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1.
In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1. 相似文献
2.
This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation. 相似文献
3.
Hirohisa Masuda 《BioEssays : news and reviews in molecular, cellular and developmental biology》1995,17(1):45-51
The mitotic spindle contains the machinery responsible for sister chromatid segregation. It is composed of a complex and dynamic array of microtubules, which are nucleated from the spindle poles. Studies of yeast spindle functions by molecular genetic analysis and by in vitro functional analysis have identified proteins that are mitosis-specific and present at very low concentrations in the cell, and have revealed the molecular bases of several processes required for the formation and functioning of the mitotic spindle. Here I review the current knowledge of the processes that are common to most eukaryotes: microtubule nucleation at the spindle poles, bipolar spindle assembly, maintenance of the spindle structure, chromosome attachment to the spindle and chromosome separation on the spindle. 相似文献
4.
Anja K. Dunsch Dean Hammond Jennifer Lloyd Lothar Schermelleh Ulrike Gruneberg Francis A. Barr 《The Journal of cell biology》2012,198(6):1039-1054
The cytoplasmic dynein motor generates pulling forces to center and orient the mitotic spindle within the cell. During this positioning process, dynein oscillates from one pole of the cell cortex to the other but only accumulates at the pole farthest from the spindle. Here, we show that dynein light chain 1 (DYNLL1) is required for this asymmetric cortical localization of dynein and has a specific function defining spindle orientation. DYNLL1 interacted with a spindle-microtubule–associated adaptor formed by CHICA and HMMR via TQT motifs in CHICA. In cells depleted of CHICA or HMMR, the mitotic spindle failed to orient correctly in relation to the growth surface. Furthermore, CHICA TQT motif mutants localized to the mitotic spindle but failed to recruit DYNLL1 to spindle microtubules and did not correct the spindle orientation or dynein localization defects. These findings support a model where DYNLL1 and CHICA-HMMR form part of the regulatory system feeding back spindle position to dynein at the cell cortex. 相似文献
5.
Aurlien Courtois Shuhei Yoshida Osamu Takenouchi Kohei Asai Tomoya S Kitajima 《EMBO reports》2021,22(4)
In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball‐like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore–microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore–microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore–microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles. These findings suggest a role for stable kinetochore–microtubule attachments in fine‐tuning acentrosomal spindle bipolarity. 相似文献
6.
《The Journal of cell biology》1976,68(1):113-122
The metaphase spindle of haploid Dictyostelium discoideum (n = 7) is 2 mum long. It consists of some 20 microtubules which seem continuous between the spindle pole bodies and there are about 20 chromosomal microtubules at each end of the spindle. During anaphase the central spindle elongates and the chromosomal microtubules shorten. The spindle length and structure at this stage suggests that lengthening is caused by elongation as well as parallel sliding of the nonchromosomal microtubules. The nuclear envelope remains mostly intact during mitosis, and nuclear separation through medial constriction takes place when the spindle is 6 mum long. Cytokinesis occurs when the spindle is 10 mum long. At that time the kinetochores double in size. During interphase, the spindle pole body separates from the nucleus to a distance of 0.7 mum, and it returns at the onset of the next prophase when it becomes functionally double, thereby starting the formation of a central spindle. When comparing mitosis in the cellular slime molds Polysphondylium violaceum and D. discoideum, several similarities and some differences are apparent. 相似文献
7.
Marina E. Crowder Jonathan R. Flynn Karen P. McNally Daniel B. Cortes Kari L. Price Paul A. Kuehnert Michelle T. Panzica Armann Andaya Julie A. Leary Francis J. McNally 《Molecular biology of the cell》2015,26(17):3030-3046
Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin. 相似文献
8.
The impact of chromosomes and centrosomes on spindle assembly as observed in living cells 总被引:7,自引:7,他引:0
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《The Journal of cell biology》1995,129(5):1287-1300
We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video- enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears. 相似文献
9.
Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes. 相似文献
10.
We addressed the role of the G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and G-proteins (Gialpha3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Gialpha3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Gialpha3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Gialpha. The influence of LGN and Gialpha3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry. 相似文献
11.
Peyre E Jaouen F Saadaoui M Haren L Merdes A Durbec P Morin X 《The Journal of cell biology》2011,193(1):141-154
To maintain tissue architecture, epithelial cells divide in a planar fashion, perpendicular to their main polarity axis. As the centrosome resumes an apical localization in interphase, planar spindle orientation is reset at each cell cycle. We used three-dimensional live imaging of GFP-labeled centrosomes to investigate the dynamics of spindle orientation in chick neuroepithelial cells. The mitotic spindle displays stereotypic movements during metaphase, with an active phase of planar orientation and a subsequent phase of planar maintenance before anaphase. We describe the localization of the NuMA and LGN proteins in a belt at the lateral cell cortex during spindle orientation. Finally, we show that the complex formed of LGN, NuMA, and of cortically located Gαi subunits is necessary for spindle movements and regulates the dynamics of spindle orientation. The restricted localization of LGN and NuMA in the lateral belt is instructive for the planar alignment of the mitotic spindle, and required for its planar maintenance. 相似文献
12.
A functional relationship between NuMA and kid is involved in both spindle organization and chromosome alignment in vertebrate cells 总被引:3,自引:0,他引:3
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We examined spindle morphology and chromosome alignment in vertebrate cells after simultaneous perturbation of the chromokinesin Kid and either NuMA, CENP-E, or HSET. Spindle morphology and chromosome alignment after simultaneous perturbation of Kid and either HSET or CENP-E were no different from when either HSET or CENP-E was perturbed alone. However, short bipolar spindles with organized poles formed after perturbation of both Kid and NuMA in stark contrast to splayed spindle poles observed after perturbation of NuMA alone. Spindles were disorganized if Kid, NuMA, and HSET were perturbed, indicating that HSET is sufficient for spindle organization in the absence of Kid and NuMA function. In addition, chromosomes failed to align efficiently at the spindle equator after simultaneous perturbation of Kid and NuMA despite appropriate kinetochore-microtubule interactions that generated chromosome movement at normal velocities. These data indicate that a functional relationship between the chromokinesin Kid and the spindle pole organizing protein NuMA influences spindle morphology, and we propose that this occurs because NuMA forms functional linkages between kinetochore and nonkinetochore microtubules at spindle poles. In addition, these data show that both Kid and NuMA contribute to chromosome alignment in mammalian cells. 相似文献
13.
Jesse C. Gatlin Alexandre Matov Gaudenz Danuser Timothy J. Mitchison Edward D. Salmon 《The Journal of cell biology》2010,188(4):481-489
Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination. 相似文献
14.
During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)-dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit. 相似文献
15.
During cell division, the molecular motor Eg5 crosslinks overlapping antiparallel microtubules and pushes them apart to separate mitotic spindle poles. Dynein has been proposed as a direct antagonist of Eg5 at the spindle equator, pulling on antiparallel microtubules and favoring spindle collapse. Some of the experiments supporting this hypothesis relied on endpoint quantifications of spindle phenotypes rather than following individual cell fates over time. Here, we present a mathematical model and proof-of-principle experiments to demonstrate that endpoint quantifications can be fundamentally misleading because they overestimate defective phenotypes. Indeed, live-cell imaging reveals that, while depletion of dynein or the dynein binding protein Lis1 enables spindle formation in presence of an Eg5 inhibitor, the activities of dynein and Eg5 cannot be titrated against each other. Thus, dynein most likely antagonizes Eg5 indirectly by exerting force at different spindle locations rather than through a simple push-pull mechanism at the spindle equator. 相似文献
16.
Edwin W. Taylor 《The Journal of cell biology》1959,6(2):193-196
The formation of the mitotic spindle of the newt cell in tissue culture has been studied, using polarized light. The rate of formation was measured and it was shown that the spindle increased in length at a constant rate until the maximum was attained. During metaphase the spindle shortened to about 50 to 60 per cent of its original length, reaching a minimum just before anaphase. No birefringence was detected in late anaphase in the spindle region after the chromosome masses had separated. The effects of certain compounds which are believed to inhibit protein synthesis were investigated. Chloramphenicol added in early prophase prevented the formation of a spindle of normal length. The possible relation of chloramphenicol to the synthesis of spindle proteins is discussed. 相似文献
17.
Xiangduo Kong Alexander R. Ball Jr. Eiichiro Sonoda Jie Feng Shunichi Takeda Tatsuo Fukagawa Tim J. Yen Kyoko Yokomori 《Molecular biology of the cell》2009,20(5):1289-1301
Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions. 相似文献
18.
《Cell cycle (Georgetown, Tex.)》2013,12(12):1709-1712
At the end of the cell cycle a cell physically divides into two daughter cells in a process called cytokinesis. Cytokinesis consists of at least four steps: 1. The position of the presumptive cytokinesis furrow is specified. 2. A contractile ring is formed. 3. The contractile ring contracts, resulting in furrow ingression. 4. Cytokinesis completes with sealing of the membranes. The mitotic spindle positions the cytokinesis furrow at the cell cortex midway along the longitudinal axis of the spindle, which is both the mid-point between the two asters and the location of the spindle midzone. The mitotic spindle emits two consecutive signals that position the furrow: Microtubule asters provide a first signal; the spindle midzone provides a second signal. Our results support the view that the spindle midzone is dispensable for completion of cytokinesis. However, the spindle midzone can negatively affect aster-positioned cytokinesis, possibly because the aster- and midzone-positioned furrows compete for contractile elements. 相似文献
19.
Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases. 相似文献
20.
During female meiosis, meiotic spindles are positioned at the oocyte cortex to allow expulsion of chromosomes into polar bodies. In C. elegans, kinesin-dependent translocation of the entire spindle to the cortex precedes dynein-dependent rotation of one spindle pole toward the cortex. To elucidate the role of kinesin-1 in spindle translocation, we examined the localization of kinesin subunits in meiotic embryos. Surprisingly, kinesin-1 was not associated with the spindle and instead was restricted to the cytoplasm in the middle of the embryo. Yolk granules moved on linear tracks, in a kinesin-dependent manner, away from the cortex, resulting in their concentration in the middle of the embryo where the kinesin was concentrated. These results suggest that cytoplasmic microtubules might be arranged with plus ends extending inward, away from the cortex. This microtubule arrangement would not be consistent with direct transport of the meiotic spindle toward the cortex by kinesin-1. In maturing oocytes, the nucleus underwent kinesin-dependent migration to the future site of spindle attachment at the anterior cortex. Thus the spindle translocation defect observed in kinesin-1 mutants may be a result of failed nuclear migration, which places the spindle too far from the cortex for the spindle translocation mechanism to function. 相似文献