SEARCH · PubMed Health

Results for “Spindle”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources

PubMed2000

CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis.

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.

Animals↗

PubMed2000

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.

Animals↗

PubMed2001

The surveillance mechanism of the spindle position checkpoint in yeast.

The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother-bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions.

Cell Cycle Proteins↗

PubMed2001

The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis.

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.

Animals↗

PubMed2001

Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity.

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

Animals↗

PubMed2002

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle.

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.

Base Sequence↗

PubMed1991

Specific association of an M-phase kinase with isolated mitotic spindles and identification of two of its substrates as MAP4 and MAP1B.

Isolated mammalian (Chinese hamster ovary [CHO]) metaphase spindles were found to be enriched in a histone H1 kinase whose activity was mitotic-cycle dependent. Two substrates for the kinase were identified as MAP1B and MAP4. Partially purified spindle kinase retained activity for the spindle microtubule-associated proteins (MAPs) as well as brain and other tissue culture MAPs; on phosphorylation, spindle MAPs exhibited increased immunoreactivity with MPM-2, a monoclonal antibody specific for a subset of mitotic phosphoproteins. Immunofluorescence using an anti-thiophosphoprotein antibody localized in vitro phosphorylated spindle proteins to microtubule fibers, centrosomes, kinetochores, and midbodies. The fractionated spindle kinase was reactive with anti-human p34cdc2 antibodies and with an anti-human cyclin B but not an anti-human cyclin A antibody. We conclude that spindle MAPs undergo mitotic cycle-dependent phosphorylations in vivo and associate with a kinase that remains active on spindle isolation and may be related to p34cdc2.

Animals↗

PubMed2003

KLP-18, a Klp2 kinesin, is required for assembly of acentrosomal meiotic spindles in Caenorhabditis elegans.

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.

Animals↗

PubMed2003

The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.

Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.

Anaphase↗

PubMed2004

Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not.

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

Actins↗

PubMed2004

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo.

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

Alleles↗

PubMed2005

The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly.

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a approximately 200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to "rescue" experiments. Defects in the "early" step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the "late" step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.

Animals↗

PubMed2005

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans.

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.

Anaphase↗

PubMed2000

Visualizing the spindle checkpoint in Drosophila spermatocytes.

The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila.

Animals↗

PubMed1996

Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women.

To examine the effects of maternal ageing on the meiotic apparatus, we obtained oocytes from naturally cycling women in two age groups, including younger (aged 20-25 years) and older (aged 40-45 years) women. Using high-resolution confocal microscopy we obtained a detailed picture of the meiotic spindle and chromosome placement during various phases of meiosis. Our data revealed that the meiotic spindle in older women is frequently abnormal, both with regard to chromosome alignment and the microtubule matrix that comprise the meiotic spindle. The spindle in 79% of the oocytes from the older group exhibited abnormal tubulin placement and one or more chromosomes were displaced from the metaphase plate during the second meiotic division. In contrast, only 17% of the oocytes from the younger age group exhibited aneuploid conditions. The majority of eggs from this group possessed a well ordered, meiotic spindle containing chromosomes that were fully aligned within a distinct metaphase plate in the spindle. Chromosome management during meiosis is directed by microtubule assembly within the spindle. These data suggest that the regulatory mechanisms responsible for assembly of the meiotic spindle are significantly altered in older women, leading to the high prevalence of aneuploidy.

Adult↗

PubMed2004

Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein.

The present study was designed to investigate the localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at various stages during the first meiosis were fixed and immunostained for MAD1, kinetochores, microtubules, and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes were treated with nocodazole or Taxol before examination. The anti-MAD1 antibody was injected into the oocytes at the germinal vesicle (GV) stage for examination of chromosome alignment and spindle formation. It was found that MAD1 was present in the oocytes from the GV to prometaphase I stages around the nuclei. When the oocytes reached the metaphase I (M-I) to metaphase II (M-II) stages, MAD1 was mainly localized at the spindle poles. However, MAD1 relocated to the vicinity of the chromosomes when spindles were disassembled by nocodazole or cooling, and the relocated MAD1 moved back to the spindle poles during spindle recovery. Taxol treatment did not affect the MAD1 localization. Although anti-MAD1 antibody injection did not affect nuclear maturation, significantly higher proportions of injected oocytes had misaligned chromosomes when the oocytes reached the M-I to M-II stages. The results of the present study indicate that MAD1 is present in mouse oocytes at all stages during the first meiosis and that it participates in spindle checkpoint during meiosis. However, MAD1 could not check misaligned chromosomes during spindle recovery after the spindles were destroyed by drug or cooling, which caused some chromosomes to scatter in the oocytes.

Animals↗

PubMed2000

Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes.

The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.

Animals↗

PubMed2002

Regulation of spindle formation by active mitogen-activated protein kinase and protein phosphatase 2A during mouse oocyte meiosis.

Mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) regulate oocyte meiosis, yet little is known regarding their mechanisms of action. This study addressed the functional importance of active MAPK and PP2A in regulating oocyte meiosis. Experiments were conducted to identify MAPK activation, PP2A activity, intracellular enzyme trafficking, and ultrastructural associations during meiosis. Questions of requisite kinase and/or phosphatase activity and chromatin condensation, microtubule polymerization, and spindle formation were addressed. At the protein level, MAPK and PP2A were present in constant amounts throughout the first meiotic division. Both MAPK and PP2A were activated following germinal vesicle breakdown (GVBD) in conjunction with metaphase I development. Immunocytochemical studies confirmed the absence of active MAPK in germinal vesicle-intact (GVI) and GVBD oocytes. At metaphase I and during the metaphase I/metaphase II transition, activated MAPK colocalized with microtubules, poles, and plates of meiotic spindles. Protein phosphatase 2A was dispersed evenly throughout the GVI oocyte cytoplasm. Throughout the metaphase I/metaphase II transition, PP2A colocalized with microtubules of meiotic spindles. Both active MAPK and PP2A associated with in vitro-polymerized microtubules, suggesting that active MAPK and PP2A locally regulate spindle formation. Inhibition of MAPK activation resulted in compromised microtubule polymerization, no spindle formation, and loosely condensed chromosomes. Treatment with okadaic acid (OA) or calyculin-A (CL-A), which inhibits oocyte cytoplasmic PP2A, caused an absence of microtubule polymerization and spindles, even though MAPK activity was increased under these treatment conditions. Thus, active MAPK is required, but is not sufficient, for normal meiotic spindle formation and chromosome condensation. In addition, the oocyte OA/CL-A-sensitive PP, presumably PP2A, is essential for microtubule polymerization and meiotic spindle formation.

Animals↗