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The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles.

Miyamoto DT, Perlman ZE, Burbank KS, Groen AC, Mitchison TJ - J. Cell Biol. (2004)

Bottom Line: This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation.Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate.Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. miyamoto@post.harvard.edu

ABSTRACT
Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

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Rapid automated measurements reveal the high variability of average flux rates in Xenopus extract spindles. (A) Schematic of cross-correlation algorithm (see Materials and methods). In brief, images (1) are filtered and background images (2) are subtracted to emphasize speckles (3), and the cross-correlation (4) is computed. The two peaks surrounding the cross-correlation maxima are fit to the sum of two Gaussians, allowing a subpixel resolution determination of the average rate of movement of speckle ensembles. (B and C) Validation of cross-correlation using simulated speckle data (see Materials and methods). See Video 4 for a sample movie. (B) Results of kymograph analysis of four sets of data simulated for a series of velocities. The linear least squares fit is described by y = 0.930x + 0.0705, R2 = 0.995. (C) Results of cross-correlation analysis of the same four sets of data. The linear least squares fit is described by y = 1.01x + .0122, R2 = 0.999. (D) Average flux rates in control spindles from four different extract days, measured using cross-correlation analysis. Each point represents a separate spindle.
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fig2: Rapid automated measurements reveal the high variability of average flux rates in Xenopus extract spindles. (A) Schematic of cross-correlation algorithm (see Materials and methods). In brief, images (1) are filtered and background images (2) are subtracted to emphasize speckles (3), and the cross-correlation (4) is computed. The two peaks surrounding the cross-correlation maxima are fit to the sum of two Gaussians, allowing a subpixel resolution determination of the average rate of movement of speckle ensembles. (B and C) Validation of cross-correlation using simulated speckle data (see Materials and methods). See Video 4 for a sample movie. (B) Results of kymograph analysis of four sets of data simulated for a series of velocities. The linear least squares fit is described by y = 0.930x + 0.0705, R2 = 0.995. (C) Results of cross-correlation analysis of the same four sets of data. The linear least squares fit is described by y = 1.01x + .0122, R2 = 0.999. (D) Average flux rates in control spindles from four different extract days, measured using cross-correlation analysis. Each point represents a separate spindle.

Mentions: We developed an automated method based on cross-correlation (Westerweel, 1997) to enable the rapid measurement of average whole spindle flux rates in large numbers of spindles. In brief, if a group of speckles in a spindle move poleward at similar velocities, their positions relative to each other will not vary over short times. The cross-correlation algorithm uses this persistent pattern to find the change in position of a group of speckles over some time interval. In a bipolar spindle with antiparallel microtubules sliding poleward, two populations of speckles move away from each other. This results in two cross-correlation maxima, and the distance between these reports on the average flux rate (Fig. 2 A and see Materials and methods). The average flux rates measured by cross-correlation and estimated from kymography were similar in a wide range of control and Eg5-inhibited spindles (Fig. 1, B and C, E and F, and H and I; and Fig. 2, B and C). This fast ensemble-based method samples a larger data set from each spindle than manual kymography and can analyze short movies to calculate speckle velocities (20 s for control spindles and 90 s for slowed spindles), whereas visual kymograph analysis generally requires significantly longer movies. Thus, we were able to considerably increase the number of spindles and perturbation conditions we could sample and accurately measure in each experiment.


The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles.

Miyamoto DT, Perlman ZE, Burbank KS, Groen AC, Mitchison TJ - J. Cell Biol. (2004)

Rapid automated measurements reveal the high variability of average flux rates in Xenopus extract spindles. (A) Schematic of cross-correlation algorithm (see Materials and methods). In brief, images (1) are filtered and background images (2) are subtracted to emphasize speckles (3), and the cross-correlation (4) is computed. The two peaks surrounding the cross-correlation maxima are fit to the sum of two Gaussians, allowing a subpixel resolution determination of the average rate of movement of speckle ensembles. (B and C) Validation of cross-correlation using simulated speckle data (see Materials and methods). See Video 4 for a sample movie. (B) Results of kymograph analysis of four sets of data simulated for a series of velocities. The linear least squares fit is described by y = 0.930x + 0.0705, R2 = 0.995. (C) Results of cross-correlation analysis of the same four sets of data. The linear least squares fit is described by y = 1.01x + .0122, R2 = 0.999. (D) Average flux rates in control spindles from four different extract days, measured using cross-correlation analysis. Each point represents a separate spindle.
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Related In: Results  -  Collection

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fig2: Rapid automated measurements reveal the high variability of average flux rates in Xenopus extract spindles. (A) Schematic of cross-correlation algorithm (see Materials and methods). In brief, images (1) are filtered and background images (2) are subtracted to emphasize speckles (3), and the cross-correlation (4) is computed. The two peaks surrounding the cross-correlation maxima are fit to the sum of two Gaussians, allowing a subpixel resolution determination of the average rate of movement of speckle ensembles. (B and C) Validation of cross-correlation using simulated speckle data (see Materials and methods). See Video 4 for a sample movie. (B) Results of kymograph analysis of four sets of data simulated for a series of velocities. The linear least squares fit is described by y = 0.930x + 0.0705, R2 = 0.995. (C) Results of cross-correlation analysis of the same four sets of data. The linear least squares fit is described by y = 1.01x + .0122, R2 = 0.999. (D) Average flux rates in control spindles from four different extract days, measured using cross-correlation analysis. Each point represents a separate spindle.
Mentions: We developed an automated method based on cross-correlation (Westerweel, 1997) to enable the rapid measurement of average whole spindle flux rates in large numbers of spindles. In brief, if a group of speckles in a spindle move poleward at similar velocities, their positions relative to each other will not vary over short times. The cross-correlation algorithm uses this persistent pattern to find the change in position of a group of speckles over some time interval. In a bipolar spindle with antiparallel microtubules sliding poleward, two populations of speckles move away from each other. This results in two cross-correlation maxima, and the distance between these reports on the average flux rate (Fig. 2 A and see Materials and methods). The average flux rates measured by cross-correlation and estimated from kymography were similar in a wide range of control and Eg5-inhibited spindles (Fig. 1, B and C, E and F, and H and I; and Fig. 2, B and C). This fast ensemble-based method samples a larger data set from each spindle than manual kymography and can analyze short movies to calculate speckle velocities (20 s for control spindles and 90 s for slowed spindles), whereas visual kymograph analysis generally requires significantly longer movies. Thus, we were able to considerably increase the number of spindles and perturbation conditions we could sample and accurately measure in each experiment.

Bottom Line: This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation.Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate.Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. miyamoto@post.harvard.edu

ABSTRACT
Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

Show MeSH