Limits...
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.

Show MeSH
Pharmacological inhibition of Eg5 decreases the flux rate in a dose–responsive manner. (A) First frame of a FSM movie showing labeled tubulin in a metaphase spindle in the presence of 1% DMSO (control; see Video 1). Bar, 10 μm. Blue lines outline the region for which kymograph analysis (B) was performed. Red dotted lines are representative traces of speckle streaks, revealing flux. Average flux rate estimated by kymography is 2.21 ± 0.45 μm/min. Bar, 10 μm. (C, left) Average cross-correlation graph for the spindle in A for intervals of 25 s between frame pairs. The two peaks reflect two speckle populations that moved apart due to poleward flux. (right) Blue line is the correlation value along the plane bisecting the two maxima of the cross-correlation graph. Red line is the best fit curve using a two-Gaussian distribution. The distance between the centers of the two peaks reports on the flux rate. Average flux rate measured by cross-correlation is 1.97 ± 0.16 μm/min. (D–F) Same as A–C, in the presence of 25 μM (S)-quinazolinone (see Video 2). Flux rate is slowed (1.32 ± 0.37 μm/min by kymography, 1.02 ± 0.01 μm/min by cross-correlation). (G–I) Same as A–C, in the presence of 100 μM (S)-quinazolinone (see Video 3). Flux rate is almost completely inhibited (0.32 ± 0.33 μm/min by kymography, <0.2 μm/min by cross-correlation). Videos are available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172449&req=5

fig1: Pharmacological inhibition of Eg5 decreases the flux rate in a dose–responsive manner. (A) First frame of a FSM movie showing labeled tubulin in a metaphase spindle in the presence of 1% DMSO (control; see Video 1). Bar, 10 μm. Blue lines outline the region for which kymograph analysis (B) was performed. Red dotted lines are representative traces of speckle streaks, revealing flux. Average flux rate estimated by kymography is 2.21 ± 0.45 μm/min. Bar, 10 μm. (C, left) Average cross-correlation graph for the spindle in A for intervals of 25 s between frame pairs. The two peaks reflect two speckle populations that moved apart due to poleward flux. (right) Blue line is the correlation value along the plane bisecting the two maxima of the cross-correlation graph. Red line is the best fit curve using a two-Gaussian distribution. The distance between the centers of the two peaks reports on the flux rate. Average flux rate measured by cross-correlation is 1.97 ± 0.16 μm/min. (D–F) Same as A–C, in the presence of 25 μM (S)-quinazolinone (see Video 2). Flux rate is slowed (1.32 ± 0.37 μm/min by kymography, 1.02 ± 0.01 μm/min by cross-correlation). (G–I) Same as A–C, in the presence of 100 μM (S)-quinazolinone (see Video 3). Flux rate is almost completely inhibited (0.32 ± 0.33 μm/min by kymography, <0.2 μm/min by cross-correlation). Videos are available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1.

Mentions: We used time-lapse fluorescent speckle microscopy (FSM; Waterman-Storer et al., 1998) to visualize the poleward movement of microtubules in metaphase spindles assembled in cell-free Xenopus egg extracts (Desai et al., 1999; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1). We first estimated flux velocities in the presence of Eg5 inhibitors by kymography (Waterman-Storer et al., 1998), in which intensities of speckles along a line in an image are used to generate a distance versus time plot from a time-lapse movie (Fig. 1, A and B). To maintain spindle bipolarity, we relied on the resistance of spindles to collapse in standard coverslip squashes prepared immediately after drug addition (Kapoor and Mitchison, 2001).


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)

Pharmacological inhibition of Eg5 decreases the flux rate in a dose–responsive manner. (A) First frame of a FSM movie showing labeled tubulin in a metaphase spindle in the presence of 1% DMSO (control; see Video 1). Bar, 10 μm. Blue lines outline the region for which kymograph analysis (B) was performed. Red dotted lines are representative traces of speckle streaks, revealing flux. Average flux rate estimated by kymography is 2.21 ± 0.45 μm/min. Bar, 10 μm. (C, left) Average cross-correlation graph for the spindle in A for intervals of 25 s between frame pairs. The two peaks reflect two speckle populations that moved apart due to poleward flux. (right) Blue line is the correlation value along the plane bisecting the two maxima of the cross-correlation graph. Red line is the best fit curve using a two-Gaussian distribution. The distance between the centers of the two peaks reports on the flux rate. Average flux rate measured by cross-correlation is 1.97 ± 0.16 μm/min. (D–F) Same as A–C, in the presence of 25 μM (S)-quinazolinone (see Video 2). Flux rate is slowed (1.32 ± 0.37 μm/min by kymography, 1.02 ± 0.01 μm/min by cross-correlation). (G–I) Same as A–C, in the presence of 100 μM (S)-quinazolinone (see Video 3). Flux rate is almost completely inhibited (0.32 ± 0.33 μm/min by kymography, <0.2 μm/min by cross-correlation). Videos are available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172449&req=5

fig1: Pharmacological inhibition of Eg5 decreases the flux rate in a dose–responsive manner. (A) First frame of a FSM movie showing labeled tubulin in a metaphase spindle in the presence of 1% DMSO (control; see Video 1). Bar, 10 μm. Blue lines outline the region for which kymograph analysis (B) was performed. Red dotted lines are representative traces of speckle streaks, revealing flux. Average flux rate estimated by kymography is 2.21 ± 0.45 μm/min. Bar, 10 μm. (C, left) Average cross-correlation graph for the spindle in A for intervals of 25 s between frame pairs. The two peaks reflect two speckle populations that moved apart due to poleward flux. (right) Blue line is the correlation value along the plane bisecting the two maxima of the cross-correlation graph. Red line is the best fit curve using a two-Gaussian distribution. The distance between the centers of the two peaks reports on the flux rate. Average flux rate measured by cross-correlation is 1.97 ± 0.16 μm/min. (D–F) Same as A–C, in the presence of 25 μM (S)-quinazolinone (see Video 2). Flux rate is slowed (1.32 ± 0.37 μm/min by kymography, 1.02 ± 0.01 μm/min by cross-correlation). (G–I) Same as A–C, in the presence of 100 μM (S)-quinazolinone (see Video 3). Flux rate is almost completely inhibited (0.32 ± 0.33 μm/min by kymography, <0.2 μm/min by cross-correlation). Videos are available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1.
Mentions: We used time-lapse fluorescent speckle microscopy (FSM; Waterman-Storer et al., 1998) to visualize the poleward movement of microtubules in metaphase spindles assembled in cell-free Xenopus egg extracts (Desai et al., 1999; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1). We first estimated flux velocities in the presence of Eg5 inhibitors by kymography (Waterman-Storer et al., 1998), in which intensities of speckles along a line in an image are used to generate a distance versus time plot from a time-lapse movie (Fig. 1, A and B). To maintain spindle bipolarity, we relied on the resistance of spindles to collapse in standard coverslip squashes prepared immediately after drug addition (Kapoor and Mitchison, 2001).

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