J Cell Biol
December 6, 2004;
The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles.
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.
J Cell Biol
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Figure 1. 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.
Figure 2. 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.
Figure 3. Flux rates show similar dose–response to inhibition of Eg5 when spindle collapse is suppressed by either mechanical or biochemical means. (A) Dose–response of flux to (S)-quinazolinone in physically trapped spindles (n =189 spindles from three dosage series done on three different days; each point represents the average flux rate from >20 spindles). (B) Dose–response of flux to (S)-quinazolinone when spindle collapse is prevented in solution by addition of p50/dynamitin (n = 114 spindles from two dosage series done on two different days; each point represents the average flux rate from >10 spindles). Error bars show SD. The gray area indicates the lower bound of velocity resolvable by the cross-correlation method (see Materials and methods). The line is a best-fit hyperbolic inhibition curve, R2 = 0.98 for A and R2 = 0.94 for B.
Figure 4. Immunodepletion of Eg5 significantly decreases the flux rate. (A) Western blot for Eg5 in Xenopus extracts over three sequential rounds of immunodepletion using either anti-Eg5 antibody or nonimmune IgG. Right-most lane shows extract triple-depleted of Eg5 and supplemented with full-length, recombinant Eg5 (twice the endogenous concentration). α-Tubulin Western blot is a loading control. (B) Percentage of spindles that are bipolar when assembled in mock-depleted extract, Eg5-depleted extract, and Eg5-depleted extract supplemented with recombinant full-length Eg5. Values are the averages from three independent experiments, with >100 spindles per condition counted for each experiment. Error bars show SD. (C) Flux rates measured by cross-correlation for p50 spindles assembled in mock-depleted extract, Eg5-depleted extract, and Eg5-depleted extract supplemented with recombinant full-length Eg5 (n = 29 spindles for mock+p50, n = 35 spindles for ΔEg5+p50, and n = 26 spindles for ΔEg5+p50+Eg5, from three independent experiments). Error bars show SD.
Brust-Mascher, Microtubule flux and sliding in mitotic spindles of Drosophila embryos. 2002, Pubmed