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Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles: implications for spindle mechanics.

Maddox P, Straight A, Coughlin P, Mitchison TJ, Salmon ED - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Division Group, Marine Biological Laboratory, Woods Hole, MA 02543, USA.

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.

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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: A–C, 5 μm; E and F, 2 μm.
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fig2: 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: A–C, 5 μm; E and F, 2 μm.

Mentions: Metaphase spindles with replicated chromosomes in Xenopus egg extracts (Desai et al., 1998) were labeled with a low level of X-rhodamine tubulin to produce fluorescent speckled microtubules (Waterman-Storer et al., 1998; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200301088/DC1), and also with a nonperturbing fluorescent antibody directed to the inner kinetochore protein CENP-A. Fig. 2 A shows one frame from a time-lapse sequence where optical sections were recorded at 10-s intervals using spinning-disk confocal microscopy. The open arrow in Fig. 2 A points to one pair of sister kinetochores kept in focus during the movie sequence. Note that spinning-disk confocal imaging resolved bundles of microtubules attached to kinetochores as well as bundles of nonkinetochore microtubules that pass by the chromosomes at the equator, and microtubule fluorescent speckles within these fibers (see also Fig. S1 and Video 1). Kinetochore microtubule bundles contain gaps in fluorescence between the plus ends of sister kinetochore fibers; gaps were absent in adjacent interpolar microtubule bundles (closed arrow). In electron micrographs, kinetochores exhibit a trilaminar structure with attached bundles of microtubules, typical of vertebrates (Fig. S2; Rieder and Salmon, 1998).


Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles: implications for spindle mechanics.

Maddox P, Straight A, Coughlin P, Mitchison TJ, Salmon ED - J. Cell Biol. (2003)

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: A–C, 5 μm; E and F, 2 μm.
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fig2: 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: A–C, 5 μm; E and F, 2 μm.
Mentions: Metaphase spindles with replicated chromosomes in Xenopus egg extracts (Desai et al., 1998) were labeled with a low level of X-rhodamine tubulin to produce fluorescent speckled microtubules (Waterman-Storer et al., 1998; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200301088/DC1), and also with a nonperturbing fluorescent antibody directed to the inner kinetochore protein CENP-A. Fig. 2 A shows one frame from a time-lapse sequence where optical sections were recorded at 10-s intervals using spinning-disk confocal microscopy. The open arrow in Fig. 2 A points to one pair of sister kinetochores kept in focus during the movie sequence. Note that spinning-disk confocal imaging resolved bundles of microtubules attached to kinetochores as well as bundles of nonkinetochore microtubules that pass by the chromosomes at the equator, and microtubule fluorescent speckles within these fibers (see also Fig. S1 and Video 1). Kinetochore microtubule bundles contain gaps in fluorescence between the plus ends of sister kinetochore fibers; gaps were absent in adjacent interpolar microtubule bundles (closed arrow). In electron micrographs, kinetochores exhibit a trilaminar structure with attached bundles of microtubules, typical of vertebrates (Fig. S2; Rieder and Salmon, 1998).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Cell Division Group, Marine Biological Laboratory, Woods Hole, MA 02543, USA.

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.

Show MeSH
Related in: MedlinePlus