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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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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.
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fig4: 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.

Mentions: These results suggest that Eg5 drives flux, but could also be explained if pharmacological inhibition of Eg5 induces a braking effect on microtubule sliding. We consider such an effect unlikely because monastrol-inhibited Eg5 does not dramatically slow microtubule gliding driven in vitro by conventional kinesin (Crevel et al., 2004). To directly address the possibility of braking and to further test specificity, we measured the effects on flux of Eg5 immunodepletion in p50 spindles (Fig. 4). Three sequential rounds of depletions were required to remove >99% of Eg5 from extracts. As expected, cycled spindle assembly in Eg5-depleted extract primarily gave monoasters, whereas mock depletion gave mostly bipolar spindles (Fig. 4 B). Addition of p50/dynamitin to Eg5-depleted extracts rescued bipolarity (Fig. S3 C, available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1; unpublished data). Measurement by kymography and cross-correlation showed that mock-depleted spindles fluxed at a normal rate (not depicted), as did mock-depleted spindles treated with p50/dynamitin (Fig. 4 C; and Fig. S3, A and B). However, p50 spindles assembled in Eg5-depleted extract showed a significantly slowed flux rate despite the rescue of spindle bipolarity (Fig. 4 C; and Fig. S3, C and D). Purified recombinant full-length Eg5, which restored spindle bipolarity in Eg5-depleted extract (Fig. 4, A and B), partially rescued flux in Eg5-depleted p50 spindles (Fig. 4 C; and Fig. S3, E and F).


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)

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.
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fig4: 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.
Mentions: These results suggest that Eg5 drives flux, but could also be explained if pharmacological inhibition of Eg5 induces a braking effect on microtubule sliding. We consider such an effect unlikely because monastrol-inhibited Eg5 does not dramatically slow microtubule gliding driven in vitro by conventional kinesin (Crevel et al., 2004). To directly address the possibility of braking and to further test specificity, we measured the effects on flux of Eg5 immunodepletion in p50 spindles (Fig. 4). Three sequential rounds of depletions were required to remove >99% of Eg5 from extracts. As expected, cycled spindle assembly in Eg5-depleted extract primarily gave monoasters, whereas mock depletion gave mostly bipolar spindles (Fig. 4 B). Addition of p50/dynamitin to Eg5-depleted extracts rescued bipolarity (Fig. S3 C, available at http://www.jcb.org/cgi/content/full/jcb.200407126/DC1; unpublished data). Measurement by kymography and cross-correlation showed that mock-depleted spindles fluxed at a normal rate (not depicted), as did mock-depleted spindles treated with p50/dynamitin (Fig. 4 C; and Fig. S3, A and B). However, p50 spindles assembled in Eg5-depleted extract showed a significantly slowed flux rate despite the rescue of spindle bipolarity (Fig. 4 C; and Fig. S3, C and D). Purified recombinant full-length Eg5, which restored spindle bipolarity in Eg5-depleted extract (Fig. 4, A and B), partially rescued flux in Eg5-depleted p50 spindles (Fig. 4 C; and Fig. S3, E and F).

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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