Chromosome Movement in Mitosis Requires Microtubule Anchorage at Spindle Poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.

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ULTRASTRUCTURE AND TIME COURSE OF MITOSIS IN THE FUNGUS FUSARIUM OXYSPORUM

The Journal of Cell Biology ◽

10.1083/jcb.55.2.368 ◽

1972 ◽

Vol 55 (2) ◽

pp. 368-389 ◽

Cited By ~ 72

Author(s):

James R. Aist ◽

P. H. Williams

Keyword(s):

Electron Microscopy ◽

Fusarium Oxysporum ◽

Time Course ◽

Light And Electron Microscopy ◽

Spindle Pole Body ◽

Spindle Pole ◽

Metaphase Chromosomes ◽

Mitotic Apparatus ◽

Spindle Poles ◽

Spindle Microtubules

Mitosis in Fusarium oxysporum Schlect. was studied by light and electron microscopy. The average times required for the stages of mitosis, as determined from measurements made on living nuclei, were as follows: prophase, 70 sec; metaphase, 120 sec; anaphase, 13 sec; and telophase, 125 sec, for a total of 5.5 min. New postfixation procedures were developed specifically to preserve the fine-structure of the mitotic apparatus. Electron microscopy of mitotic nuclei revealed a fibrillo-granular, extranuclear Spindle Pole Body (SPB) at each pole of the intranuclear, microtubular spindles. Metaphase chromosomes were attached to spindle microtubules via kinetochores, which were found near the spindle poles at telophase. The still-intact, original nuclear envelope constricted around the incipient daughter nuclei during telophase.

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Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.488 ◽

1985 ◽

Vol 101 (2) ◽

pp. 488-499 ◽

Cited By ~ 48

Author(s):

M Vantard ◽

A M Lambert ◽

J De Mey ◽

P Picquot ◽

L J Van Eldik

Keyword(s):

Animal Cell ◽

Regulatory Mechanisms ◽

Present Observation ◽

Chromosome Movement ◽

Animal Cells ◽

Higher Plant ◽

Mitotic Apparatus ◽

Immunogold Staining ◽

Staining Methods ◽

Spindle Development

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

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The regulation of mitotic spindle function

Biochemistry and Cell Biology ◽

10.1139/o88-061 ◽

1988 ◽

Vol 66 (6) ◽

pp. 490-514 ◽

Cited By ~ 20

Author(s):

Stephen M. Wolniak

Keyword(s):

Morphological Changes ◽

Cytosolic Calcium ◽

Spindle Pole ◽

Chromosome Movement ◽

Mitotic Apparatus ◽

Physiological Regulation ◽

New Findings ◽

Controlling Elements ◽

Spindle Elongation ◽

Spindle Function

The process of mitosis includes a series of morphological changes in the cell in which the directional movements of chromosomes are the most prominent. The presence of a microtubular array, known as the spindle or mitotic apparatus, provides at least a scaffold upon which these movements take place. The precise mechanism for chromosome movement remains obscure, but new findings suggest that the kinetochore may play a key role in chromosome movement toward the spindle pole, and that sliding interactions between or among adjacent microtubules may provide the mechanochemical basis for spindle elongation. The physiological regulation of the anaphase motors and of spindle operation either before or after anaphase remains equally elusive. Elicitors that may serve as controlling elements in spindle function include shifts in cytosolic calcium activity and perhaps the activation or inactivation of protein kinases, which in turn produce changes in the state of phosphorylation of specific spindle components.

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The Drosophila Kinesin-13, KLP59D, Impacts Pacman- and Flux-based Chromosome Movement

Molecular Biology of the Cell ◽

10.1091/mbc.e09-07-0557 ◽

2009 ◽

Vol 20 (22) ◽

pp. 4696-4705 ◽

Cited By ~ 20

Author(s):

Uttama Rath ◽

Gregory C. Rogers ◽

Dongyan Tan ◽

Maria Ana Gomez-Ferreria ◽

Daniel W. Buster ◽

...

Keyword(s):

Electron Microscopy ◽

Family Member ◽

Somatic Cells ◽

Spindle Pole ◽

The Other ◽

Chromosome Movement ◽

Spindle Microtubule ◽

Important Regulator ◽

Chromosome Movements

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole–associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D's impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.

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BUB-1 promotes amphitelic chromosome biorientation via multiple activities at the kinetochore

eLife ◽

10.7554/elife.40690 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 2

Author(s):

Frances Edwards ◽

Gilliane Maton ◽

Nelly Gareil ◽

Julie C Canman ◽

Julien Dumont

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Underlying Mechanism ◽

Microtubule Assembly ◽

Spindle Poles ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Chromatid Segregation ◽

Associated Proteins ◽

Chromosome Attachment

Accurate chromosome segregation relies on bioriented amphitelic attachments of chromosomes to microtubules of the mitotic spindle, in which sister chromatids are connected to opposite spindle poles. BUB-1 is a protein of the Spindle Assembly Checkpoint (SAC) that coordinates chromosome attachment with anaphase onset. BUB-1 is also required for accurate sister chromatid segregation independently of its SAC function, but the underlying mechanism remains unclear. Here we show that, in Caenorhabditis elegans embryos, BUB-1 accelerates the establishment of non-merotelic end-on kinetochore-microtubule attachments by recruiting the RZZ complex and its downstream partner dynein-dynactin at the kinetochore. In parallel, BUB-1 limits attachment maturation by the SKA complex. This activity opposes kinetochore-microtubule attachment stabilisation promoted by CLS-2CLASP-dependent kinetochore-microtubule assembly. BUB-1 is therefore a SAC component that coordinates the function of multiple downstream kinetochore-associated proteins to ensure accurate chromosome segregation.

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Anti-tubulin antibodies locate the blepharoplast during spermatogenesis in the fern Platyzoma microphyllum R.Br.: a correlated immunofluorescence and electron-microscopic study

Journal of Cell Science ◽

10.1242/jcs.81.1.243 ◽

1986 ◽

Vol 81 (1) ◽

pp. 243-265

Author(s):

J.H. Doonan ◽

C.W. Lloyd ◽

J.G. Duckett

Keyword(s):

Basal Body ◽

De Novo ◽

Spindle Pole ◽

Electron Microscopic ◽

Mitotic Apparatus ◽

Strong Argument ◽

Body Formation ◽

Spindle Poles ◽

Tubulin Antibodies ◽

Cytoplasmic Microtubules

The discovery that the monoclonal anti-tubulin antibody YOL 1/34 recognizes a microtubule organizing centre, the blepharoplast (which arises de novo during the latter stages of spermatogenesis in the fern, Platyzoma microphyllum), has enabled us to follow it and associated microtubules throughout most of its ontogeny. By correlating electron-microscopic and immunofluorescence observations, YOL 1/34 is seen to stain the blepharoplast uniformly at a time when no microtubules are present within the organelle. Later, staining becomes intense at the surface, concomitant with the re-location of cylindrical channels to the periphery of the blepharoplast. During anaphase of the ultimate division of the spermatid mother cell the blepharoplast moves to the spindle poles and sharpens the otherwise barrel-shaped mitotic apparatus. Prior to this stage the blepharoplast is, however, off-centre and at variable positions around the poles. Later still, in the differentiating spermatids, the blepharoplast is the focus for radiating cytoplasmic microtubules that abut directly onto the electron-dense organelle, penetrating the ribosome-free halo. The three main conclusions are: that tubulin in a pre-microtubular form is associated with the cylindrical channels that arise de novo within the previously amorphous blepharoplast and act as a template in basal body formation; that the late appearance of the blepharoplast as a focus for the spindle poles during the final mitosis provides strong argument against its functioning during spindle pole initiation (despite its ability to sharpen the poles at anaphase); that the blepharoplast does seem to act as a microtubule organizing centre in the mitotically quiescent spermatid.

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Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms of Chromosome Congression and Anaphase Onset

The Journal of Cell Biology ◽

10.1083/jcb.137.7.1567 ◽

1997 ◽

Vol 137 (7) ◽

pp. 1567-1580 ◽

Cited By ~ 143

Author(s):

Bruce F. McEwen ◽

Amy B. Heagle ◽

Grisel O. Cassels ◽

Karolyn F. Buttle ◽

Conly L. Rieder

Keyword(s):

Time Course ◽

Spindle Pole ◽

Metaphase Chromosomes ◽

Spindle Poles ◽

Anaphase Onset ◽

Chromosome Congression ◽

Early Anaphase ◽

Full Complement ◽

Spindle Microtubules ◽

Chromosome Motion

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK1 cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late metaphase 24.3 ± 4.9 (n = 62), and at early anaphase 27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 μM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.

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Evidence for anaphase pulling forces during C. elegans meiosis

The Journal of Cell Biology ◽

10.1083/jcb.202005179 ◽

2020 ◽

Vol 219 (12) ◽

Cited By ~ 2

Author(s):

Brennan M. Danlasky ◽

Michelle T. Panzica ◽

Karen P. McNally ◽

Elizabeth Vargas ◽

Cynthia Bailey ◽

...

Keyword(s):

Spindle Pole ◽

Outer Face ◽

Chromosome Movement ◽

Female Meiosis ◽

Homologous Chromosomes ◽

C Elegans ◽

Spindle Poles ◽

Pulling Forces ◽

Spindle Elongation ◽

Anaphase B

Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.

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CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e14-09-1366 ◽

2015 ◽

Vol 26 (7) ◽

pp. 1225-1237 ◽

Cited By ~ 3

Author(s):

Shinya Ohta ◽

Laura Wood ◽

Iyo Toramoto ◽

Ken-Ichi Yagyu ◽

Tatsuo Fukagawa ◽

...

Keyword(s):

Chromosome Segregation ◽

Additional Data ◽

Weak Interactions ◽

Metaphase Plate ◽

Spindle Pole ◽

Animal Cells ◽

Central Spindle ◽

Spindle Poles ◽

Spindle Microtubules ◽

Bipolar Spindles

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.

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The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.72.2.368 ◽

1977 ◽

Vol 72 (2) ◽

pp. 368-379 ◽

Cited By ~ 39

Author(s):

S Brenner ◽

A Branch ◽

S Meredith ◽

M W Berns

Keyword(s):

Electron Microscopy ◽

Light And Electron Microscopy ◽

Time Lapse ◽

Spindle Pole ◽

Reduction Division ◽

Daughter Cells ◽

Spindle Poles ◽

Spindle Apparatus ◽

Time Lapse Photography

Light and electron microscopy were used to study somatic cell reduction division occurring spontaneously in tetraploid populations of rat kangaroo Potorous tridactylis (PtK2) cells in vitro. Light microscopy coupled with time-lapse photography documented the pattern of reduction division which includes an anaphase-like movement of double chromatid chromosomes to opposite spindle poles followed by the organization of two separate metaphase plates and synchronous anaphase division to form four poles and four daughter nuclei. The resulting daughter cells were isolated and cloned, showing their viability, and karyotyped to determine their ploidy. Ultrastructural analysis of cells undergoing reduction consistently revealed two duplexes of centrioles (one at each of two spindle poles) and two spindle poles in each cell that lacked centrioles but with microtubules terminating in a pericentriolar-like cloud of material. These results suggest that the centriole is not essential for spindle pole formation and division and implicate the could region as a necessary component of the spindle apparatus.

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