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  • 2010-2014  (3)
  • 1
    Publication Date: 2022-05-25
    Description: © The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 23 (2012): 3133-3142, doi:10.1091/mbc.E12-04-0314.
    Description: We report on experiments directly in living cells that reveal the regulation of kinetochore function by tension. X and Y sex chromosomes in crane fly (Nephrotoma suturalis) spermatocytes exhibit an atypical segregation mechanism in which each univalent maintains K-fibers to both poles. During anaphase, each maintains a leading fiber (which shortens) to one pole and a trailing fiber (which elongates) to the other. We used this intriguing behavior to study the motile states that X-Y kinetochores are able to support during anaphase. We used a laser microbeam to either sever a univalent along the plane of sister chromatid cohesion or knock out one of a univalent's two kinetochores to release one or both from the resistive influence of its sister's K-fiber. Released kinetochores with attached chromosome arms moved poleward at rates at least two times faster than normal. Furthermore, fluorescent speckle microscopy revealed that detached kinetochores converted their functional state from reverse pac-man to pac-man motility as a consequence of their release from mechanical tension. We conclude that kinetochores can exhibit pac-man motility, even though their normal behavior is dominated by traction fiber mechanics. Unleashing of kinetochore motility through loss of resistive force is further evidence for the emerging model that kinetochores are subject to tension-sensitive regulation.
    Description: This work was supported by a grant to R.O. from the National Institute of Biomedical Imaging and Bioengineering (R01EB002045).
    Repository Name: Woods Hole Open Access Server
    Type: Article
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  • 2
    Publication Date: 2022-05-25
    Description: © The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 22 (2011): 4801-4808, doi:10.1091/mbc.E11-06-0494.
    Description: The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is “park,” in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.
    Description: This study was supported by a grant to R.O. from the National Institute of Biomedical Imaging and Bioengineering (R01EB002583-17).
    Repository Name: Woods Hole Open Access Server
    Type: Article
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  • 3
    Publication Date: 2022-05-26
    Description: © The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 25 (2014): 1437-1445, doi:10.1091/mbc.E14-01-0008.
    Description: We use liquid crystal polarized light imaging to record the life histories of single kinetochore (K-) fibers in living crane-fly spermatocytes, from their origins as nascent K-fibers in early prometaphase to their fully matured form at metaphase, just before anaphase onset. Increased image brightness due to increased retardance reveals where microtubules are added during K-fiber formation. Analysis of experimentally generated bipolar spindles with only one centrosome, as well as of regular, bicentrosomal spindles, reveals that microtubule addition occurs at the kinetochore-proximal ends of K-fibers, and added polymer expands poleward, giving rise to the robust K-fibers of metaphase cells. These results are not compatible with a model for K-fiber formation in which microtubules are added to nascent fibers solely by repetitive “search and capture” of centrosomal microtubule plus ends. Our interpretation is that capture of centrosomal microtubules—when deployed—is limited to early stages in establishment of nascent K-fibers, which then mature through kinetochore-driven outgrowth. When kinetochore capture of centrosomal microtubules is not used, the polar ends of K-fibers grow outward from their kinetochores and usually converge to make a centrosome-free pole.
    Description: This work was supported by Grant EB002045 from the National Institute of Biomedical Imaging and Bioengineering awarded to R.O.
    Repository Name: Woods Hole Open Access Server
    Type: Article
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