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PubMed1999

Monastral bipolar spindles in meiosis II of male Trichosia pubescens (Sciaridae): early stages of spindle formation and chromosome orientation.

The metaphase spindle of male meiosis II in fungus gnats (Sciaridae) is one example of naturally occurring monastral bipolar spindles. To gain further insights into how the bipolar spindle is formed in the presence of only one polar center, prometaphase of male meiosis II was investigated in the sciarid Trichosia pubescens by means of anti-tubulin immunofluorescence, DAPI chromosome staining, and electron microscopy of ultrathin serial sections. The first step in spindle formation after interkinesis seems to be the organization of an astral half-spindle, probably by MTOC activity of the astral region. With the exception of the non-disjunctional X chromosome, which always lies close to the aster, the chromosomes are found to occupy various positions with respect to the astral region, revealing different orientations of their chromatid kinetochores. It was observed that some of the mal-oriented kinetochores are associated with microtubules that, due to their orientation perpendicular to the spindle axis, are unlikely to originate from the astral region. Therefore, these mal-oriented microtubules are taken as an indication of a dispersed MTOC activity near the chromosomes or at kinetochores. According to recent models of chromosome-induced spindle self-organization [e.g., Merdes et al., 1997: J. Cell Biol. 138:953-956], they could be responsible for the formation of the other (anastral) half-spindle and for amphitelic (bipolar) orientation of the chromosomes.

Animals↗

PubMed2006

Asymmetric recruitment of dynein to spindle poles and microtubules promotes proper spindle orientation in yeast.

The orientation of the mitotic spindle plays a key role in determining whether a polarized cell will divide symmetrically or asymmetrically. In most cell types, cytoplasmic dynein plays a critical role in spindle orientation. However, how dynein directs opposite spindle poles toward distinct and predetermined cell ends is poorly understood. Here, we show that dynein distributes preferentially to the spindle pole bodies (SPB) and astral microtubules (MTs) proximal to the bud in metaphase yeast cells. Dynein asymmetry depended on the bud neck kinases Elm1, Hsl1, and Gin4, on the spindle pole components Cnm67 and Cdk1, and on the B-type cyclins Clb1 and Clb2. Furthermore, phenotypic and genetic studies both indicated that dynein is unable to orient the spindle when it localizes to both poles and associated microtubules. Together, our data indicate that proper orientation of the spindle requires dynein to act on a single spindle pole.

CDC2 Protein Kinase↗

PubMed1990

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.

Anaphase↗

PubMed2001

Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix.

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.

Adenosine Triphosphatases↗

PubMed2004

TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly.

The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336-341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by approximately 20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by approximately 20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.

Cell Separation↗

PubMed2004

Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces.

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.

Alleles↗

PubMed2002

[Meiosis spindle formation and chromosome behavior in diploid potatoes with 'fused spindles' mutation].

Chromosomal behaviour and spindle morphology were studied in microsporogenesis of two kinds of diploid potato clones: with normal meiosis, and with "fused spindles" (fs) occurring during the second meiotic division from prometaphase II (proMII) to telophase II (TII). For the first time, morphological effect of fs was found at the late proMII stage to be expressed as two interrelated processes: 1) abnormal chromosome movement, which resulted in joining two groups of chromosomes in the central zone of meiocytes, and 2) abnormal formation of two spindles in the direction to two division poles instead of four poles that actually led to the formation of a united bipolar spindle. Thus, it is not the fusion of two parallel spindles but the formation of united bipolar spindle that constitutes fs abnormality, while the parallel co-orientation of two spatially separated meiotic spindles is a norm in diploid potato. These primary abnormalities detected at proMII resulted in abnormalities at its subsequent meiotic stages: formation of fused spindle and united metaphase plate at MII, bipolar chromosome segration at anaphase II, formation of two telophase nuclei at TII and dyads at the tetrad stage. The results obtained evidence the polar division disturbance in diploid potato clones with fs abnormality.

Chromosomes, Plant↗

PubMed1978

Evoked K-complexes and cardiovascular responses to spindle-synchronous and spindle-asynchronous stimulus clicks during NREM sleep.

The hypothesis that the functional role of the sleep spindle is to preserve sleep by inhibiting sensory input (Yamadori 1971) was examined. Series of 44 dB, 10 msec, 1000 c/sec 'clicks' were presented to 12 subjects at a 30-sec ISI during stage 2 sleep either during spindle bursts (i.e. spindle-synchronous clicks) or during interburst periods (i.e. spindle-asynchronous clicks). Contrary to the spindle inhibitory hypothesis, cortical EEG and cardiovascular responses showed no evidence of spindle 'suppression'. Evoked K-complexes were potentiated by the spindle-synchronous stimulation. A second study with 7 subjects replicated this result and extended the finding to include stage 3--4 sleep. It was suggested that the potentiation of evoked K-complexes was due to phasic reductions in inhibitory action during sleep spindles resulting in increased transmission of sensory events or, perhaps, an increase in the lability of certain EEG response systems.

Adolescent↗

PubMed1993

MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae.

The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.

Alleles↗

PubMed2005

Spindle checkpoint signaling requires the mis6 kinetochore subcomplex, which interacts with mad2 and mitotic spindles.

The spindle checkpoint coordinates cell cycle progression and chromosome segregation by inhibiting anaphase promoting complex/cyclosome until all kinetochores interact with the spindle properly. During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. Here, we assess the requirement of various kinetochore components for the accumulation of Mad2 and Bub1 on the kinetochore in fission yeast and show that the necessity of the Mis6-complex and the Nuf2-complex is an evolutionarily conserved feature in the loading of Mad2 onto the kinetochore. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed N-terminal fragments of Mis6 localize along the mitotic spindle, highlighting the potential binding ability of Mis6 not only to the centromeric chromatin but also to the spindle microtubules. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle-kinetochore attachment state and acts as a platform for Mad2 to accumulate at unattached kinetochores.

Autoantigens↗

PubMed1984

Spindle membranes in mitosis and meiosis of the heteropteran insect Dysdercus intermedius. A study of the interrelationship of spindle architecture and the kinetic organization of chromosomes.

The fate of cytoplasmic membranes has been traced from spermatogonial interphase to the telophase of the second meiotic division with the aid of osmium ferrocyanide staining. At prophase, the nuclear membrane is indented to a funnel shape due to microtubules which radiate from the poles. These indentations open at prometaphase admitting microtubules to the nuclear interior. Since the nuclear envelope widens at prometaphase with only few lateral defects, organelles are excluded and the spindle is essentially intranuclear. Membranes surround the spindle until late anaphase. There are no cytoplasmic vesicles within the spindle, but there is an abundance of vesicles around the spindle poles following the radiating microtubules. At telophase, small vesicles around the chromosomes contribute to the new nuclear envelope while others fuse into large blisters which disappear gradually. A new vesicle system is formed in the daughter cells. At meiosis, the nuclear membrane is not indented by the polar microtubules which follow the contour of the nucleus together with vesicles. At late diakinesis, the bivalents are individually ensheathed by vesicles and lamellae which separate them from the remainder of the nucleus. In building up the metaphase configuration, the chromosomal sheaths become interconnected with the X1/X2-pseudotetrad in the center. Lamellae from these sheaths extend to the poles following the kinetochore microtubule bundles. In contrast to mitosis, the spindle body is pervaded by a structural framework of membranes with mitochondria in between. Essentially the same spindle architecture exists in the second meiotic division with the exception of the tandemly joined X1 and X2 chromosomes which are ensheathed by endoplasmic reticulum but lie outside the autosome group as they form spindle connections to one pole only. Since the chromosomes form a holocentric group at mitosis but behave as individual telocentrics at meiosis, their ensheathment by membranes during meiosis is thought to be essential for their orientation and orderly disjunction in the absence of true kinetochore plates.

Animals↗

PubMed1978

[Orientation of the cleavage spindles in pulmonate mollusks. I. The role of the form of the blastomeres in 2d cleavage spindle orientation].

In the normal two-celled embryos of various pulmonate molluscs, the orientation of spindles characteristic of metaanaphase is being frequently established gradually, in the process of transition from pro- to metaphase accompained by the growth of spindle and asters. The typical growth of contact zone between the blastomeres of the common pond snail embryos was inhibited to a different extent under their cultivation after the 1 cleavage division in the calcium-free media or after trypsinization. At the same time the orientation of meta-anaphase spindles was markedly affected (as judged by an angle alpha between the spindle axis and the plane of contact zone in the equatorial projection). When analyzing the model distributions of the angles between the two spindle axes (in the same projection), it was shown that the empirical distributions of these angles corresponded to the principle of stochastic combination of two alpha. A conclusion is drawn that the orientation of one spindle does not depend on that of another but the position of each of them depends on the size of the contact zone and, hence, on the general form of the adjacent blastomere region. Some other processes determining the spindle orientation are discussed.

Anaphase↗

PubMed1978

[Orientation of the cleavage spindles in pulmonate mollusks. II. The role of the architecture of the intercellular contacts in III and IV cleavage spindle orientation].

In the normal development of pulmonate molluscs, the variety of orientations of the III and IV cleavage spindles markedly reduces in the process of transition from pro- to meta- and anaphase. Even prior to the completion of spindle growth in these cleavage divisions the external faces of blastomeres become asymmetrical due to intercellular interactions but the whole system of external intercellular contacts (edges) is characterized by a certain symmetry. This symmetry coincides partially or fully with that of the system of definitive spindles. In the artificially obtained four-celled common pond snail embryos with the chain-like position of blastomeres, the III cleavage spindles were oriented at right angles to the polar axis, rather than in parallel with it (as in the normal development). The eight-celled embryos with symmetrical external faces of macromeres were also obtained. The variety of orientation of the IV cleavage definitive spindles in such embryos was markedly widened and in the macromeres with inverse asymmetry the inversion of the sign of declination of the spindles was observed. The spindle orientation depends, thus, on the form of adjacent region of the external face. This form as a whole is determined by the mutual position of blastomeres, curvature of surface and relative length of the face edges.

Anaphase↗

PubMed1999

Proportions of slow myosin heavy chain-positive fibers in muscle spindles and adjoining extrafusal fascicles, and the positioning of spindles relative to these fascicles.

Chicken leg muscles were examined to calculate the percentages of slow myosin heavy chain (MHC)-positive fibers in spindles and in adjacent extrafusal fascicles, and to clarify how the encapsulated portions of muscle spindles are positioned relative to these fascicles. Unlike mammals, in chicken leg muscles slow-twitch MHC and slow-tonic MHC are expressed in intrafusal fibers and in extrafusal fibers, suggesting a close developmental connection between the two fiber populations. In 8-week-old muscles the proportions of slow MHC-positive extrafusal fibers that ringed muscle spindles ranged from 0-100%. In contrast, proportions of slow MHC-positive intrafusal fibers in spindles ranged from 0-57%. Similar proportions in fiber type composition between intrafusal fibers and surrounding extrafusal fibers were apparent at embryonic days 15 and 16, demonstrating early divergence of extrafusal and intrafusal fibers. Muscle spindles were rarely located within single fascicles. Instead, they were commonly placed where several fascicles converged. The frequent extrafascicular location of spindles suggests migration of intrafusal myoblasts from developing clusters of extrafusal fibers toward the interstitium, perhaps along a neurotrophic gradient established by sensory axons that are advancing in the connective tissue matrix that separates adjoining fascicles.

Animals↗

PubMed1983

The spindle potential in the frog muscle spindle does not require external Na+.

Spindle potential recorded from the sensory nerve terminal of isolated frog muscle spindles disappeared within 20-30 min after the spindle receptor was perfused with Na+-free (Li, Tris or choline) Ringer's solution, whereas the amplitude of spindle potential was not attenuated for periods up to 60 min when the spindles were perfused in a Na+-free Ringer's solution containing both 10 mM TEA and 0.1 mM 4-aminopyridine after being washed with a normal Ringer's solution containing both the K+-channel blockers. It is concluded that the time-dependent decrease in the amplitude of spindle potential during the application of Na+-free solution is not ascribable to a decrease in the inward current carried by Na+, but is due to an increase in an outward current carried by K+.

Animals↗

PubMed1985

Reversal of the static component of spindle potential by imposed depolarizing current in the frog muscle spindle.

The static component of the spindle potential provoked during stretch of isolated muscle spindles of the frog was reversed during the application of depolarizing currents ranging from 0.2 to 5 nA in normal Ringer solution and also in Na+-free Ringer solution. In the same range of current intensities, spontaneous rhythmic hyperpolarizations due to [Ca2+]i-activated GK, an attenuation of membrane impedance, and an anomalous decrease in amplitude of the afferent spikes were observed. All 4 phenomena were abolished by K+ channel blockers (10 mM CsCl, 1-2 mM 4-aminopyridine (4-AP), or 20 mM tetraethylammonium chloride (TEA], Ca2+ channel blockers (5-10 mM CoCl2, MnCl2, 1-2 mM CdCl2 or 0.5 mM verapamil) or 0.1 mM quinine. The amplitude of the static component of the spindle potential was markedly increased at threshold concentration of the K+ channel blockers (5 mM CsCl, 0.1-0.5 mM 4-AP or 5-10 mM TEA), but the component disappeared at that of the Ca2+ channel blockers. The rhythmic hyperpolarizations are associated with the spindle potential, except for its dynamic component, which often triggers a hyperpolarizing deflection. We suggest that both the static component of the spindle potential and rhythmic hyperpolarizations are due to GK(Ca) in the intracapsular axon, either along the terminal or at the branching nodes, or both; and that the receptor potential contributes to, but is not the same as, the spindle potential.

Animals↗

PubMed2003

Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment.

Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends. In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis. We find that Kar9 localizes to the prospective daughter cell spindle pole. Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud. Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell. Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud. Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole. In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud. Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.

Actins↗

PubMed2001

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

Animals↗