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  • American Society for Cell Biology  (4)
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  • 1
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    Pac-man motility of kinetochores unleashed by laser microsurgery (2012)
    American Society for Cell Biology
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    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
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  • 2
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    Maloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other (2007)
    American Society for Cell Biology
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    Publication Date: 2022-05-25
    Description: Author Posting. © American Society for Cell Biology, 2004. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 15 (2004): 5346-5355, doi:10.1091/mbc.E04-06-0524.
    Description: To test the "traction fiber" model for metaphase positioning of bivalents during meiosis, kinetochore fibers of maloriented bivalents, induced during recovery from cold arrest, were analyzed with a liquid crystal polarizing microscope. The measured birefringence retardation of kinetochore fibers is proportional to the number of microtubules in a fiber. Five of the 11 maloriented bivalents analyzed exhibited bipolar malorientations that had at least four times more kinetochore microtubules to one pole than to the other pole, and two had microtubules directed to only one pole. Yet all maloriented bivalents had positions at or near the spindle equator. The traction fiber model predicts such maloriented bivalents should be positioned closer to the pole with more kinetochore microtubules. A metaphase position at the spindle equator, according to the model, requires equal numbers of kinetochore microtubules to both poles. Data from polarizing microscope images were not in accord with those predictions, leading to the conclusion that other factors, in addition to traction forces, must be involved in metaphase positioning in crane-fly spermatocytes. Although the identity of additional factors has not been established, one possibility is that polar ejection forces operate to exert away-from-the-pole forces that could counteract pole-directed traction forces. Another is that kinetochores are "smart," meaning they embody a position-sensitive mechanism that controls their activity.
    Description: J.R.L. is supported by grant MCB-0235934 from the National Science Foundation. R.O. is supported by grants GM49210 from the National Institute of General Medical Sciences and EB002045 from the National Institute of Biomedical Imaging and Bioengineering.
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  • 3
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    Chromosome malorientations after meiosis II arrest cause nondisjunction (2007)
    American Society for Cell Biology
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    Publication Date: 2022-05-25
    Description: Author Posting. © American Society for Cell Biology, 2007. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 18 (2007): 1645-1656, doi:10.1091/mbc.E06-10-0963.
    Description: This study investigated the basis of meiosis II nondisjunction. Cold arrest induced a fraction of meiosis II crane fly spermatocytes to form (n + 1) and (n – 1) daughters during recovery. Live-cell liquid crystal polarized light microscope imaging showed nondisjunction was caused by chromosome malorientation. Whereas amphitely (sister kinetochore fibers to opposite poles) is normal, cold recovery induced anaphase syntely (sister fibers to the same pole) and merotely (fibers to both poles from 1 kinetochore). Maloriented chromosomes had stable metaphase positions near the equator or between the equator and a pole. Syntelics were at the spindle periphery at metaphase; their sisters disconnected at anaphase and moved all the way to a centrosome, as their strongly birefringent kinetochore fibers shortened. The kinetochore fibers of merotelics shortened little if any during anaphase, making anaphase lag common. If one fiber of a merotelic was more birefringent than the other, the less birefringent fiber lengthened with anaphase spindle elongation, often permitting inclusion of merotelics in a daughter nucleus. Meroamphitely (near amphitely but with some merotely) caused sisters to move in opposite directions. In contrast, syntely and merosyntely (near syntely but with some merotely) resulted in nondisjunction. Anaphase malorientations were more frequent after longer arrests, with particularly long arrests required to induce syntely and merosyntely.
    Description: This study was supported by National Science Foundation grant MCB-0235934 (to J.L.) and grants GM 49210 from the National Institute of General Medical Sciences and EB002045 from the National Institutes of Biomedical Imaging and Bioengineering (to R.O.).
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  • 4
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    Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes (2014)
    American Society for Cell Biology
    Details
    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
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