LaFountain James R.

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LaFountain

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James R.

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

( 2002-11) LaFountain, James R. ; Oldenbourg, Rudolf

The events of meiosis II in two living spermatocytes obtained from the testis of a crane-fly larva are recorded in this time-lapse sequence beginning at prophase II through telophase II to the near completion of cytokinesis following meiosis II.
Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes

(American Society for Cell Biology, 2014-02-26) LaFountain, James R. ; Oldenbourg, Rudolf

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

( 2002-11) LaFountain, James R. ; Oldenbourg, Rudolf

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.
Functional states of kinetochores revealed by laser microsurgery and fluorescent speckle microscopy

(American Society of Cell Biology, 2011-10-26) LaFountain, James R. ; Cohan, Christopher S. ; Oldenbourg, Rudolf

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.
Chromosome malorientations after meiosis II arrest cause nondisjunction

(American Society for Cell Biology, 2007-02-21) Janicke, Marie A. ; Lasko, Loren ; Oldenbourg, Rudolf ; LaFountain, James R.

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.
Maloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other

(American Society for Cell Biology, 2004-09-22) LaFountain, James R. ; Oldenbourg, Rudolf

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.
Pac-man motility of kinetochores unleashed by laser microsurgery

(American Society for Cell Biology, 2012-06-27) LaFountain, James R. ; Cohan, Christopher S. ; Oldenbourg, Rudolf

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

(Cambridge University Press, 2007-08-05) Shribak, Michael ; LaFountain, James R. ; Biggs, David ; Inoue, Shinya

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.

LaFountain James R.

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