Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, gamma-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. gamma-tubulin translocated into the two poles of the transient spindles, while no accumulated gamma-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, gamma-tubulin was translocated to the spindle poles. The distribution of gamma-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. gamma-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as pericentriolar material surrounding a pair of centrioles, is degraded in the 1-cell reconstituted embryos after activation; (2) components of donor cell centrosomes contribute to the formation of the transient spindle and normal functional mitotic spindle, although the contribution of centrosomal material stored in the recipient ooplasm is not excluded; and (3) components of donor cell centrosomes involved in spindle assembly may not be species-specific.     

Development of interspecies cloned embryos in yak and dog

Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.     

PACSIN proteins bind tubulin and promote microtubule assembly

PACSINs are intracellular adapter proteins involved in vesicle transport, membrane dynamics and actin reorganisation. In this study, we report a novel role for PACSIN proteins as components of the centrosome involved in microtubule dynamics. Glutathione S-transferase (GST)-tagged PACSIN proteins interacted with protein complexes containing α- and γ-tubulin in brain homogenate. Analysis of cell lysates showed that all three endogenous PACSINs co-immunoprecipitated dynamin, α-tubulin and γ-tubulin. Furthermore, PACSINs bound only to unpolymerised tubulin, not to microtubules purified from brain. In agreement, the cellular localisation of endogenous PACSIN 2 was not affected by the microtubule depolymerising reagent nocodazole. By light microscopy, endogenous PACSIN 2 localised next to γ-tubulin at purified centrosomes from NIH 3T3 cells. Finally, reduction of PACSIN 2 protein levels with small-interfering RNA (siRNA) resulted in impaired microtubule nucleation from centrosomes, whereas microtubule centrosome splitting was not affected, suggesting a role for PACSIN 2 in the regulation of tubulin polymerisation. These findings suggest a novel function for PACSIN proteins in dynamic microtubuli nucleation.     

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes

Loss of the tumor suppressor PTEN is observed in many human cancers that display increased chromosome instability and aneuploidy. The subcellular fractions of PTEN are associated with different functions that regulate cell growth, invasion and chromosome stability. In this study, we show a novel role for PTEN in regulating mitotic centrosomes. PTEN localization at mitotic centrosomes peaks between prophase and metaphase, paralleling the centrosomal localization of PLK-1 and γ-tubulin and coinciding with the time frame of centrosome maturation. In primary keratinocytes, knockdown of PTEN increased whole-cell levels of γ-tubulin and PLK-1 in an Akt-dependent manner and had little effect on recruitment of either protein to mitotic centrosomes. Conversely, knockdown of PTEN reduced centrosomal levels of pericentrin in an Akt-independent manner. Inhibition of Akt activation with MK2206 reduced the whole-cell and centrosome levels of PLK-1 and γ-tubulin and also prevented the recruitment of PTEN to mitotic centrosomes. This reduction in centrosome-associated proteins upon inhibition of Akt activity may contribute to the increase in defects in centrosome number and separation observed in metaphase cells. Concomitant PTEN knockdown and Akt inhibition reduced the frequency of metaphase cells with centrosome defects when compared with MK2206 treatment alone, indicating that both PTEN and pAkt are required to properly regulate centrosome composition during mitosis. The findings presented in this study demonstrate a novel role for PTEN and Akt in controlling centrosome composition and integrity during mitosis and provide insight into how PTEN functions as a multifaceted tumor suppressor.     

未经休眠处理的体细胞用于异种核移植

自“多莉”诞生以来,在全世界掀起了一场体细胞克隆的浪潮,许多体细胞克隆动物,如小鼠、山羊、牛、猪等纷纷问世。围绕体细胞克隆的供体细胞周期问题,学术界存在两种不同的观点,一是Wilmut等认为体细胞必须经过休眠处理,使细胞停滞在G0/G1期,或者采用以G0/G1期为主的活体细胞作为供体,这是克隆成功的关键,这一方面的报道已有很多。第二是Cibelli等认为不必对细胞作     

未经休眠处理的体细胞用于异种核移植（简报）

It is the point at issue in intraspecies nuclear transfer whether quiescence is necessary for development of nuclear transfer reconstructed embryos. In the interspecies nuclear transfer, some reports have proved that quiescent cell is able to support preimplantation development of the interspecies reconstructed embryos. Are non-quiescent cells able to support preimplantation development of the interspecies reconstructed embryos? We used non-quiescent somatic cells from C57BL/6 mice and giant pandas as donors to transfer into enucleated rabbit oocytes. After electrofusion (the electrofusion rates were 62.2% and 71.6%, respectively) and electrical activation, 5.1% of those mouse-rabbit reconstructed embryos developed to blastocyst in vitro, and 4.2% of panda-rabbit reconstructed embryos developed to blastocyst after transferring into ligated rabbit oviduct. These results indicate that non-quiescent cell from C57BL/6 mouse and giant panda could be dedifferentiated in enucleated rabbit oocytes and support early embryo development.     

γ-微管蛋白在猪卵母细胞成熟和活化中的分布

微管蛋白(tubulin)是一蛋白质超家族,其中α-,β-微管蛋白是主要的微管蛋白,而γ-微管蛋白主要在微管组装中起作用. 我们利用蛋白质印迹和激光共聚焦技术研究了γ-微管蛋白在猪卵母细胞成熟、受精和活化中的分布. γ-微管蛋白存在于猪卵母细胞中,并且在减数分裂成熟各个时期的量保持不变. 它聚集在微管上,特别是中期纺锤体的两极和后末期的中板. 体外受精和孤雌活化后,γ-微管蛋白聚集在雌雄原核的周围.另外它也存在于精子的顶体帽和颈部.在早期卵裂中,γ-微管蛋白聚集在胚胎的细胞核周围.实验结果表明,γ-微管蛋白在猪卵母细胞、精子和胚胎的微管组装中起重要的调节作用,在猪受精过程中,精子和卵子都向受精卵贡献中心体物质.     

Chromatin and microtubule morphology during the first cell cycle in bovine zygotes

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage. © 1993 Wiley-Liss, Inc.     

The role of γ-tubulin in centrosomal microtubule organization

As part of a multi-subunit ring complex, γ-tubulin has been shown to promote microtubule nucleation both in vitro and in vivo, and the structural properties of the complex suggest that it also seals the minus ends of the polymers with a conical cap. Cells depleted of γ-tubulin, however, still display many microtubules that participate in mitotic spindle assembly, suggesting that γ-tubulin is not absolutely required for microtubule nucleation in vivo, and raising questions about the function of the minus end cap. Here, we assessed the role of γ-tubulin in centrosomal microtubule organisation using three-dimensional reconstructions of γ-tubulin-depleted C. elegans embryos. We found that microtubule minus-end capping and the PCM component SPD-5 are both essential for the proper placement of microtubules in the centrosome. Our results further suggest that γ-tubulin and SPD-5 limit microtubule polymerization within the centrosome core, and we propose a model for how abnormal microtubule organization at the centrosome could indirectly affect centriole structure and daughter centriole replication.     

Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome

Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.     

HOPS is an essential constituent of centrosome assembly

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.     

Distinct cell cycle-dependent roles for dynactin and dynein at centrosomes

Centrosomal dynactin is required for normal microtubule anchoring and/or focusing independently of dynein. Dynactin is present at centrosomes throughout interphase, but dynein accumulates only during S and G2 phases. Blocking dynein-based motility prevents recruitment of dynactin and dynein to centrosomes and destabilizes both centrosomes and the microtubule array, interfering with cell cycle progression during mitosis. Destabilization of the centrosomal pool of dynactin does not inhibit dynein-based motility or dynein recruitment to centrosomes, but instead causes abnormal G1 centriole separation and delayed entry into S phase. The correct balance of centrosome-associated dynactin subunits is apparently important for satisfaction of the cell cycle mechanism that monitors centrosome integrity before centrosome duplication and ultimately governs the G1 to S transition. Our results suggest that, in addition to functioning as a microtubule anchor, dynactin contributes to the recruitment of important cell cycle regulators to centrosomes.     

Analysis of centrosome localization of BRCA1 and its activity in suppressing centrosomal aster formation

BRCA1, a product of a familial breast and ovarian cancer susceptibility gene, localizes to centrosomes and physically interacts with γ-tubulin, a key centrosomal protein for microtubule nucleation and anchoring at centrosomes. Here, we performed a rigorous analysis of centrosome localization of BRCA1, and found that BRCA1 is specifically associated with mother centrioles in unduplicated centrosomes, and daughter centrioles acquire BRCA1 prior to initiation of duplication, and thus duplicated centrosomes are both bound by BRCA1. We further found that BRCA1 suppresses centrosomal aster formation. In addition, we identified a new domain of BRCA1 critical for γ-tubulin binding, which confers not only its localization to centrosomes, but also its activity to suppress centrosomal aster formation.     

Excess centrosomes disrupt endothelial cell migration via centrosome scattering

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome–MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.     

Asymmetric localization of the CDC25B phosphatase to the mother centrosome during interphase

The Cyclin-Dependent Kinase (CDK)-activating phosphatase CDC25B, localises to the centrosomes where its activity is both positively and negatively regulated by several kinases including Aurora A and CHK1. Our recent data also demonstrate a role for CDC25B in the centrosome duplication cycle and microtubule nucleation in interphase that appears to involve the recruitment of γ-tubulin to the centrosomes. In the present study, we report that CDC25B, along with CHK1, CDK1 and WEE1, localise asymmetrically around the mother centrosome from S to G2-phases, and gradually become evenly distributed to the two centrosomes by late G2 phase, concomitant with centrosome maturation. We further demonstrate that siRNA inhibition of CDC25B results in an accumulation of cells in G2 phase with two separated centrosomes, each containing only a single centriole, suggesting a requirement for CDC25B in centriole duplication. We propose that the localisation of key cell cycle regulators to the mother centrosome ensures synchrony between the centrosome duplication and cell division cycles.     

Development of embryos reconstructed by interspecies nuclear transfer of adult fibroblasts between buffalo (Bubalus bubalis) and cattle (Bos indicus)

The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 microg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media+0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 microM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and cattle developed to blastocysts, but bovine cytoplasts may direct embryonic development more effectively than buffalo cytoplasts, regardless of donor cell species.     

Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1-Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation.     

Nek9 Phosphorylation of NEDD1/GCP-WD Contributes to Plk1 Control of γ-Tubulin Recruitment to the Mitotic Centrosome

The accumulation of γ-tubulin at the centrosomes during maturation is a key mechanism that ensures the formation of two dense microtubule (MT) asters in cells entering mitosis, defining spindle pole positioning and ensuring the faithful outcome of cell division ([1] and references herein; [2]). Centrosomal γ-tubulin recruitment depends on the adaptor protein NEDD1/GCP-WD [3, 4] and is controlled by the kinase Plk1 [5-8]. Surprisingly, and although Plk1 binds and phosphorylates NEDD1 at multiple sites [9, 10], the mechanism by which this kinase promotes the centrosomal recruitment of γ-tubulin has remained elusive. Using Xenopus egg extracts and mammalian cells, we now show that it involves Nek9, a NIMA-family kinase required for normal mitotic progression and spindle organization [11,?12]. Nek9 phosphorylates NEDD1 on Ser377 driving its recruitment and thereby that of γ-tubulin to the centrosome in mitotic cells. This role of Nek9 requires its activation by Plk1-dependent phosphorylation [13] but is independent from the downstream related kinases Nek6 and Nek7 [14]. Our data contribute to understand the mechanism by which Plk1 promotes the recruitment of γ-tubulin to the centrosome in dividing cells and position Nek9 as a key regulator of centrosome maturation.     

The mammalian centrosome and its functional significance

Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome's functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome's role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome's significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease.     

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.