Probing the mechanical architecture of the vertebrate meiotic spindle

Itabashi, Takeshi; Takagi, Jun; Shimamoto, Yuta; Onoe, Hiroaki; Kuwana, Kazunori; Shimoyama, Isao; Gaetz, Jedidiah; Kapoor, Tarun M.; Ishiwata, Shin’ichi

doi:10.1038/nmeth.1297

Cited by 71 publications

(74 citation statements)

References 21 publications

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“…In contrast to physical manipulation of flattened cells that have various shapes during cell division (16, 32), metaphase HeLa cells, which become spherical during mitosis due to a "rounding up" force (6), enabled us to not only compress but also stretch the mitotic spindle by applying the external force from orthogonal directions. Although morphological changes in the mitotic spindle were indirectly induced by the mechanical perturbation applied from the outside of the cell, the deformed shape of the spindle was similar to that obtained by directly compressing the meiotic spindle assembled in the cytoplasmic extracts (21). Moreover, the changes in the intercentromere distance depended on the direction and the extent of compression.…”

Section: Discussionmentioning

confidence: 52%

“…For compressing a cell, we used plate-like microfabricated cantilevers (20,21) (Fig. 1A), between which a metaphase HeLa cell expressing EGFP-tagged histone H2B was sandwiched (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The controlled mechanical perturbation of HeLa cells was performed using a dual cantilever-based system coupled with fluorescence microscopic imaging (21). The timeline of an experiment is shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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Mechanical impulses can control metaphase progression in a mammalian cell

Itabashi¹,

Terada

Kuwana

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Chromosome segregation machinery is controlled by mechanochemical regulation. Tension in a mitotic spindle, which is balanced by molecular motors and polymerization-depolymerization dynamics of microtubules, is thought to be essential for determining the timing of chromosome segregation after the establishment of the kinetochore-microtubule attachments. It is not known, however, whether and how applied mechanical forces modulate the tension balance and chemically affect the molecular processes involved in chromosome segregation. Here we found that a mechanical impulse externally applied to mitotic HeLa cells alters the balance of forces within the mitotic spindle. We identified two distinct mitotic responses to the applied mechanical force that either facilitate or delay anaphase onset, depending on the direction of force and the extent of cell compression. An external mechanical impulse that physically increases tension within the mitotic spindle accelerates anaphase onset, and this is attributed to the facilitation of physical cleavage of sister chromatid cohesion. On the other hand, a decrease in tension activates the spindle assembly checkpoint, which impedes the degradation of mitotic proteins and delays the timing of chromosome segregation. Thus, the external mechanical force acts as a crucial regulator for metaphase progression, modulating the internal force balance and thereby triggering specific mechanochemical cellular reactions.chromosome segregation | mechanobiology | mitotic force | mitotic spindle | spindle assembly checkpoint

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Section: Discussionmentioning

confidence: 52%

“…For compressing a cell, we used plate-like microfabricated cantilevers (20,21) (Fig. 1A), between which a metaphase HeLa cell expressing EGFP-tagged histone H2B was sandwiched (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Mechanical impulses can control metaphase progression in a mammalian cell

Itabashi¹,

Terada

Kuwana

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition to heuristic arguments, there is growing experimental evidence that the mitotic spindle is a viscoelastic-coupled structure mediated by microtubule crosslinking (Charlebois et al 2011;Shimamoto et al 2011;Itabashi et al 2009). Putative mechanical couplers in the mitotic spindle include the variety of microtubule cross-linking proteins like NuMA, Dynein/Dynactin, HSET/kinesin-14, or Eg5/kinesin-5, which are known to establish connections between spindle microtubules (Walczak and Heald 2008).…”

Section: Coupling and The Maturation Of Kinetochoremicrotubule Attachmentioning

confidence: 99%

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

Pereira

Maiato

2012

Chromosome Res

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Chromosome positioning at the equator of the mitotic spindle emerges out of a relatively entropic background. At this moment, termed metaphase, all kinetochores have typically captured microtubules leading to satisfaction of the spindle-assembly checkpoint, but the cell does not enter anaphase immediately. The waiting time in metaphase is related to the kinetics of securin and cyclin B1 degradation, which trigger sisterchromatid separation and promote anaphase processivity, respectively. Yet, as judged by metaphase duration, such kinetics vary widely between cell types and organisms, with no evident correlation to ploidy or cell size. During metaphase, many animal and plant spindles are also characterized by a conspicuous "flux" activity characterized by continuous poleward translocation of spindle microtubules, which maintain steady-state length and position. Whether spindle microtubule flux plays a specific role during metaphase remains arguable. Based on known experimental parameters, we have performed a comparative analysis amongst different cell types from different organisms and show that spindle length, metaphase duration and flux velocity combine within each system to obey a quasi-universal rule. As so, knowledge of two of these parameters is enough to estimate the third. This trend indicates that metaphase duration is tuned to allow approximately one kinetochore-to-pole round of microtubule flux. We propose that the time cells spend in metaphase evolved as a quality enhancement step that allows for the uniform stabilization/correction of kinetochore-microtubule attachments, thereby promoting mitotic fidelity.

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“…Recently, this has been taken to quantitative levels, examining the exact quantity of chromatin required to make a mitotic spindle, and the effect that chromatin shape has on the resulting spindles through lithographic micropatterning (Dinarina et al 2009). Other advanced techniques in Xenopus, such as the use of piezoresistive dual cantilevers to quantitatively measure the mechanical forces intrinsic to in vitro spindles may, when combined with pathway disruption through immunodepletion, assist in teasing apart the properties of populations of MTs generated by different pathways (Itabashi et al 2009).…”

Section: Part Iii: Towards An Understanding Of Spindle Formationmentioning

confidence: 99%

50 ways to build a spindle: the complexity of microtubule generation during mitosis

Duncan

Wakefield

2011

Chromosome Res

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The accurate segregation of duplicated chromosomes, essential for the development and viability of a eukaryotic organism, requires the formation of a robust microtubule (MT)-based spindle apparatus. Entry into mitosis or meiosis precipitates a cascade of signalling events which result in the activation of pathways responsible for a dramatic reorganisation of the MT cytoskeleton: through changes in the properties of MT-associated proteins, local concentrations of free tubulin dimer and through enhanced MT nucleation. The latter is generally thought to be driven by localisation and activation of γ-tubulin-containing complexes (γ-TuSC and γ-TuRC) at specific subcellular locations. For example, upon entering mitosis, animal cells concentrate γ-tubulin at centrosomes to tenfold the normal level during interphase, resulting in an aster-driven search and capture of chromosomes and bipolar mitotic spindle formation. Thus, in these cells, centrosomes have traditionally been perceived as the primary microtubule organising centre during spindle formation. However, studies in meiotic cells, plants and cell-free extracts have revealed the existence of complementary mechanisms of spindle formation, mitotic chromatin, kinetochores and nucleation from existing MTs or the cytoplasm can all contribute to a bipolar spindle apparatus. Here, we outline the individual known mechanisms responsible for spindle formation and formulate ideas regarding the relationship between them in assembling a functional spindle apparatus.

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Probing the mechanical architecture of the vertebrate meiotic spindle

Cited by 71 publications

References 21 publications

Mechanical impulses can control metaphase progression in a mammalian cell

Mechanical impulses can control metaphase progression in a mammalian cell

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

50 ways to build a spindle: the complexity of microtubule generation during mitosis

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