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PubMed2003

Asymmetrically distributed C. elegans homologs of AGS3/PINS control spindle position in the early embryo.

BACKGROUND: Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric Galpha subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown. RESULTS: Here we show that the Galpha subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). Finally, we show that the GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3. CONCLUSIONS: Our results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins.

Amino Acid Sequence↗

PubMed2006

Interactions of the meiotic spindle with mitotic chromosomes in GV mouse oocytes.

During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte's microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 h in-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division.

Anaphase↗

PubMed2005

Meiotic spindle visualization in living human oocytes.

A computer-assisted polarization microscopy system (polscope) has made it possible to analyse the meiotic spindle of oocytes subjected to intracytoplasmic sperm injection (ICSI) without affecting their viability. It has been shown that the presence of a detectable birefringent meiotic spindle inside the oocyte cytoplasm of human metaphase II (MII) prepared for ICSI is an indicator of oocyte quality, such as fertilization and developmental ability. Meiotic spindle imaging has also shown that this structure, when detectable, is not always aligned with the first polar body (PB1) in fresh MII oocytes. The relationship between the degree of meiotic spindle deviation from the PB1 location and ICSI outcomes is discussed in this paper. When the meiotic spindle of in-vitro matured oocytes is analysed, it is always found to be aligned with the PB1, suggesting that the misalignment observed in the oocytes matured in vivo results from the PB1 displacement during the manipulations for the cumulus and corona removal. Furthermore, polscope analysis of meiotic spindle changes in living MII oocytes subjected to freezing and thawing procedures has shown that the current techniques of oocyte cryopreservation cause meiotic spindle destruction. The polscope system may assist in the selection of fresh and thawed oocytes for ICSI.

Cryopreservation↗

PubMed1995

F-actin is required for spindle anchoring and rotation in Xenopus oocytes: a re-examination of the effects of cytochalasin B on oocyte maturation.

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.

Actins↗

PubMed2004

Visualization and examination of the meiotic spindle in human oocytes with polscope.

PURPOSE: The effect of detectability/location of meiotic spindle to fertilization of human MII oocytes and outcome of clinical IVF/ICSI-ET cycles was studied. METHODS: The location of spindle relative to polar body positioned to 6 o'clock was detected with polscope prior to ICSI in oocytes. Data of 83 IVF/ICSI-ET cycle, including pregnancy results, and a total of 1033 oocytes examined were analyzed retrospectively. RESULTS: Polar body position does not always accurately predict spindle position. Similar fertilization was observed in the polscope detected and in the control group. The main number of oocytes and spindle positive oocytes collected was higher in the pregnant than in the non-pregnant group. Higher fertilization was found in that group where 2/3-d of the oocytes had detected spindle. The main number of oocytes and spindle positive oocytes obtained decreased with increasing age of patients. CONCLUSIONS: The presence of birefringent spindle predicts higher fertilization and development and greater probability of pregnancy.

Adult↗

PubMed2001

Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin.

Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells. This often involves interactions between spindle and astral microtubules and cortical actin. In yeast and Caenorhabditis elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious. Here, we report that the tumour suppressor homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos. Armadillo, alpha-catenin and APC2 all localize to sites of cortical spindle attachment. APC2-Armadillo complexes often localize with interphase microtubules. Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2-Armadillo complexes. Together, these data suggest that APC2, Armadillo and alpha-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase.

Actins↗

PubMed2003

Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint.

The spindle checkpoint delays anaphase until all chromosomes are properly attached to spindle microtubules. When the spindle checkpoint is activated at unattached kinetochores, the checkpoint proteins BubR1, Bub3 and Mad2 bind and inhibit Cdc20, an activator of the anaphase-promoting complex (APC). Here, we show that Xenopus laevis Cdc20 is phosphorylated at Ser 50, Thr 64, Thr 68 and Thr 79 during mitosis and that mitogen-activated protein kinase (MAPK) contributes to the phosphorylation at Thr 64 or Thr 68. Cdc20 mutants that are phosphorylation-deficient are able to activate the APC in X. laevis egg extracts. However, Cdc20 mutants in which any of the four phosphorylation sites were altered to Ala or Val failed to respond to the spindle checkpoint signal, owing to their reduced affinity for the spindle checkpoint proteins. This study demonstrates that the spindle checkpoint stops anaphase by inhibiting fully-phosphorylated Cdc20. Our results also have implications for the spindle checkpoint silencing mechanism.

Animals↗

PubMed2004

The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint.

The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2001

A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization.

Asymmetric cell division requires the orientation of mitotic spindles along the cell-polarity axis. In Drosophila neuroblasts, this involves the interaction of the proteins Inscuteable (Insc) and Partner of inscuteable (Pins). We report here that a human Pins-related protein, called LGN, is instead essential for the assembly and organization of the mitotic spindle. LGN is cytoplasmic in interphase cells, but associates with the spindle poles during mitosis. Ectopic expression of LGN disrupts spindle-pole organization and chromosome segregation. Silencing of LGN expression by RNA interference also disrupts spindle-pole organization and prevents normal chromosome segregation. We found that LGN binds the nuclear mitotic apparatus protein NuMA, which tethers spindles at the poles, and that this interaction is required for the LGN phenotype. Anti-LGN antibodies and the LGN-binding domain of NuMA both trigger microtubule aster formation in mitotic Xenopus egg extracts, and the NuMA-binding domain of LGN blocks aster assembly in egg extracts treated with taxol. Thus, we have identified a mammalian Pins homologue as a key regulator of spindle organization during mitosis.

Animals↗

PubMed2002

Astral microtubules monitor metaphase spindle alignment in fission yeast.

Segregating genetic material along the longest axis of the cell ensures that there is a sufficient distance between daughter chromosomes at the point of cytokinesis. Monitoring the orientation of the mitotic spindle can be subjected to cell cycle controls. In the fission yeast Schizosaccharomyces pombe, the existence of such a cell-cycle checkpoint has been proposed to delay the metaphase to anaphase transition when spindle poles are not properly oriented with respect to the actomyosin ring. Here we show, by using a fission yeast mutant compromised in its assembly of astral microtubules, that in the absence of astral microtubules short metaphase spindles are unable to orient themselves with respect to the long axis of the cell and are delayed in spindle elongation. This astral defect engages a spindle orientation checkpoint because deletion of the transcription factor Atf1, which is involved in maintaining this checkpoint, allows misaligned asterless metaphase spindles to elongate. We propose that astral microtubules are involved directly in monitoring orientation of the metaphase spindle and in controlling the timing of elongation in fission yeast.

Cell Compartmentation↗

PubMed2004

Cyclin B degradation leads to NuMA release from dynein/dynactin and from spindle poles.

The protein NuMA localizes to mitotic spindle poles where it contributes to the organization of microtubules. In this study, we demonstrate that NuMA loses its stable association with the spindle poles after anaphase onset. Using extracts from Xenopus laevis eggs, we show that NuMA is dephosphorylated in anaphase and released from dynein and dynactin. In the presence of a nondegradable form of cyclin B (Delta90), NuMA remains phosphorylated and associated with dynein and dynactin, and remains localized to stable spindle poles that fail to disassemble at the end of mitosis. Inhibition of NuMA or dynein allows completion of mitosis, despite inducing spindle pole abnormalities. We propose that NuMA functions early in mitosis during the formation of spindle poles, but is released from the spindle after anaphase, to allow spindle disassembly and remodelling of the microtubule network.

Anaphase↗

PubMed2005

A pre-anaphase role for a Cks/Suc1 in acentrosomal spindle formation of Drosophila female meiosis.

Conventional centrosomes are absent from a female meiotic spindle in many animals. Instead, chromosomes drive spindle assembly, but the molecular mechanism of this acentrosomal spindle formation is not well understood. We have screened female sterile mutations for defects in acentrosomal spindle formation in Drosophila female meiosis. One of them, remnants (rem), disrupted bipolar spindle morphology and chromosome alignment in non-activated oocytes. We found that rem encodes a conserved subunit of Cdc2 (Cks30A). As Drosophila oocytes arrest in metaphase I, the defect represents a new Cks function before metaphase-anaphase transition. In addition, we found that the essential pole components, Msps and D-TACC, were often mislocalized to the equator, which may explain part of the spindle defect. We showed that the second cks gene cks85A, in contrast, has an important role in mitosis. In conclusion, this study describes a new pre-anaphase role for a Cks in acentrosomal meiotic spindle formation.

Amino Acid Sequence↗

PubMed1997

Expression of myosin heavy chain isoforms in regenerated muscle spindle fibres after muscle grafting in young and adult rats--plasticity of intrafusal satellite cells.

Satellite cells are the myogenic precursor cells of postnatal skeletal muscles. After muscle injury they can proliferate, differentiate, fuse and form myofibres. We have analysed regeneration of distinctly different types of intrafusal fibres in rat muscle spindles. We have introduced the new technique of heterochronous allotransplantation and compared it with the previously used standard autografting method. The allotransplantation method enables one to graft muscles from very young animals; we have used the extensor digitorum longus (EDL) muscles from 2- to 28-day-old rats, which were grafted into EDL muscles of adult inbred recipients. The regenerated "intrafusal" fibres did not express the spindle-specific slow tonic and alpha cardiac-like myosin heavy chain (MyHC) isoforms and they did not exhibit the dual mATPase reaction typical of the nuclear bag2 fibres and the characteristic regional differences in MyHC expression and in the mATPase reaction of nuclear bag1 and nuclear bag2 fibres. On the other hand, they expressed either fast twitch or slow twitch/beta cardiac MyHC isoforms and exhibited an alkali or acid stable mATPase reaction along their whole length, like extrafusal fast type 2 and slow type 1 muscle fibres, respectively. In all regenerated muscle spindles only motor, but no sensory axons were found. More than 85% of muscle spindles in our sample contained regenerated spindle fibres of the same extrafusal fibre type (either type 2 or type 1), in contrast to control muscle spindles, which always contained intrafusal fibres of three different intrafusal fibre types (nuclear bag1, nuclear bag2 and nuclear chain fibres). There were no differences in MyHC expression and mATPase activity between spindle fibres regenerated in grafts taken from young rats of various ages or between allotransplanted and autotransplanted EDL muscles. The present results demonstrate that regenerated "intrafusal" fibres resemble, according to MyHC expression, extrafusal fast or slow muscle fibres. It can thus be concluded that intrafusal satellite cells derived from distinctly different nuclear bag1, nuclear bag2 and nuclear chain fibres show great plasticity, as their MyHC expression can be respecified towards the extrafusal muscle fibre phenotype by foreign alpha-motor innervation.

Animals↗

PubMed2002

The influence of increased muscle spindle sensitivity on Achilles tendon jerk and H-reflex in relaxed human subjects.

Whether the fusimotor system contributes to reflex gain changes during reinforcement maneuvers is re-examined in the light of new data. Recently, from direct recordings of spindle afferent activity originating from ankle flexor muscles, we showed that mental computation increased the muscle spindle mechanical sensitivity in completely relaxed human subjects without concomitant alpha-motoneuron activation, providing evidence for selective fusimotor drive activation. In the present study, the effects of mental computation were investigated on monosynaptic reflexes elicited in non-contracting soleus muscle either by direct nerve stimulation (Hoffmann reflex, H) or by tendon tap (Tendinous reflex, T). The aim was to relate the time course of the changes in reflex size to the increase in spindle sensitivity during mental task in order to explore whether fusimotor activation can influence the size of the monosynaptic reflex. The results show changes in reflex amplitude that parallel the increase in muscle spindle sensitivity. When T-reflex is consistently facilitated during mental effort, the H-reflex is either depressed or facilitated, depending on the subjects. These findings suggest that the increased activity in muscle spindle primary endings may account for mental computation-induced changes in both tendon jerk and H-reflex. The facilitation of T-reflex is attributed to the enhanced spindle mechanical sensitivity and the inhibition of H-reflex is attributed to post-activation depression following the increased Ia ongoing discharge. This study supports the view that the fusimotor sensitization of muscle spindles is responsible for changes in both the mechanically and electrically elicited reflexes. It is concluded that the fusimotor drive contributed to adjustment of the size of tendon jerk and H-reflex during mental effort. The possibility that a mental computation task may also operate by reducing the level of presynaptic inhibition is discussed on the basis of H-reflex facilitation.

Achilles Tendon↗

PubMed1986

The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis.

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.

Benzimidazoles↗

PubMed1995

Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

Adenosine Triphosphate↗

PubMed1997

XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts.

We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central alpha-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor-arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.

Amino Acid Sequence↗

PubMed1997

Astral microtubule dynamics in yeast: a microtubule-based searching mechanism for spindle orientation and nuclear migration into the bud.

Localization of dynein-green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at approximately 0.3-1.5 micron/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

Anaphase↗