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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/ 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).
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fig3: 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).

Mentions: Anaphase was initiated (Desai et al., 1998) and time-lapse series were recorded (Video 3) to test if Xenopus kinetochores pull their chromosomes poleward along stationary kinetochore microtubules (Fig. 1, model 1), become parked on the lattice of fluxing kinetochore microtubules (Fig. 1, model 2) or switch to depolymerization while flux persists (Fig. 1, model 3). Kymographs generated through aligned kinetochores and kinetochore fibers (Fig. 3, A and B) allowed measurement of polymerization (speckle slopes away from kinetochores) versus depolymerization (speckle slopes toward kinetochores) of microtubule plus ends at kinetochores (Fig. 3 C). Because the position of the right-hand sister kinetochore in Fig. 3 A was fixed in the alignment procedure, it appears as a vertical line in the kymograph, and the sister moved to the left after anaphase onset. In Fig. 3 B, the left-hand kinetochore was fixed, resulting in the sister moving to the right during anaphase. When sister chromosomes separated at anaphase onset, polymerization at the kinetochore slowed. When polymerization was slow enough, fluorescent speckles on kinetochore microtubules abruptly switched to movement toward the kinetochore, indicating microtubule depolymerization at kinetochores (Fig. 3, A and B). The velocity of plus end depolymerization added to the velocity of microtubule poleward flux, resulting in transient anaphase A rates greater than that of poleward flux. Variable switching between persistent polymerization and persistent depolymerization was observed for many kinetochores (Fig. 3 A). This switching likely accounts for the periods of fast and slow velocity seen in plots of kinetochore-to-pole movement, and likely explains why kinetochores move, on average, ∼40% faster than poleward flux of kinetochore microtubules in anaphase (Fig. 3, C and D). Only rarely and for brief periods did we see kinetochores appear to become fixed in position at the ends of their kinetochore microtubules in anaphase. Thus, model 3 is correct and the hypothetical park state does not play a significant role in spindle mechanics in this system.


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/ 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).
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fig3: 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).
Mentions: Anaphase was initiated (Desai et al., 1998) and time-lapse series were recorded (Video 3) to test if Xenopus kinetochores pull their chromosomes poleward along stationary kinetochore microtubules (Fig. 1, model 1), become parked on the lattice of fluxing kinetochore microtubules (Fig. 1, model 2) or switch to depolymerization while flux persists (Fig. 1, model 3). Kymographs generated through aligned kinetochores and kinetochore fibers (Fig. 3, A and B) allowed measurement of polymerization (speckle slopes away from kinetochores) versus depolymerization (speckle slopes toward kinetochores) of microtubule plus ends at kinetochores (Fig. 3 C). Because the position of the right-hand sister kinetochore in Fig. 3 A was fixed in the alignment procedure, it appears as a vertical line in the kymograph, and the sister moved to the left after anaphase onset. In Fig. 3 B, the left-hand kinetochore was fixed, resulting in the sister moving to the right during anaphase. When sister chromosomes separated at anaphase onset, polymerization at the kinetochore slowed. When polymerization was slow enough, fluorescent speckles on kinetochore microtubules abruptly switched to movement toward the kinetochore, indicating microtubule depolymerization at kinetochores (Fig. 3, A and B). The velocity of plus end depolymerization added to the velocity of microtubule poleward flux, resulting in transient anaphase A rates greater than that of poleward flux. Variable switching between persistent polymerization and persistent depolymerization was observed for many kinetochores (Fig. 3 A). This switching likely accounts for the periods of fast and slow velocity seen in plots of kinetochore-to-pole movement, and likely explains why kinetochores move, on average, ∼40% faster than poleward flux of kinetochore microtubules in anaphase (Fig. 3, C and D). Only rarely and for brief periods did we see kinetochores appear to become fixed in position at the ends of their kinetochore microtubules in anaphase. Thus, model 3 is correct and the hypothetical park state does not play a significant role in spindle mechanics in this system.

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