the vegetal pole through the first two cleavages. Inhibition of Arp2/3 affected PL formation and partitioning of cytoplasm into the two daughter cells, suggesting that Arp2/3 plays a functional role in defining the zone of cortex to be sequestered into the polar lobe. Together, these data offer for the first time a mechanism by which a cytoskeletal specialization defines the polar lobe in this atypical form of asymmetric cell division.
... 34 In addition to Ran, the Cdc42 protein regulates actin polymerization to promote polar body emission during meiosis. 35 Our results indicated that RhoA regulated porcine oocyte maturation and early embryo development through its regulation of actin assembly. ...
Article
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Mammalian oocyte maturation is distinguished by asymmetric division that is regulated primarily by cytoskeleton, including microtubules and microfilaments. Small Rho GTPase RhoA is a key regulator of cytoskeletal organization which regulates cell polarity, migration, and division. In this study, we investigated the roles of RhoA in mammalian oocyte meiosis and early embryo cleavage. (1) Disrupting RhoA activity or knock down the expression of RhoA caused the failure of polar body emission. This may have been due to decreased actin assembly and subsequent spindle migration defects. The involvement of RhoA in this process may have been though its regulation of actin nucleators ROCK, p-Cofilin, and ARP2 expression. (2) In addition, spindle morphology was also disrupted and p-MAPK expression decreased in RhoA inhibited or RhoA KD oocytes, which indicated that RhoA also regulated MAPK phosphorylation for spindle formation. (3) Porcine embryo development was also suppressed by inhibiting RhoA activity. Two nuclei were observed in one blastomere, and actin expression was reduced, which indicated that RhoA regulated actin-based cytokinesis of porcine embryo. Thus, our results demonstrated indispensable roles for RhoA in regulating porcine oocyte meiosis and cleavage during early embryo development.
... However, the actin cytoskeleton is not strictly required for polarization of the worm zygote, which can also be triggered by a microtubule-based mechanism (Motegi et al., 2011;Zonies et al., 2010). In Drosophila and mammalian oocytes, the actin cytoskeleton is polarized and plays an important role with Cdc42 in breaking symmetry (Leblanc et al., 2011;Leibfried et al., 2013;Ma et al., 2006;Wang et al., 2013;Yi et al., 2011Yi et al., , 2013Zhang et al., 2008). In epithelial cells, which of these two mechanisms, oligomeric clustering of determinants versus cytoskeletal transport of determinants, is responsible for directing polarity remains a fundamental unsolved problem. ...
Article
Full-text available
Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. We use the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins - Merlin, Moesin and Expanded - whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.
... By imaging fluorescently labelled actin, a clear accumulation of actin at the position where the polar body is formed becomes visible (Fig 8F and S11 Video). This suggests that actin polymerisation is the underlying mechanism to generate polar body formation, similar to the mechanism of polar body extrusion in Xenopus [38]. ...
Article
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Author summary As already noted by Aristotle, life is motion. On the molecular scale, thermal motion leads to diffusive transport. On cellular scales, however, diffusion starts to become inefficient, due to the general property of random walks that their spatial excursions grow less than linear with time. Therefore more directed transport processes are needed on cellular scales, including transport by molecular motors or by hydrodynamic flows. This is especially true for oocytes and eggs in early animal development, which often have to be large in order to store sufficient amounts of nutrients. Here we use starfish oocytes as a convenient model system to investigate the nature and function of cytoplasmic flows in early development. These cells are very large and optically transparent, and therefore ideal for live cell imaging that here we combine with image processing and mathematical modelling. This approach allows us to demonstrate that the experimentally observed cytoplasmic flows during early development are a direct consequence of surface contraction waves that deform the soft and contractile eggs. Additionally we show that despite its microscopic complexity, the cytoplasm behaves like a Newtonian fluid on the cellular scale. Our findings impose strong physical limits on the potential biological function of these flows and suggest that also other cellular systems that are soft and contractile might experience large cytoplasmic flows upon cell shape changes, for example during cell migration or division.
... The cortical actin cap, which forms near the approaching spindle and promotes spindle positioning, contains a dense actin network (Yi et al., 2013;Yi et al., 2011). Cortical actin cap formation is dependent on the ARP2/3 complex, Rho family GTPases, and various nucleation-promoting factors (Leblanc et al., 2011;Wang et al., 2013). Previously, a CAP1-null mutation and mutations in the ARP2/3 complex pathway were shown to synergistically enhance morphological defects, suggesting that CAP1 is actively involved in ARP2/3-dependent actin regulation, or that CAP1 and ARP2/3 are involved in the same process (Deeks et al., 2007). ...
Article
Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cellular events, and various actin-regulatory proteins modulate actin polymerization and depolymerization. Adenylyl cyclase-associated proteins (CAPs), highly conserved actin monomer-binding proteins, have been known to promote actin disassembly by enhancing the actin-severing activity of the ADF/cofilin protein family. In this study, we found that CAP1 regulated actin remodeling during mouse oocyte maturation. Efficient actin disassembly during oocyte maturation is essential for asymmetric division and cytokinesis. CAP1 knockdown impaired meiotic spindle migration and asymmetric division, and resulted in an accumulation of excessive actin filaments near the spindles. In contrast, CAP1 overexpression reduced actin mesh levels. CAP1 knockdown also rescued a decrease in cofilin family protein overexpression-mediated actin levels, and simultaneous expression of human CAP1 (hCAP1) and cofilin synergistically decreased cytoplasmic actin levels. Overexpression of hCAP1 decreased the amount of phosphorylated cofilin, indicating that CAP1 facilitated actin depolymerization via interaction with ADF/cofilin during mouse oocyte maturation. Taken together, our results provide evidence for the importance of dynamic actin recycling by CAP1 and cofilin in the asymmetric division of mouse female gametes. This article has an associated First Person interview with the first author of the paper.
... When Cdc42 was deleted in oocytes, chromosome segregation occurred, but polar body extrusion failed, which indicated a defect in contractile ring constriction [48]. Moreover, a similar Cdc42 function was found in Xenopus oocytes, for which Cdc42 was inhibited, leading to blockage of polar body extrusion and failure of contractile ring formation, although the oocytes could initiate anaphase [108,109]. Cdc42 was also shown to coordinate with RhoA during oocyte polar body extrusion. In this study, Cdc42 promoted membrane protrusion, and RhoA restricted the region with the outpocket to initiate contractile ring constriction [55]. ...
Article
During mitosis, cells undergo symmetrical cell division, while oocyte meiotic maturation undergoes two consecutive, asymmetric divisions that generate a totipotent haploid oocyte and two small polar bodies not involved in DNA replication. This specialized division allows most maternal components be maintained in the oocytes for early embryo development. Nuclear positioning, germinal vesicle breakdown, spindle migration, spindle rotation, chromosome segregation, and polar body extrusion are the most critical cellular processes during oocyte meiosis I and II, and a growing number of studies primarily using the mouse oocyte model revealed that actin filaments were critical for these processes, especially for spindle migration. Several important molecules have been reported to be involved in these processes. One family of molecules are the small GTPases, such as Rho GTPases, Ran GTPases, and Rab GTPases and another are the actin nucleators, such as the formin family and the Arp2/3 complex. The present review summarizes recent progress made regarding the roles of actin filaments in the asymmetric oocyte division.
... The actomyosin-ring contraction leads to protrusion of the cortex cap overlying the spindle. These events are regulated by the small GTPase, CDC42 and its downstream affecter Arp2/3 complex, a major actin regulation complex, both detected in the cortex cap (Leblanc et al., 2011). ...
... Cdc42 is a vital factor in cell phagocytosis, which is an actin-dependent process essential to the host defense mechanism (9). Cdc42 plays a role in actin polymerization (10)(11)(12) and can regulate gene transcription in various signaling pathways, such as the NF-B, NOD1, c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase (MAPK) pathways (7,13). Alternatively, several RNA virus species, including human immunodeficiency virus type 1 (HIV-1), respiratory syncytial virus (RSV), and Ebola virus (EBOV), have developed a diverse repertoire of mechanisms to hijack cellular actin-regulating signaling pathways as part of their cell entry processes. ...
Article
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Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration and cell movement. Several RNA viruses employ Cdc42 hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 gene in the kuruma shrimp Marsupenaeus japonicus and named it Mj Cdc42. Mj Cdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of Mj Cdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that Mj Cdc42 interacted with an arginine kinase ( Mj AK). By analyzing the binding activity and enzyme activity of Mj AK and its mutant Δ Mj AK, we found that Mj AK could enhance the replication of WSSV in shrimp. Mj AK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of Mj AK in WSSV replication. Further study demonstrated that the binding of Mj Cdc42 and Mj AK depends on Cys ²⁷¹ of Mj AK and suppresses the WSSV replication-promoting effect of Mj AK. By interacting with the active site of Mj AK and suppressing its enzyme activity, Mj Cdc42 inhibits WSSV replication in shrimp. Our results demonstrated a new function of Cdc42 in the cellular defense against viral infection in addition to regulating actin and phagocytosis, which have been reported in previous studies. IMPORTANCE: The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates.
... Constitutively activated Cdc42 leads to cytokinesis failure in HeLa cells and Drosophila embryos (Crawford et al., 1998;Dutartre et al., 1996). In mouse oocytes, Cdc42 is required for polar body protrusion and asymmetric cytokinesis (Bielak-Zmijewska et al., 2008;Dehapiot et al., 2013;Leblanc et al., 2011;Liu, 2012;Ma et al., 2006;Maddox et al., 2012;Zhang et al., 2008a). In Xenopus embryos, both constitutively active and dominant negative forms of Cdc42 lead to cytokinetic failure (Drechsel et al., 1997). ...
Article
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Rho family GTPase, Cdc42 regulates cell polarity and localizes to the cell division site. Cdc42 is activated by Guanine nucleotide exchange factors (GEFs). We report that, Cdc42 promotes cytokinesis via a unique spatiotemporal activation pattern due to the distinct action of its GEFs, Gef1 and Scd1 in fission yeast. Cdc42 activation, prior to cytokinetic ring constriction, is Gef1-dependent; and post ring constriction, it is Scd1-dependent. Gef1 localizes to the actomyosin ring immediately after ring assembly and promotes timely onset of ring constriction. Gef1 is required for proper actin organization during cytokinesis, distribution of type V myosin Myo52 to the division site, and timely recruitment of septum protein Bgs1. In contrast, Scd1 localizes to the broader region of ingressing membrane during cytokinetic furrowing. Scd1 promotes normal septum formation and scd1Δ cells display aberrant septa with reduced Bgs1 localization. Thus, we define unique roles of the GEFs Gef1 and Scd1 in the regulation of distinct events during cytokinesis. Gef1 localizes first to the cytokinetic ring and promotes timely constriction, while Scd1 localizes later to the ingressing membrane and promotes septum formation. Our findings are consistent with reports that, complexity in GTPase signaling patterns enable exquisite precision over the control of cellular processes.
... Also, actin filaments ensure the targeting of microtubule spindle positioning at the periphery of an oocyte concomitant with chromosome segregation [11,12]. Additionally, a fine network of microfilaments is found throughout the entire cortex and is responsible for constructing the classical contractile ring, which constricts at the base of the protrusion for pinching off a polar body [2,13]. Although several molecules have been proposed to contribute to cytoskeletal regulation during oocyte meiosis in mammals, the molecular mechanisms that modulate the meiotic apparatus remain to be determined. ...
Article
Mammalian diaphanous1 (mDia1) is a homologue of Drosophila diaphanous and belongs to the Formin-homology family of proteins that catalyze actin nucleation and polymerization. Although Formin family proteins, such as Drosophila diaphanous, have been shown to be essential for cytokinesis, whether and how mDia1 functions during meiosis remain uncertain. In this study, we explored possible roles and the signaling pathway involved for mDia1 using a mouse oocyte model. mDia1 depletion reduced polar body extrusion, which may have been due to reduced cortical actin assembly. mDia1 and Profilin1 had similar localization patterns in mouse oocytes and mDia1 knockdown resulted in reduced Profilin1 expression. Depleting FMNL1, another Formin family member, resulted in reduced mDia1 expression, while RhoA inhibition did not alter mDia1 expression, which indicated that there was a FMNL1-mDia1-Profilin1 signaling pathway in mouse oocytes. Additionally, mDia1 knockdown resulted in disrupting oocyte spindle morphology, which was confirmed by aberrant p-MAPK localization. Thus, these results demonstrated indispensable roles for mDia1 in regulating mouse oocyte meiotic maturation through its effects on actin assembly and spindle organization.
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In contrast to symmetric division in mitosis, mammalian oocyte maturation is characterized by asymmetric cell division that produces a large egg and a small polar body. The asymmetry results from oocyte polarization, which includes spindle positioning, migration, and cortical reorganization, and this process is critical for fertilization and the retention of maternal components for early embryo development. Although actin dynamics are involved in this process, the molecular mechanism underlying this remained unclear until the use of confocal microscopy and live cell imaging became widespread in recent years. Information obtained through a PubMed database search of all articles published in English between 2000 and 2012 that included the phrases "oocyte, actin, spindle migration," "oocyte, actin, polar body," or "oocyte, actin, asymmetric division" was reviewed. The actin nucleation factor actin-related protein 2/3 complex and its nucleation-promoting factors, formins and Spire, and regulators such as small GTPases, partitioning-defective/protein kinase C, Fyn, microRNAs, cis-Golgi apparatus components, myosin/myosin light-chain kinase, spindle stability regulators, and spindle assembly checkpoint regulators, play critical roles in asymmetric cell division in oocytes. This review summarizes recent findings on these actin-related regulators in mammalian oocyte asymmetric division and outlines a complete signaling pathway.
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The mammalian oocyte undergoes an asymmetric division during meiotic maturation, producing a small polar body and a haploid gamete. This process involves the dynamics of actin filaments, and the guanosine triphosphatase (GTPase) protein superfamily is a major regulator of actin assembly. In the present study, the small GTPase CDC42 was shown to participate in the meiotic maturation of porcine oocytes. Immunofluorescent staining showed that CDC42 was mainly localized at the periphery of the oocytes, and accumulated with microtubules. Deactivation of CDC42 protein activity with the effective inhibitor ML141 caused a decrease in actin distribution in the cortex, which resulted in a failure of polar body extrusion. Moreover, western blot analysis revealed that besides the Cdc42-N-WASP pathway previously reported in mouse oocytes, the expression of ROCK and p-cofilin, two molecules involved in actin dynamics, was also decreased after CDC42 inhibition during porcine oocyte maturation. Thus, our study demonstrates that CDC42 is an indispensable protein during porcine oocyte meiosis, and CDC42 may interact with N-WASP, ROCK, and cofilin in the assembly of actin filaments during porcine oocyte maturation.
Article
Oocyte meiosis is accomplished through two successive rounds of cellular divisions, without DNA replication, allowing for gamete haploidization necessary for parental genome fusion after fertilization. These divisions are highly asymmetric and allow extra-DNA expulsion, in small polar bodies, while retaining most of the cytoplasmic resources needed for early embryo development. Studies in mouse oocyte have demonstrated the capabilities of the gamete to autonomously break his symmetry by positioning the spindle near the cortex. By doing so, the spindle is able to induce a cortical polarization that is dependent on a Ran-GTP gradient emanating from the chromosomes. This polarization will be necessary for delimiting extrusion sites of the future polar bodies. A polarized accumulation of Arp2/3 actin filaments is one of the most evident features of oocyte polarization. We have shown that polarization of Cdc42-GTP, trough N-WASP activation, is an essential intermediate between Ran-GTP and the polarized polymerization of actin filaments. We also investigated ERM (Ezrin Radixin Moesin) proteins localization that are known to promote microvilli assembly. According to our data, microvilli and ERM are excluded from the polarized cortex in a Ran-GTP dependent manner. Finally, we studied cortical acto-myosin dynamics during the second meiotic division which requires spindle rotation. We demonstrated the existence of two cortical myosin 2 sub-populations which depend either on chromosomes (Ran-GTP/Cdc42-GTP) or on the central spindle (Ect2/RhoA).
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Tubulobulbar complexes (TBCs), evaginations of mature spermatids, penetrate into the surrounding Sertoli cell cytoplasm of testis seminiferous epithelium during rat spermatogenesis. These structures prepare mature spermatids for their release into the seminiferous tubular lumen via a process called spermiation. Based on their functions of transient attachment and endocytosis, many actin-regulatory and endocytic proteins are associated with TBCs. Previously, exogenous 17β-estradiol administration to adult male rats showed spermiation failure that was attributed to TBC disruption. To determine the molecular basis of estrogen-induced TBC disruption, we examined the expressions and localizations of actin-regulatory proteins, endocytic proteins, Rho-GTPases, and phosphorylation in TBCs during sperm release. Results demonstrated absence of neural Wiscott Aldrich syndrome protein, cortactin, adaptor-related protein complex 2 sigma-1 subunit, dynamin 2, cell division control protein 42, and phosphocortactin in the concavity of spermatid head where TBCs are present without change in their protein expression levels. Absence of these proteins could have led to collapse of the TBC structure which is involved in its formation and function.
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Rho GTPases are molecular switches that elicit distinct effects on the actomyosin cytoskeleton to accurately promote cytokinesis. Although they represent less than 1% of the human genome, Rho GTPases exert disproportionate control over cell division. Crucial to this master regulatory role is their localized occupation of specific domains of the cell to ensure the assembly of a contractile ring at the proper time and place. RhoA occupies the division plane and is the central positive Rho family regulator of cytokinesis. Rac1 is a negative regulator of cytokinesis and is inactivated within the division plane while active Rac1 occupies the cell poles. Cdc42 regulation during cytokinesis is less studied, but thus far a clear role has only been shown during polar body emission. Here we review what is known about the function of Rho family GTPases during cell division, as well as their upstream regulators and known downstream cytokinetic effectors. © 2012 Wiley Periodicals, Inc.
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In mammals, journey from metaphase-I (M-I) to metaphase-II (M-II) is important since oocyte extrude first polar body (PB-I) and gets converted into haploid gamete. The molecular and cellular changes associated with meiotic cell cycle progression from M-I to M-II stage and extrusion of PB-I remain ill understood. Several factors drive oocyte meiosis from M-I to M-II stage. The mitogen-activated protein kinase3/1 (MAPK3/1), signal molecules and Rho family GTPases act through various pathways to drive cell cycle progression from M-I to M-II stage. The down regulation of MOS/MEK/MAPK3/1 pathway results in the activation of anaphase-promoting complex/cyclosome (APC/C). The active APC/C destabilizes maturation promoting factor (MPF) and induces meiotic resumption. Several signal molecules such as, c-Jun N-terminal kinase (JNK2), SENP3, mitotic kinesin-like protein 2 (MKlp2), regulator of G-protein signalling (RGS2), Epsin2, polo-like kinase 1 (Plk1) are directly or indirectly involved in chromosomal segregation. Rho family GTPase is another enzyme that along with cell division cycle (Cdc42) to form actomyosin contractile ring required for chromosomal segregation. In the presence of origin recognition complex (ORC4), eccentrically localized haploid set of chromosomes trigger cortex differentiation and determine the division site for polar body formation. The actomyosin contractile activity at the site of division plane helps to form cytokinetic furrow that results in the formation and extrusion of PB-I. Indeed, oocyte journey from M-I to M-II stage is coordinated by several factors and pathways that enable oocyte to extrude PB-I. Quality of oocyte directly impact fertilization rate, early embryonic development and reproductive outcome in mammals.
Article
LIM kinases (LIMK1/2) are LIM domain-containing serine/threonine/tyrosine kinases that mediate multiple cellular processes in mitosis. In the present study, we explored the functional roles and potential signaling pathway of LIMK1/2 during mouse oocyte meiosis. Disruption of LIMK1/2 activity and expression significantly decreased oocyte polar body extrusion. Live-cell imaging revealed that spindle migration was disturbed after both LIMK1 and LIMK2 knock down, and this might be due to aberrant distribution of actin filaments in the oocyte cytoplasm and cortex. Meanwhile, our results demonstrated that the function of LIMK1 and LIMK2 in actin assembly was related to cofilin phosphorylation levels. In addition, disruption of LIMK1/2 activity significantly increased the percentage of oocytes with abnormal spindle morphologies, which was confirmed by the abnormal p-MAPK localization. We further explored the upstream molecules of LIMK1/2, and we found that after depletion of ROCK, phosphorylation of LIMK1/2 and cofilin were significantly decreased. Moreover, RhoA inhibition caused the decreased expression of ROCK, p-LIMK1/2 and cofilin. In summary, our results indicated that the small GTPase RhoA regulated LIMK1/2-cofilin to modulate cytoskeletal dynamics during mouse oocyte meiosis. This article is protected by copyright. All rights reserved
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Polar body emission is a special form of cytokinesis in oocyte meiosis that ensures the correct number of chromosomes in reproduction-competent eggs. The molecular mechanism of the last step, polar body abscission, is poorly understood. While it has been proposed that Ca2+ signaling plays important roles in embryonic cytokinesis, to date transient increases in intracellular free Ca2+ have been difficult to document in oocyte meiosis except for the global Ca2+ wave induced by sperm at fertilization. Here, we find that microinjection of the calcium chelator dibromo-BAPTA inhibits polar body abscission in Xenopus laevis oocytes. Using a novel, microtubule-targeted ratio-metric calcium sensor, we detected a calcium transient that is focused at the contractile ring-associated plasma membrane and which occurred after anaphase and constriction of the contractile ring but prior to abscission. This calcium transient was confirmed by mobile calcium probes. Further, the Ca2+-sensitive protein kinase Cβ C2 domain transiently translocated to the contractile ring-associated membrane simultaneously with the calcium transient. Collectively, these results demonstrate that a calcium transient, apparently originating at the contractile ring-associated plasma membrane, promotes polar body abscission.
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Polar body extrusion (PBE) is the specialized asymmetric division by which oocytes accomplish reduction in ploidy and retention of cytoplasm. During maternal gametogenesis, as in male meiosis and mitosis, cytokinesis is accomplished by a ring rich in active Rho, myosin, and formin-nucleated F-actin [1-7]. However, unlike mitosis, wherein the contractile ring encircles the cell equator, the polar body ring assembles as a discoid cortical washer. Here we show that in Caenorhabditis elegans, the meiotic contractile ring transforms during closure from a disc above the spindle to a cylinder around the spindle midzone. The meiotic midbody tube comprises stacked cytoskeletal rings. This topological transition suggests a novel mechanism for constriction of an initially discoid cytokinetic ring. Analysis of mouse PBE indicates that midbody tube formation is a conserved process. Depletion of the scaffold protein anillin (ANI-1) from C. elegans results in large and unstable polar bodies that often fuse with the oocyte. Anillin is dispensable for contractile ring assembly, initiation, and closure but is required for the meiotic contractile ring to transform from a disc into a tube. We propose that cytoskeletal bundling by anillin promotes formation of the midbody tube, which ensures the fidelity of PBE.
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Completion of the first meiosis in oocytes is achieved by the extrusion of the first polar body (PBI), a particular example of cell division. In mitosis, the small GTPase RhoA, which is activated by epithelial cell transforming protein 2 (ECT2), orchestrates contractile ring constriction, thus enabling cytokinesis. However, the involvement of this pathway in mammalian oocytes has not been established. To characterize the role of ECT2 in PBI emission in mouse oocytes, the small interfering RNA approach was employed. We found that ECT2 depletion significantly reduces PBI emission, induces first metaphase arrest, and generates oocytes containing two properly formed spindles of the second metaphase. Moreover, we describe, for the first time, that before PBI emission, RhoA forms a ring that is preceded by a dome-like accumulation at the oocyte cortex, next to the spindle. This unique mode of RhoA translocation failed to occur in the absence of ECT2. We further found that the Rho-dependent kinase, a main RhoA effector, is essential for PBI emission. In addition, we demonstrate herein that ECT2 is subjected to phosphorylation/dephosphorylation throughout meiosis in oocytes and further reveal that PBI emission is temporally associated with ECT2 dephosphorylation. Our data provide the first demonstration that an active cyclin-dependent kinase 1, the catalytic subunit of the maturation-promoting factor, phosphorylates ECT2 during the first meiotic metaphase and that cyclin-dependent kinase 1 inactivation at anaphase allows ECT2 dephosphorylation. In conclusion, our study demonstrates the indispensable role of the maturation-promoting factor/ECT2/RhoA pathway in PBI extrusion in mouse oocytes.
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Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion.
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In animal cells, cytokinesis is powered by a contractile ring of actin filaments (F-actin) and myosin-2. Formation of the contractile ring is dependent on the small GTPase RhoA, which is activated in a precise zone at the cell equator. It has long been assumed that cytokinesis and other Rho-dependent processes are controlled in a sequential manner, whereby Rho activation by guanine nucleotide exchange factors (GEFs) initiates a particular event, and Rho inactivation by GTPase activating proteins (GAPs) terminates that event. MgcRacGAP is a conserved cytokinesis regulator thought to be required only at the end of cytokinesis. Here we show that GAP activity of MgcRacGAP is necessary early during cytokinesis for the formation and maintenance of the Rho activity zone. Disruption of GAP activity by point mutation results in poorly focused Rho activity zones, whereas complete removal of the GAP domain results in unfocused zones that show lateral instability and/or rapid side-to-side oscillations. We propose that the GAP domain of MgcRacGAP has two unexpected roles throughout cytokinesis: first, it transiently anchors active Rho, and second, it promotes local Rho inactivation, resulting in the constant flux of Rho through the GTPase cycle.
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Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.
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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.
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The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2ts) grown at the restrictive temperature. In addition, Cdc42p from a cdc42C188S mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in alpha-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.
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E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. Cdc42, a member of the Rho family of small GTPases, participates in cytoskeletal rearrangement and cell cycle progression. Recent evidence reveals that members of the Rho family modulate E-cadherin function. To further examine the role of Cdc42 in E-cadherin-mediated cell-cell adhesion, we developed an assay for active Cdc42 using the GTPase-binding domain of the Wiskott-Aldrich syndrome protein. Initiation of E-cadherin-mediated cell-cell attachment significantly increased in a time-dependent manner the amount of active Cdc42 in MCF-7 epithelial cell lysates. By contrast, Cdc42 activity was not increased under identical conditions in MCF-7 cells incubated with anti-E-cadherin antibodies nor in MDA-MB-231 (E-cadherin negative) epithelial cells. By fusing the Wiskott-Aldrich syndrome protein/GTPase-binding domain to a green fluorescent protein, activation of endogenous Cdc42 by E-cadherin was demonstrated in live cells. These data indicate that E-cadherin activates Cdc42, demonstrating bi-directional interactions between the Rho- and E-cadherin signaling pathways.
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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.
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Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine nucleotide exchange factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.
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The actin filament (F-actin) cytoskeleton associates dynamically with the plasma membrane and is thus ideally positioned to participate in endocytosis. Indeed, a wealth of genetic and biochemical evidence has confirmed that actin interacts with components of the endocytic machinery, although its precise function in endocytosis remains unclear. Here, we use 4D microscopy to visualize the contribution of actin during compensatory endocytosis in Xenopus laevis eggs. We show that the actin cytoskeleton maintains exocytosing cortical granules as discrete invaginated compartments, such that when actin is disrupted, they collapse into the plasma membrane. Invaginated, exocytosing cortical granule compartments are directly retrieved from the plasma membrane by F-actin coats that assemble on their surface. These dynamic F-actin coats seem to drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments. Active Cdc42 and N-WASP are recruited to exocytosing cortical granule membranes before F-actin coat assembly and coats assemble by Cdc42-dependent, de novo actin polymerization. Thus, F-actin may power fusion pore resealing and function in two novel endocytic capacities: the maintenance of invaginated compartments and the processing of endosomes.
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Ca(2+) is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca(2+) in mitosis. In contrast, there has been a long-standing debate as to whether Ca(2+) signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca(2+) (Ca(2+)(cyt)) plays a dual role during Xenopus oocyte maturation. Ca(2+) signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca(2+)(cyt) signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca(2+)-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca(2+)(cyt) acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca(2+)(cyt) delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca(2+) modulates both the cell cycle machinery and nuclear maturation during meiosis.
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Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility-previously assumed-was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis.
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Cytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.
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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.
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The RhoA GTPase plays a vital role in assembly of contractile actin-myosin filaments (stress fibers) and of associated focal adhesion complexes of adherent monolayer cells in culture. GEF-H1 is a microtubule-associated guanine nucleotide exchange factor that activates RhoA upon release from microtubules. The overexpression of GEF-H1 deficient in microtubule binding or treatment of HeLa cells with nocodazole to induce microtubule depolymerization results in Rho-dependent actin stress fiber formation and contractile cell morphology. However, whether GEF-H1 is required and sufficient to mediate nocodazole-induced contractility remains unclear. We establish here that siRNA-mediated depletion of GEF-H1 in HeLa cells prevents nocodazole-induced cell contraction. Furthermore, the nocodazole-induced activation of RhoA and Rho-associated kinase (ROCK) that mediates phosphorylation of myosin regulatory light chain (MLC) is impaired in GEF-H1-depleted cells. Conversely, RhoA activation and contractility are rescued by reintroduction of siRNA-resistant GEF-H1. Our studies reveal a critical role for a GEF-H1/RhoA/ROCK/MLC signaling pathway in mediating nocodazole-induced cell contractility.
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The relationship between the microtubules and a massive system of vesicles associated with the sea urchin mitotic apparatus was examined by light and electron microscopy. Astral rays made up of elongated vesicles and associated tracts of microtubules continue to grow toward the cell surface during late anaphase and telophase at the same time the aster center appears to be breaking down in preparation for the next division. On the basis of current knowledge of the requirements for microtubule polymerization and the known presence of a calcium-dependent ATPase in the mitotic apparatus, it is proposed that the vesicle system functions to control the polymerization and depolymerization of microtubules by calcium ion regulation. A model for such a system is proposed.
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The Rho GTPases-Rho, Rac, and Cdc42-regulate the dynamics of F-actin (filamentous actin) and myosin-2 with considerable subcellular precision. Consistent with this ability, active Rho and Cdc42 occupy mutually exclusive zones during single-cell wound repair and asymmetric cytokinesis, suggesting the existence of mechanisms for local crosstalk, but how local Rho GTPase crosstalk is controlled is unknown. Using a candidate screen approach for Rho GTPase activators (guanine nucleotide exchange factors; GEFs) and Rho GTPase inactivators (GTPase-activating proteins; GAPs), we find that Abr, a protein with both GEF and GAP activity, regulates Rho and Cdc42 during single-cell wound repair. Abr is targeted to the Rho activity zone via active Rho. Within the Rho zone, Abr promotes local Rho activation via its GEF domain and controls local crosstalk via its GAP domain, which limits Cdc42 activity within the Rho zone. Depletion of Abr attenuates Rho activity and wound repair. Abr is the first identified Rho GTPase regulator of single-cell wound healing. Its novel mode of targeting by interaction with active Rho allows Abr to rapidly amplify local increases in Rho activity using its GEF domain while its ability to inactivate Cdc42 using its GAP domain results in sharp segregation of the Rho and Cdc42 zones. Similar mechanisms of local Rho GTPase activation and segregation enforcement may be employed in other processes that exhibit local Rho GTPase crosstalk.
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An oocyte matures into an egg by extruding half of the chromosomes in a small polar body. This extremely asymmetric division enables the oocyte to retain sufficient storage material for the development of the embryo after fertilization. To divide asymmetrically, mammalian oocytes relocate the spindle from their center to the cortex. In all mammalian species analyzed so far, including human, mouse, cow, pig, and hamster, spindle relocation depends on filamentous actin (F-actin). However, even though spindle relocation is essential for fertility, the involved F-actin structures and the mechanism by which they relocate the spindle are unknown. Here we show in live mouse oocytes that spindle relocation requires a continuously reorganizing cytoplasmic actin network nucleated by Formin-2 (Fmn2). We found that the spindle poles were enriched in activated myosin and pulled on this network. Inhibition of myosin activation by myosin light chain kinase (MLCK) stopped pulling and spindle relocation, indicating that myosin pulling creates the force that drives spindle movement. Based on these results, we propose the first mechanistic model for asymmetric spindle positioning in mammalian oocytes and validate five of its key predictions experimentally.
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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.
Article
Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.
Article
The actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing. Recent evidence suggests that Ca2+ triggers the contraction and that cell-cycle kinases regulate the timing of cytokinesis. New work is required to identify the signals from the mitotic spindle that specify the position of the furrow.
Article
We used confocal immunofluorescence microscopy to examine spindle migration, morphology and orientation during the maturation of Xenopus oocytes, in the presence or absence of cytochalasin B (CB), an inhibitor of actin assembly. Treatment with CB during maturation (10-50 micrograms/ml beginning 0-3 h prior to addition of progesterone) disrupted the normal organisation of the novel MTOC and transient microtubule array (MTOC-TMA complex) that serves as the immediate precursor of the first meiotic spindle, suggesting that F-actin plays an important role in the assembly or maintenance of this complex. However, CB treatment did not block translocation of the MTOC-TMA complex to the oocyte cortex, suggesting that MTOC-TMA translocation is not dependent on an actin-based mechanism. Bipolar spindles were observed in CB-treated oocytes fixed during both M1 and M2. However, rotation of the M1 and M2 spindles into an orientation orthogonal to the oocyte surface was inhibited by CB. Rhodamine-phalloidin revealed a concentration of F-actin at the site of M1 spindle attachment, further suggesting that cortical actin is required for anchoring and rotation of the meiotic spindles. Finally, the incidence of M1 monasters was significantly increased in CB-treated oocytes, suggesting that interactions between the nascent M1 spindle and cortex are dependent on F-actin.
Article
The endoplasmic reticulum (ER) of live metaphase II mouse eggs and prophase I-arrested oocytes was compared using the fluorescent, lipophilic dicarbocyanine dye, DiI. DiL, dissolved in soybean oil, was microinjected into oocytes and eggs; the dye diffused throughout the cytoplasm to label the ER, which was imaged by confocal microscopy. The mature egg had a fine reticular network of ER throughout the cell and numerous dense accumulations of membrane in the cortex. These ER accumulations, 1-2 microns in diameter, were generally absent deeper in the cytoplasm. A similar staining pattern was observed when the eggs were fixed within 1 min of injection, providing evidence that the cortical accumulations of membrane are part of a continuous ER membrane system, since membrane trafficking could not occur in a fixed egg. Cortical ER accumulations were localized to the same region of the egg as the cortical granules and were not observed in the cortical granule-free region adjacent to the meiotic spindle. In contrast, ER accumulations were rarely found in the cortex of the immature, prophase I-arrested oocyte, but larger and less well-defined membrane clusters were found throughout the deeper cytoplasm of the oocyte. The appearance of ER clusters in the egg cortex following oocyte maturation correlates with an increased ability of the mature egg to release calcium at fertilization. Since the ER is a calcium store, structural reorganization of the ER may be necessary to permit the large release of calcium and resulting cortical granule exocytosis at fertilization.
Article
In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and beta-COP, expression of a GTPase-deficient form of ARF (ARF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (; ). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.
Article
During cytokinesis in animal cells, an equatorial actomyosin-based contractile ring divides the cell into two daughter cells. The position of the contractile ring is specified by a signal that emanates from the mitotic spindle. This signal has not been identified and it is not understood how the components of the contractile ring assemble. It is also unclear how the ring constricts or how new plasma membrane inserts specifically behind the leading edge of the constricting furrow. The Rho family of small GTPases regulate polarized changes in cell growth and cell shape by affecting the formation of actin structures beneath the plasma membrane, but their role in cytokinesis is unclear. We have studied the function of two Rho family members during the early cell divisions of Xenopus embryos by injecting modified forms of Rho and Cdc42. Both inhibition and constitutive activation of either GTPase blocked cytokinesis. Furrow specification occurred normally, but ingression of the furrow was inhibited. Newly inserted cleavage membranes appeared aberrantly on the outer surface of the embryo. Microinjected Rho localized to the cortex and regulated the levels of cortical F-actin. These results show that Rho regulates the assembly of actin filaments in the cortex during cytokinesis, that local activation of Rho is important for proper constriction of the contractile furrow, and that Cdc42 plays a role in furrow ingression. Moreover, our observations reveal that furrow ingression and membrane insertion are not strictly linked. Neither Rho nor Cdc42 appear to be required for establishment of the cell-division plane.
Article
Soluble factors from serum such as lysophosphatidic acid (LPA) are thought to activate the small GTP-binding protein Rho based on their ability to induce actin stress fibers and focal adhesions in a Rho-dependent manner. Cell adhesion to extracellular matrices (ECM) has also been proposed to activate Rho, but this point has been controversial due to the difficulty of distinguishing changes in Rho activity from the structural contributions of ECM to the formation of focal adhesions. To address these questions, we established an assay for GTP-bound cellular Rho. Plating Swiss 3T3 cells on fibronectin-coated dishes elicited a transient inhibition of Rho, followed by a phase of Rho activation. The activation phase was greatly enhanced by serum. In serum-starved adherent cells, LPA induced transient Rho activation, whereas in suspended cells Rho activation was sustained. Furthermore, suspended cells showed higher Rho activity than adherent cells in the presence of serum. These data indicate the existence of an adhesion-dependent negative-feedback loop. We also observed that both cytochalasin D and colchicine trigger Rho activation despite their opposite effects on stress fibers and focal adhesions. Our results show that ECM, cytoskeletal structures and soluble factors all contribute to regulation of Rho activity.
Article
The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releasability of Ca(2+) by IP(3). The clusters dispersed during the Ca(2+) wave at activation. Possible relationships of ER structure and Ca(2+) regulation are discussed.
Article
Proper development of neurons depends on synaptic activity, but the mechanisms of activity-dependent neuronal growth are not well understood. The small GTPases, RhoA, Rac, and Cdc42, regulate neuronal morphogenesis by controlling the assembly and stability of the actin cytoskeleton. We report an in situ method to determine endogenous Rho GTPase activity in intact Xenopus brain. We use this method to provide evidence for crosstalk between Rho GTPases in optic tectal cells. Moreover, crosstalk between the Rho GTPases appears to affect dendritic arbor development in vivo. Finally, we demonstrate that optic nerve stimulation regulates Rho GTPase activity in a glutamate receptor-dependent manner. These data suggest a link between glutamate receptor function, Rho GTPase activity, and dendritic arbor growth in the intact animal.
Article
During mitosis, the mitotic spindle, a bipolar structure composed of microtubules (MTs) and associated motor proteins, segregates sister chromatids to daughter cells. Initially some MTs emanating from one centrosome attach to the kinetochore at the centromere of one of the duplicated chromosomes. This attachment allows rapid poleward movement of the bound chromosome. Subsequent attachment of the sister kinetochore to MTs growing from the other centrosome results in the bi-orientation of the chromosome, in which interactions between kinetochores and the plus ends of MTs are formed and stabilized. These processes ensure alignment of chromosomes during metaphase and their correct segregation during anaphase. Although many proteins constituting the kinetochore have been identified and extensively studied, the signalling responsible for MT capture and stabilization is unclear. Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization. We now show that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.
Article
Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis.
Article
During Xenopus development, convergent extension movements mediated by cell intercalation drive axial elongation. While many genes required for convergent extension have been identified, little is known of regulation of the cytoskeleton during these cell movements. Although microtubules are required for convergent extension, this applies only to initial stages of gastrulation, between stages 10 and 10.5. To examine the cytoskeleton more directly during convergent extension, we visualized actin and microtubules simultaneously in live explants using spinning disk confocal fluorescence microscopy. Microtubule depolymerization by nocodazole inhibits lamellipodial protrusions and cell-cell contact, thereby inhibiting convergent extension. However, neither taxol nor vinblastine, both of which block microtubule dynamics while stabilizing a polymer form of tubulin, inhibits lamellipodia or convergent extension. This suggests an unusual explanation: the mass of polymerized tubulin, not dynamics of the microtubule cytoskeleton, is crucial for convergent extension. Because microtubule depolymerization elicits striking effects on actin-based protrusions, the role of Rho-family GTPases was tested. The effects of nocodazole are partially rescued using dominant negative Rho, Rho-kinase inhibitor, or constitutively active Rac, suggesting that microtubules regulate small GTPases, possibly via a guanine-nucleotide exchange factor. We cloned full-length XLfc, a microtubule-binding Rho-GEF. Nucleotide exchange activity of XLfc is required for nocodazole-mediated inhibition of convergent extension; constitutively active XLfc recapitulates the effects of microtubule depolymerization. Morpholino knockdown of XLfc abrogates the ability of nocodazole to inhibit convergent extension. Therefore, we believe that XLfc is a crucial regulator of cell morphology during convergent extension, and microtubules limit its activity through binding to the lattice.
Article
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.
Article
Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.
Article
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.
Article
The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Previous studies demonstrated that the asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. Here we show that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. We hypothesize that a Ran(GTP) gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin.
Article
Oocyte maturation in mouse is associated with a dramatic reorganisation of the endoplasmic reticulum (ER) from a network of cytoplasmic accumulations in the germinal vesicle-stage oocyte (GV) to a network of distinctive cortical clusters in the metaphase II egg (MII). Multiple lines of evidence suggest that this redistribution of the ER is important to prepare the oocyte for the generation of repetitive Ca2+ transients which trigger egg activation at fertilisation. The aim of the current study was therefore to investigate the timecourse and mechanism of ER reorganisation during oocyte maturation. The ER is first restructured at the time of GV-breakdown (GVBD) into a dense network of membranes which envelop and invade the developing meiotic spindle. GVBD is essential for the initiation of ER reorganisation, since ER structure does not change in GV-arrested oocytes. ER reorganisation is also prevented by the microtubule inhibitor nocodazole and by the inhibition of cytoplasmic dynein, a microtubule-associated motor protein. ER redistribution at GVBD is therefore dynein-driven and cell cycle-dependent. Following GVBD the dense network of ER surrounds the spindle during its migration to the oocyte cortex. Cortical clusters of ER are formed close to the time of, but independently of the metaphase I-metaphase II transition. Formation of the characteristic ER clusters is prevented by the depolymerisation of microfilaments, but not of microtubules. These experiments reveal that ER reorganisation during oocyte maturation is a complex multi-step process involving distinct microtubule- and microfilament-dependent phases and indicate a role for dynein in the cytoplasmic changes which prepare the oocyte for fertilisation.
Article
Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Consequently, filopodia have an important role in cell migration, neurite outgrowth and wound healing and serve as precursors for dendritic spines in neurons. The initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and crosslinking of actin filaments. The increased understanding of the functions of various actin-associated proteins during the initiation and elongation of filopodia has provided new information on the mechanisms of filopodia formation in distinct cell types.
  • Ll Satterwhite
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Filopodia: molecular architecture and cellular functions
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Mattila PK, Lappalainen P. Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol 2008;9:446-454.