scholarly journals A Functional Relationship between NuMA and Kid Is Involved in Both Spindle Organization and Chromosome Alignment in Vertebrate Cells

2003 ◽  
Vol 14 (9) ◽  
pp. 3541-3552 ◽  
Author(s):  
Aime A. Levesque ◽  
Louisa Howard ◽  
Michael B. Gordon ◽  
Duane A. Compton
Keyword(s):  
Mammalian Cells ◽  
Functional Relationship ◽  
Spindle Pole ◽  
Chromosome Movement ◽  
Spindle Poles ◽  
Chromosome Alignment ◽  
Stark Contrast ◽  
Bipolar Spindles

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.

scholarly journals CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can Be Achieved via Redundant Mechanisms in Mammalian Cells

2001 ◽  
Vol 12 (9) ◽  
pp. 2776-2789 ◽  
Author(s):  
Bruce F. McEwen ◽  
Gordon K.T. Chan ◽  
Beata Zubrowski ◽  
Matthew S. Savoian ◽  
Matthew T. Sauer ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Localization ◽  
Mitotic Arrest ◽  
Spindle Pole ◽  
Critical Determinant ◽  
Microtubule Binding ◽  
Checkpoint Protein ◽  
Spindle Poles ◽  
Chromosome Position ◽  
Chromosome Alignment

CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with a mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescence, video, and electron microscopy to demonstrate that depletion of CENP-E from kinetochores via antibody microinjection reduces kinetochore microtubule binding by 23% at aligned chromosomes, and severely reduces microtubule binding at unaligned chromosomes. Disruption of CENP-E function also reduces tension across the centromere, increases the incidence of spindle pole fragmentation, and results in monooriented chromosomes approaching abnormally close to the spindle pole. Nevertheless, chromosomes show typical patterns of congression, fast poleward motion, and oscillatory motions. Furthermore, kinetochores of aligned and unaligned chromosomes exhibit normal patterns of checkpoint protein localization. These data are explained by a model in which redundant mechanisms enable kinetochore microtubule binding and checkpoint monitoring in the absence of CENP-E at kinetochores, but where reduced microtubule-binding efficiency, exacerbated by poor positioning at the spindle poles, results in chronically monooriented chromosomes and mitotic arrest. Chromosome position within the spindle appears to be a critical determinant of CENP-E function at kinetochores.

scholarly journals Direct kinetochore–spindle pole connections are not required for chromosome segregation

2014 ◽  
Vol 206 (2) ◽  
pp. 231-243 ◽  
Author(s):  
Vitali Sikirzhytski ◽  
Valentin Magidson ◽  
Jonathan B. Steinman ◽  
Jie He ◽  
Maël Le Berre ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Genetic Material ◽  
Spindle Pole ◽  
Chromosome Movement ◽  
Parallel Mechanisms ◽  
Laser Microbeam ◽  
Kinetochore Assembly ◽  
Spindle Poles

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes′ kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.

scholarly journals Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

2019 ◽  
Vol 30 (22) ◽  
pp. 2802-2813 ◽  
Author(s):  
Yutaka Shirasugi ◽  
Masamitsu Sato
Keyword(s):  
Fission Yeast ◽  
Motor Proteins ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Double Knockout ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Bipolar Spindles ◽  
Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-­dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

scholarly journals Aster-free spindle poles in insect spermatocytes: evidence for chromosome-induced spindle formation?

1986 ◽  
Vol 102 (5) ◽  
pp. 1679-1687 ◽  
Author(s):  
W Steffen ◽  
H Fuge ◽  
R Dietz ◽  
M Bastmeyer ◽  
G Müller
Keyword(s):  
Spindle Pole ◽  
Serial Sections ◽  
Spindle Formation ◽  
Microtubule Polymerization ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Lysis Buffer ◽  
Bipolar Spindles

Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.

scholarly journals Centriole number and the reproductive capacity of spindle poles.

1985 ◽  
Vol 100 (3) ◽  
pp. 887-896 ◽  
Author(s):  
G Sluder ◽  
C L Rieder
Keyword(s):  
Spindle Pole ◽  
Reproductive Capacity ◽  
Centriole Duplication ◽  
Voltage Electron ◽  
Spindle Poles ◽  
Mother And Daughter ◽  
Pericentriolar Material ◽  
History Of ◽  
Bipolar Spindles

The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.

Centrosomenfreie Spindelpole in Tipuliden-Spermatocyten

1959 ◽  
Vol 14 (12) ◽  
pp. 749-752b ◽  
Author(s):  
Roland Dietz
Keyword(s):  
Chromosome Movement ◽  
Experimental Conditions ◽  
Size And Shape ◽  
Spindle Poles ◽  
Multipolar Spindles ◽  
Stable Orientation ◽  
Bipolar Spindles

In spermatocytes I of the crane-fly Pales crocata under experimental conditions the normal relation between spindle poles and centrosomes can be disturbed. Bipolar spindles can be formed without participation of one or both centrosomes. Multipolar spindles often arise by formation of acentric (centrosomeless) poles in addition to the two centric ones. The effect of centric and acentric poles on chromosome movement is identical. Co-oriented bivalents which (in contrast to univalents and trivalents) exhibit stable orientation in normal spindles, are able to re-orient in multipolar figures. Size and shape of monocentric, bipolar spindles often do not differ from normal bicentric spindles and chromosome movement in them also is normal. Sometimes the spindles are not pointed at the acentric pole and occasionally fan-shaped monocentric, monopolar spindles are formed. Centrosomes which remain unconnected with the spindle keep more or less their diacinetic appearance.These and other results demonstrate that in tipuline spermatocytes the spindle is—at least mainly—organized by the chromosomes, the centrosomes having at best a subsidiary function in focussing the spindle poles.

scholarly journals Evidence for anaphase pulling forces during C. elegans meiosis

2020 ◽  
Vol 219 (12) ◽  
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.

scholarly journals Three-dimensional structure of the kinetochore-fibers in human mitotic spindles

2021 ◽  
Author(s):  
Robert Kiewisz ◽  
Gunar Fabig ◽  
William Conway ◽  
Daniel Needleman ◽  
Thomas Muller-Reichert
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Spindle Pole ◽  
Dimensional Structure ◽  
Biophysical Modeling ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Physical Linkage

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the spindle poles and the chromosomes. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers in detail, and we find that they exhibit remarkable variation. K-fibers display differences in circumference and KMT density along their length, with the pole-facing side showing a splayed-out appearance. We further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for models of KMT behavior and k-fiber self-organization as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2021). The presented data will also serve as a resource for further studies on mitosis in human cells.

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

2015 ◽  
Vol 26 (7) ◽  
pp. 1225-1237 ◽  
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.

scholarly journals Measurements of forces produced by the mitotic spindle using optical tweezers

2013 ◽  
Vol 24 (9) ◽  
pp. 1375-1386 ◽  
Author(s):  
Jessica Ferraro-Gideon ◽  
Rozhan Sheykhani ◽  
Qingyuan Zhu ◽  
Michelle L. Duquette ◽  
Michael W. Berns ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Mitotic Spindle ◽  
Theoretical Calculations ◽  
Single Point ◽  
Spindle Pole ◽  
Chromosome Movement ◽  
Speed Of Light ◽  
Spindle Poles ◽  
Ptk2 Cell ◽  
Chromosome Movements

We used a trapping laser to stop chromosome movements in Mesostoma and crane-fly spermatocytes and inward movements of spindle poles after laser cuts across Potorous tridactylus (rat kangaroo) kidney (PtK2) cell half-spindles. Mesostoma spermatocyte kinetochores execute oscillatory movements to and away from the spindle pole for 1–2 h, so we could trap kinetochores multiple times in the same spermatocyte. The trap was focused to a single point using a 63× oil immersion objective. Trap powers of 15–23 mW caused kinetochore oscillations to stop or decrease. Kinetochore oscillations resumed when the trap was released. In crane-fly spermatocytes trap powers of 56–85 mW stopped or slowed poleward chromosome movement. In PtK2 cells 8-mW trap power stopped the spindle pole from moving toward the equator. Forces in the traps were calculated using the equation F = Q′P/c, where P is the laser power and c is the speed of light. Use of appropriate Q′ coefficients gave the forces for stopping pole movements as 0.3–2.3 pN and for stopping chromosome movements in Mesostoma spermatocytes and crane-fly spermatocytes as 2–3 and 6–10 pN, respectively. These forces are close to theoretical calculations of forces causing chromosome movements but 100 times lower than the 700 pN measured previously in grasshopper spermatocytes.

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