Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract??? Microtubule plus ends dynamically attach to kinetochores on mitotic chromosomes. We directly imaged this dynamic interface using high resolution fluorescent speckle microscopy and direct labeling of kinetochores in Xenopus extract spindles. During metaphase, kinetochores were stationary and under tension while plus end polymerization and poleward microtubule flux (flux) occurred at velocities varying from 1.5-2.5 micro m/min. Because kinetochore microtubules polymerize at metaphase kinetochores, the primary source of kinetochore tension must be the spindle forces that produce flux and not a kinetochore-based mechanism. We infer that the kinetochore resists translocation of kinetochore microtubules through their attachment sites, and that the polymerization state of the kinetochore acts a "slip-clutch" mechanism that prevents detachment at high tension. At anaphase onset, kinetochores switched to depolymerization of microtubule plus ends, resulting in chromosome-to-pole rates transiently greater than flux. Kinetochores switched from persistent depolymerization to persistent polymerization and back again during anaphase, bistability exhibited by kinetochores in vertebrate tissue cells. These results provide the most complete description of spindle microtubule poleward flux to date, with important implications for the microtubule-kinetochore interface and for how flux regulates kinetochore function.
Figure 1. Three models based on different contributions of kinetochore motility and kinetochore microtubule flux to metaphase kinetochore tension and anaphase A. For each model, only a single kinetochore microtubule (KMT) is shown with the polar minus end (â) at the left and the plus end (+) attached to the kinetochore on the right. Arrows indicate sites of polymerization or depolymerization. Metaphase includes sequential times, t1 and t2; anaphase onset occurs at t2 and continues for sequential times t2, t3, and t4. At metaphase, the centromeric linkage between sister kinetochores and polar ejection forces on the arms support tension at kinetochores (large blue arrow). Tension is lost at anaphase onset when sisters separate and the polar ejection forces on the arms are inactivated (Funabiki and Murray, 2000). See text for details.
Figure 2. Confocal FSM of microtubule polymerization at metaphase kinetochores and centromere stretch. (A) Selected frame from a time-lapse movie showing the polymerization at kinetochores labeled with fluorescent CENP-A antibodies (red) and the poleward flux of kinetochore microtubule fluorescent speckles (green). The open arrow marks a kinetochore pair that was aligned for kymograph analysis (see Materials and methods); closed arrow marks a nonkinetochore fiber. (B) Kymograph of the kinetochore pair marked with open arrow in A. The kinetochores move relatively little with respect to each other while speckles on microtubules appear at the kinetochores and flux poleward. Flux velocity is proportional to the slope of the speckle trajectories away from the vertical direction in B. (C) Kymograph of nonkinetochore microtubules shows that speckles move poleward at similar rates in all microtubules. Note the gap of tubulin fluorescence between the sister kinetochores in B. There is no such gap for the interpolar bundles of microtubules (C). For the interpolar fibers, fluorescent speckle trajectories are seen toward both poles at most positions along the fiber, in contrast to kinetochore fibers, which exhibit trajectories primarily toward the pole faced by the kinetochore. D shows a histogram of flux velocities for kinetochore fibers (red) and for interpolar spindle fibers (green) obtained from the slopes of kymographs such as those in B and C. Note they have a similar distribution, with nonkinetochore flux being slightly faster. Colored arrows point to average values (see text). Image of sister kinetochores labeled with fluorescent CENP-A antibody in unfixed extracts at metaphase (E) or where spindles were disassembled with 10 μM nocodazole (F). Bars: AâC, 5 μm; E and F, 2 μm.
Figure 3. Confocal FSM of microtubule polymerization/ depolymerization at anaphase kinetochores. Time-lapse images were aligned so that a selected kinetochore was fixed in position relative to the rest of the spindle (see text). A and B are kymographs made from two different aligned spindles. Dotted white lines in each kymograph highlight speckle movements relative to the aligned kinetochores. In both examples, polymerization at the kinetochores slows as sisters begin to separate in anaphase (e.g., slopes of black lines in A become more vertical). When polymerization is slow enough, the kinetochore switches to depolymerization, where speckles are seen to move toward and disappear at the kinetochores. The kinetochore in A persists in depolymerization, whereas the kinetochore in B switches back to polymerization during the interval analyzed. (C) Histograms of velocities measured for polymerization and depolymerization at kinetochores and flux during anaphase. Arrows mark the average values. Depolymerization = 1.2 ± 0.6 μm/min (n = 27); polymerization = 0.9 ± 0.3 μm/min (n = 24 measurements); flux = 1.6 ± 0.4 μm/min (n = 27). We were unable to obtain values as chromosomes approached their poles because of the curvature of the kinetochore fibers. (D) Poleward movements of three kinetochores during the first two thirds of anaphase A. Notice that the kinetochores exhibit asynchronous periods of fast and slow movement. The average velocity over this period was 2.4 μm/min (n = 20 kinetochores from six spindles).
Figure 4. Updated models for the kinetochoreâmicrotubule interface that include contributions from flux. Drawings in AâC are modified from Rieder and Salmon (1998). OP is a cross section of one microtubule attachment site in the outer plate of the kinetochore, and IP is a cross section of the inner plate. The stretch of the centromere beyond its rest length indicates the tension generated. Microtubule attachment and resistance to translocation through the attachment site may be provided by the microtubule motors CENP-E and cytoplasmic dynein, the nonmotor microtubuleâbinding domain of CENP-E, the microtubule binding domain of the p150 component of the dynactin complex bound to dynein, and unknown microtubule-binding proteins within the attachment site (see text for details).
Brust-Mascher,
Microtubule flux and sliding in mitotic spindles of Drosophila embryos.
2002, Pubmed
Brust-Mascher,
Microtubule flux and sliding in mitotic spindles of Drosophila embryos.
2002,
Pubmed
Desai,
Anaphase A chromosome movement and poleward spindle microtubule flux occur At similar rates in Xenopus extract spindles.
1998,
Pubmed
,
Xenbase
Funabiki,
The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement.
2000,
Pubmed
,
Xenbase
Grego,
Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.
2001,
Pubmed
He,
Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast.
2000,
Pubmed
Hill,
Theoretical problems related to the attachment of microtubules to kinetochores.
1985,
Pubmed
Inoué,
Force generation by microtubule assembly/disassembly in mitosis and related movements.
1995,
Pubmed
Kapoor,
Searching for the middle ground: mechanisms of chromosome alignment during mitosis.
2002,
Pubmed
Maddox,
Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos.
2002,
Pubmed
Maddox,
The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae.
2000,
Pubmed
Maddox,
Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells.
2003,
Pubmed
Mallavarapu,
A switch in microtubule dynamics at the onset of anaphase B in the mitotic spindle of Schizosaccharomyces pombe.
1999,
Pubmed
Margolis,
Microtubule treadmills--possible molecular machinery.
1981,
Pubmed
Mitchison,
Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence.
1989,
Pubmed
Mitchison,
Mitosis: a history of division.
2001,
Pubmed
Pearson,
Budding yeast chromosome structure and dynamics during mitosis.
2001,
Pubmed
Rieder,
The vertebrate cell kinetochore and its roles during mitosis.
1998,
Pubmed
Skibbens,
Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push-pull mechanism.
1993,
Pubmed
Tirnauer,
EB1 targets to kinetochores with attached, polymerizing microtubules.
2002,
Pubmed
Waterman-Storer,
Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.
1998,
Pubmed
,
Xenbase
Waters,
The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work.
1996,
Pubmed
