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PubMed2004

Phallacidin stains the mitotic spindle of the diatom Pinnularia spp.

Mitotic spindles of the diatom Pinnularia viritiformis stained with the fluorescent actin-labelling reagent bodipyphallacidin revealed actin among the chromosomes and extending along the spindle to the poles. This is the first report of actin's presence within spindles of this ecologically important group of organisms. Since diatom mitoses have a number of marked differences compared to that of many other eukaryotes (Int Rev Cytol 128 (1991) 63; Cell 14 (1978) 455), the present observations substantially extend the diversity of mitotic spindle types in which there is evidence of spindle actin.

Actins↗

PubMed2004

Control of embryonic spindle positioning and Galpha activity by C. elegans RIC-8.

Asymmetric spindle positioning is of fundamental importance for generating cell diversity during development. In the C. elegans 1 cell embryo, spindle positioning has been shown to depend on heterotrimeric G protein signaling. Two Galpha subunits, GOA-1 and GPA-16 (hereafter Galpha), and receptor independent activators of G protein signaling GPR-1 and GPR-2 (GPR-1/2) are required for proper regulation of spindle positioning . However, it remains unclear whether Galpha regulates spindle positioning in its GDP or GTP bound form. Here, we investigate the role of RIC-8 in this pathway. RIC-8 was genetically shown to act in concert with goa-1 to regulate centrosome movements in C. elegans . Interestingly, mammalian RIC-8 was recently found to behave as a GEF for Galpha subunits in vitro . We show that reduction of function of ric-8 results in a 1 cell embryo phenotype very similar to the phenotype of embryos depleted of Galpha. RIC-8 is able to directly bind to GOA-1, preferentially to GOA-1-GDP, consistent with a GEF role. RIC-8 is localized at the embryo cortex, and its activity is essential for the asymmetric localization of GPR-1/2. We suggest that RIC-8 directly modulates Galpha activity and that Galpha-GTP is the signaling molecule regulating spindle positioning in the early embryo.

Animals↗

PubMed2004

XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly.

BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.

Animals↗

PubMed2004

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes.

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.

Anaphase↗

PubMed2005

Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly.

The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts . Recently, we have shown that AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis . However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear . Here, we report the development of functional assays that allow us to show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads , AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs.

Animals↗

PubMed2004

Multiple Wnt signaling pathways converge to orient the mitotic spindle in early C. elegans embryos.

How cells integrate the input of multiple polarizing signals during division is poorly understood. We demonstrate that two distinct Caenorhabditis elegans Wnt pathways contribute to the polarization of the ABar blastomere by differentially regulating its duplicated centrosomes. Contact with the C blastomere orients the ABar spindle through a nontranscriptional Wnt spindle alignment pathway, while a Wnt/beta-catenin pathway controls the timing of ABar spindle rotation. The three C. elegans Dishevelled homologs contribute to these processes in different ways, suggesting that functional distinctions may exist among them. We also find that CKI (KIN-19) plays a role not only in the Wnt/beta-catenin pathway, but also in the Wnt spindle orientation pathway as well. Based on these findings, we establish a model for the coordination of cell-cell interactions and distinct Wnt signaling pathways that ensures the robust timing and orientation of spindle rotation during a developmentally regulated cell division event.

Alleles↗

PubMed2005

Spindle positioning by cortical pulling forces.

Proper spatial control of the cell division plane is essential to any developing organism. In most cell types, the relative size of the two daughter cells is determined by the position of the mitotic spindle within the geometry of the mother cell. We review the underlying mechanisms responsible for positioning of the mitotic spindle, both in cases where the spindle is placed in the center of the cell and in cases where the spindle is placed away from the center of the cell. We discuss the idea that cortical pulling forces are sufficient to provide a general mechanism for spindle positioning within symmetrically and asymmetrically dividing cells.

Cell Division↗

PubMed2005

An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles.

In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.

Amino Acid Sequence↗

PubMed2006

The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division.

During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the G alpha binding partner Pins, but how the Pins-G alphai complex interacts with the mitotic spindle is unclear. Here, we show that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of NuMA. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. In the absence of Mud, mitotic spindles in Drosophila neuroblasts fail to align with the polarity axis. This can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the mis-specification of daughter cell fates and tumor-like over proliferation in the Drosophila nervous system. Our data suggest a model in which asymmetrically localized Pins-G alphai complexes regulate spindle orientation by directly binding to Mud.

Amino Acid Motifs↗

PubMed2007

Reconstruction of ooplasm recipient oocytes with frozen-thawed donor microcytoplasts and influence on the microtubular spindle.

OBJECTIVE(S): To cryopreserve micromanipulated ooplast segments (microcytoplasts) from mouse oocytes, compare microcytoplast and parent or recipient oocyte fusion performed within or without the zona pellucida, compare electrofusion of fresh or frozen oocyte with frozen-thawed microcytoplasts, and assess spindle integrity after reconstruction of oocytes. DESIGN: Prospective experimental study. SETTING: University-based experimental laboratory. ANIMAL(S): Mouse (MII) oocytes obtained after superovulation (n = 363). INTERVENTION(S): Micromanipulation of oocytes (n = 363) into microcytoplasts (n = 181), cryopreservation of microcytoplasts along with parent and sibling control oocytes (n = 182), reconstruction by electrofusion of microcytoplast and parent or recipient oocyte performed with (group A, n = 35) or without a zona pellucida (group B, n = 32), comparison of electrofusion of fresh oocyte (group C, n = 40) or frozen oocyte (group D, n = 36) with frozen-thawed microcytoplasts fused within zona, and assessment of spindle morphology of reconstructed oocyte. MAIN OUTCOME MEASURE(S): Post-thaw survival, success of fusion, and spindle integrity as assessed by immunostaining. RESULT(S): Higher success of post-thaw fusion was seen in group A (91.4%) compared with group B (56.2%). The post-thaw fusion of microcytoplasts with either fresh or frozen oocytes was not significantly different. Spindle integrity was 82.5% in group C as compared with 47.2% in group D. CONCLUSION(S): Microcytoplasts created from oocytes can be successfully cryopreserved, thawed, and used to reconstruct oocytes with intact spindles.

Animals↗

PubMed2004

Trichlorfon predisposes to aneuploidy and interferes with spindle formation in in vitro maturing mouse oocytes.

The pesticide trichlorfon (TCF) has been implicated in human trisomy 21, and in errors in chromosome segregation at male meiosis II in the mouse. We previously provided evidence that TCF interferes with spindle integrity and cell-cycle control during murine oogenesis. To assess the aneugenic activity of TCF in oogenesis, we presently analysed maturation, spindle assembly, and chromosome constitution in mouse oocytes maturing in vitro in the presence of 50 or 100 microg/ml TCF for 16 h or in pulse-chase experiments. TCF stimulated maturation to meiosis II at 50 microg/ml, but arrested meiosis in some oocytes at 100 microg/ml. TCF at 100 microg/ml was aneugenic causing non-disjunction of homologous chromosomes at meiosis I, a significant increase of the hyperploidy rate at metaphase II, and a significant rise in the numbers of oocytes that contained a 'diploid' set of metaphase II chromosomes (dyads). TCF elevated the rate of precocious chromatid segregation (predivision) at 50 and 100 microg/ml. Pulse-chase experiments with 100 microg/ml TCF present during the first 7 h or the last 9 h of maturation in vitro did not affect meiotic progression and induced intermediate levels of hyperploidy at metaphase II. Exposure to > or =50 microg/ml TCF throughout maturation in vitro induced severe spindle aberrations at metaphase II, and over one-third of the oocytes failed to align all chromosomes at the spindle equator (congression failure). These observations suggest that exposure to high concentrations of TCF induces non-disjunction at meiosis I of oogenesis, while lower doses may preferentially cause errors in chromosome segregation at meiosis II due to disturbances in spindle function, and chromosome congression as well as precocious separation of chromatids prior to anaphase II. The data support evidence from other studies that TCF has to be regarded as a germ cell aneugen.

Aneuploidy↗

PubMedDate not supplied

Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes.

Trisomies due to nondisjunction in oogenesis are still a major cause of genetic diseases in humans. In this study, we analysed spindle morphology of in vitro matured nocodazole-exposed mouse oocytes by novel non-invasive Polscope-microscopy, and compared images to those obtained by anti-tubulin immunofluorescence of fixed oocytes. Polscope revealed a reduction in the numbers of oocytes expressing a birefringent spindle, and alterations in spindle morphology at concentrations of nocodazole below those inducing detectable aberrations in immunofluorescence. Hyperploidy increased significantly at a concentration of 40 nM nocodazole in mouse metaphase II oocytes, similar to thresholds inducing nondisjunction in cultured human lymphocytes. In conclusion, Polscope represents a novel highly sensitive, non-invasive method to identify chemicals inducing severe spindle aberrations that predispose mammalian oocytes to nondisjunction. Polscope may provide information on the functionality of the spindle in experimental studies but is also compatible with clinical trials in human assisted reproduction due to its non-invasive nature.

Aneuploidy↗

PubMed2004

E pluribus unum: towards a universal mechanism for spindle assembly.

Recent data have revealed that the mitotic spindle might form by centrosome-independent mechanisms, even in centrosome-containing cells. This suggests that spindle assembly might proceed by a generally conserved acentrosomal mechanism in all higher eukaryotes, regardless of the presence of centrosomes. Thus, what is the role of centrosomes in mitosis? We propose that these organelles are needed to generate radial arrays of microtubules that integrate preassembled (by centrosome-independent mechanisms) spindle components into a common spindle and orientate the spindle within malleable animal cells.

Animals↗

PubMed2006

Immunohistochemical localization of alpha(1a)-adrenoreceptors in muscle spindles of rabbit masseter muscle.

The expression of alpha(1a)-adrenoreceptors (alpha(1a)-ARs) within the muscle spindles of rabbit masseter muscle was investigated. The alpha(1a)-ARs were detected by immunohistochemical fluorescent method and examined along the entire length of 109 cross serially sectioned spindles. The sympathetic fibers were visualized by the immunofluorescent labeling of the noradrenaline synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). In order to recognize the intrafusal muscle fiber types, antibodies for different myosin heavy chain isoforms (MyHCI) were used. TH and DBH immunolabeled nerve fibers have been observed within the capsule lamellar layers, in the periaxial fluid space and close to intrafusal muscle fibers. The alpha(1a)-ARs were detected on the smooth muscle cells of the blood vessels coursing in the muscle and in the capsule lamellar layers or within the periaxial fluid space of the spindles. Moreover, at the polar regions of a high percentage (88.1%) of muscle spindles a strong alpha(1a)-ARs immunoreactivity was present on the intrafusal muscle fibers. In double immunostained sections for alpha(1a)-ARs and MyHCI it was evidenced that both bag, and nuclear chain fibers express alpha(1a)-ARs. The receptors that we have detected by immunofluorescence may support a direct control by adrenergic fibers on muscle spindle.

Animals↗

PubMed2004

PKC isotypes in post-activated and fertilized mouse eggs: association with the meiotic spindle.

Several isotypes of protein kinase C (PKC) have been reported to be expressed in mammalian eggs, but it is unknown whether these isotypes have a common function in the egg during or within the first few hours of fertilization. Here we show that the isotypes of PKC exhibit distinct patterns of enrichment immediately after mouse egg activation. PKCalpha and gamma accumulate in the egg cortex 25 min post-activation, while only PKCalpha accumulates at the contractile ring of the forming second polar body about 1.5 h post-activation. PKCzeta exhibits some unique features that resulted in it being the focus of more extensive analysis. PKCzeta is tightly associated with the meiotic spindle as determined by detergent extraction and is closely associated with alpha-tubulin as determined by FRET analysis in the metaphase II (MII) egg. In addition, after egg activation, PKCzeta remains associated with the spindle as it transits into anaphase II and later telophase II, becoming associated with the midzone microtubules. Antibodies to the active form of PKCzeta are enriched on the spindle poles and later in development on the midzone microtubules. Active PKCzeta also is enriched in both pronuclei in the 6-h post-fertilization and in the 14-h post-fertilization embryo as well as in the nuclei of the two-cell embryo. Inhibition of PKCzeta, but not inhibition of other isotypes of PKC, results in rapid disruption of the meiotic spindle. This study suggests that PKCzeta has a role in spindle stability, while other PKC isotypes have different roles in the conversion of the egg to the zygote.

Animals↗

PubMed2005

Differential roles of p39Mos-Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes.

Fully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathways. The latter cascade is activated upon p39(Mos) accumulation. Using U0126, a MEK1 inhibitor, and p39(Mos) antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39(Mos) accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42(Mpk1) but not p90(Rsk). Second, inhibition of either p39(Mos) accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39(Mos) or p90(Rsk) alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathway is required for bipolar organization of the meiotic spindle at the cortex.

Animals↗

PubMed2006

Asymmetrization of first cleavage by transient disassembly of one spindle pole aster in the leech Helobdella robusta.

Unequal first cleavage is characteristic of a diverse group of protostome animals. In the nematode Caenorhabditis elegans, unequal first cleavage is achieved through the interaction of an apparently symmetric mitotic spindle apparatus with a clearly polarized cell cortex. In the clitellate annelid Tubifex tubifex, by contrast, the spindle is monastral and contains only one gamma-tubulin-reactive centrosome; this monastral spindle is inherently asymmetric throughout mitosis. Here, we have used immunostaining for beta- and gamma-tubulin to follow spindle dynamics during the unequal first cleavage in another clitellate annelid, the leech Helobdella robusta. We find that the mitotic spindle is diastral and symmetric through early metaphase, then becomes asymmetric following the transient down-regulation of one centrosome, as judged by gamma-tubulin immunofluorescence. Low levels of drugs that affect microtubule dynamics can symmetrize the first cleavage without affecting the gamma-tubulin dynamics. Our results provide a striking example of the evolvability of cellular mechanisms underlying an unambiguously homologous developmental process.

Animals↗

PubMed2006

Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes.

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.

Animals↗