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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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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.
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fig3: 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.

Mentions: We used cross-correlation to measure average flux rates in short time-lapse movies of Eg5 inhibitor-treated spindles. We performed multiple dose–response series and measured multiple spindles at each dose to account for the considerable variation in flux rates within samples (Fig. 2 D). Flux rates decreased with increasing doses of both monastrol and (S)-quinazolinone (Fig. 3 A and Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1). A simple hyperbolic curve reflecting noncooperative drug binding closely fit the dose–response relationship we observed.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172449&req=5

fig3: 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.
Mentions: We used cross-correlation to measure average flux rates in short time-lapse movies of Eg5 inhibitor-treated spindles. We performed multiple dose–response series and measured multiple spindles at each dose to account for the considerable variation in flux rates within samples (Fig. 2 D). Flux rates decreased with increasing doses of both monastrol and (S)-quinazolinone (Fig. 3 A and Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1). A simple hyperbolic curve reflecting noncooperative drug binding closely fit the dose–response relationship we observed.

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