Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes

© 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.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.This work was supported by Grant EB002045 from the National Institute of Biomedical Imaging and Bioengineering awarded to R.O

Time lapse movie of meiosis I in a living spermatocyte from the crane fly, Nephrotoma suturalis, viewed with polarized light microscopy

The events of meiosis I in a living spermatocyte obtained from the testis of a crane-fly larva are recorded in this time-lapse sequence beginning at diakinesis through telophase to the near completion of cytokinesis following meiosis I

Pac-man motility of kinetochores unleashed by laser microsurgery

© 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.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.This work was supported by a grant to R.O. from the National Institute of Biomedical Imaging and Bioengineering (R01EB002045)

Quantitative orientation-independent differential interference contrast (DIC) microscopy coupled with orientation-independent Polarization microscopy

Author Posting. © Microscopy Society of America, 2007. This article is posted here by permission of Cambridge University Press for personal use, not for redistribution. The definitive version was published in Microscopy and Microanalysis 13 Suppl. 2 (2007): 10-11, doi:10.1017/S1431927607075186.Differential interference contrast (DIC) microscopy is widely used to observe structure and motion in unstained, transparent living cells and isolated organelles, producing a monochromatic shadowcast image of optical phase gradient. Polarized light microscopy (Pol) reveals structural anisotropy due to form birefringence, intrinsic birefringence, stress birefringence, etc. DIC and Pol complement each other as, for example, in a live dividing cell, the DIC image will clearly show the chromosomes while the Pol image will depict the distribution of the birefringent microtubules in the spindle. Both methods, however, have the same shortcomings: they require the proper orientation of a specimen in relation to the optical system in order to achieve best results

Filtered screens and augmented Teichmüller space

We study a new bordification of the decorated Teichmüller space for a multiply punctured surface F by a space of filtered screens on the surface that arises from a natural elaboration of earlier work of McShane–Penner. We identify necessary and sufficient conditions for paths in this space of filtered screens to yield short curves having vanishing length in the underlying surface F . As a result, an appropriate quotient of this space of filtered screens on F yields a decorated augmented Teichmüller space which is shown to admit a CW decomposition that naturally projects to the augmented Teichmüller space by forgetting decorations and whose strata are indexed by a new object termed partially oriented stratum graphs

Filtered screens and augmented Teichm\"uller space

We study a new bordification of the decorated Teichm\"uller space for a multiply punctured surface F by a space of filtered screens on the surface that arises from a natural elaboration of earlier work of McShane-Penner. We identify necessary and sufficient conditions for paths in this space of filtered screens to yield short curves having vanishing length in the underlying surface F. As a result, an appropriate quotient of this space of filtered screens on F yields a decorated augmented Teichm\"uller space which is shown to admit a CW decomposition that naturally projects to the augmented Teichm\"uller space by forgetting decorations and whose strata are indexed by a new object termed partially oriented stratum graphs.Comment: Final version to appear in Geometriae Dedicat

Mesoproterozoic surface oxygenation accompanied major sedimentary manganese deposition at 1.4 and 1.1 Ga

This research was funded by the Australian Science and Industry Endowment Fund (SIEF) as part of The Distal Footprints of Giant Ore Systems: UNCOVER Australia Project (RP04-063)—Capricorn Distal Footprints. EAS also thanks the donors of The American Chemical Society Petroleum Research Fund for partial support of this research (61017-ND2).Peer reviewedPublisher PD