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A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes.

Zou J, Hallen MA, Yankel CD, Endow SA - J. Cell Biol. (2008)

Bottom Line: We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules.A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle.By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.

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FRAP analysis of KLP10A-GFP at pole bodies. (A and C) Images from FRAP assays (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200711031/DC1) of KLP10A-GFP at a pole body (A) and NCD-GFP in a meiosis I spindle (C) before bleaching and at exponentially increasing times during recovery. PreB, prebleach. Yellow circles indicate photobleached regions of interest (radius = 0.66 μm). (B and D) Mean fluorescence of the photobleached pole bodies (B; purple) or spindles (D; green) during recovery. Fluorescence was normalized to the first prebleach value, corrected for loss during imaging, and fit to kinetic models (blue); the two-state binding model fit well. (B, inset) Pole body data fit to the single-state binding model showed deviation at early points. (D, inset) KLP10A at pole bodies compared with NCD in spindles. Bars, 2 μm.
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fig4: FRAP analysis of KLP10A-GFP at pole bodies. (A and C) Images from FRAP assays (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200711031/DC1) of KLP10A-GFP at a pole body (A) and NCD-GFP in a meiosis I spindle (C) before bleaching and at exponentially increasing times during recovery. PreB, prebleach. Yellow circles indicate photobleached regions of interest (radius = 0.66 μm). (B and D) Mean fluorescence of the photobleached pole bodies (B; purple) or spindles (D; green) during recovery. Fluorescence was normalized to the first prebleach value, corrected for loss during imaging, and fit to kinetic models (blue); the two-state binding model fit well. (B, inset) Pole body data fit to the single-state binding model showed deviation at early points. (D, inset) KLP10A at pole bodies compared with NCD in spindles. Bars, 2 μm.

Mentions: The assays showed slow turnover of KLP10A at pole bodies (Fig. 4 and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200711031/DC1). The data fit well to a two-state binding model (Fig. 4 B; Sprague et al., 2004); a single-state binding model (Bulinski et al., 2001; Sprague et al., 2004) also fit well, except for the early points (Fig. 4 B, inset). Both models indicated a relatively tight binding state that dominates the recovery with an early phase that is explained by the two-state binding model as a rapid, transient, much weaker binding state. At equilibrium, ∼50% of KLP10A is bound to the pole bodies in the tight-binding state and ∼10% in the weak transient state (Table I). NCD in the meiosis I spindle (Video 2) showed recovery that was poorly fit by a single-state binding model but well fit by a two-state binding model (Fig. 4). At equilibrium, ∼25% of NCD was bound in the slow, more tightly binding state, and ∼20% was bound in the transient weak state.


A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes.

Zou J, Hallen MA, Yankel CD, Endow SA - J. Cell Biol. (2008)

FRAP analysis of KLP10A-GFP at pole bodies. (A and C) Images from FRAP assays (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200711031/DC1) of KLP10A-GFP at a pole body (A) and NCD-GFP in a meiosis I spindle (C) before bleaching and at exponentially increasing times during recovery. PreB, prebleach. Yellow circles indicate photobleached regions of interest (radius = 0.66 μm). (B and D) Mean fluorescence of the photobleached pole bodies (B; purple) or spindles (D; green) during recovery. Fluorescence was normalized to the first prebleach value, corrected for loss during imaging, and fit to kinetic models (blue); the two-state binding model fit well. (B, inset) Pole body data fit to the single-state binding model showed deviation at early points. (D, inset) KLP10A at pole bodies compared with NCD in spindles. Bars, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234233&req=5

fig4: FRAP analysis of KLP10A-GFP at pole bodies. (A and C) Images from FRAP assays (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200711031/DC1) of KLP10A-GFP at a pole body (A) and NCD-GFP in a meiosis I spindle (C) before bleaching and at exponentially increasing times during recovery. PreB, prebleach. Yellow circles indicate photobleached regions of interest (radius = 0.66 μm). (B and D) Mean fluorescence of the photobleached pole bodies (B; purple) or spindles (D; green) during recovery. Fluorescence was normalized to the first prebleach value, corrected for loss during imaging, and fit to kinetic models (blue); the two-state binding model fit well. (B, inset) Pole body data fit to the single-state binding model showed deviation at early points. (D, inset) KLP10A at pole bodies compared with NCD in spindles. Bars, 2 μm.
Mentions: The assays showed slow turnover of KLP10A at pole bodies (Fig. 4 and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200711031/DC1). The data fit well to a two-state binding model (Fig. 4 B; Sprague et al., 2004); a single-state binding model (Bulinski et al., 2001; Sprague et al., 2004) also fit well, except for the early points (Fig. 4 B, inset). Both models indicated a relatively tight binding state that dominates the recovery with an early phase that is explained by the two-state binding model as a rapid, transient, much weaker binding state. At equilibrium, ∼50% of KLP10A is bound to the pole bodies in the tight-binding state and ∼10% in the weak transient state (Table I). NCD in the meiosis I spindle (Video 2) showed recovery that was poorly fit by a single-state binding model but well fit by a two-state binding model (Fig. 4). At equilibrium, ∼25% of NCD was bound in the slow, more tightly binding state, and ∼20% was bound in the transient weak state.

Bottom Line: We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules.A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle.By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.

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