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The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly.

Labbé JC, McCarthy EK, Goldstein B - J. Cell Biol. (2004)

Bottom Line: The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex.Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans.We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. jc.labbe@umontreal.ca

ABSTRACT
Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

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Posterior spindle displacement begins at metaphase. (A) Time-lapse images of an early C. elegans embryo expressing both γ-tubulin and histone H2B fused to GFP. (B) Kymograph analysis of spindle behavior from these time-lapse images. In both panels, arrowheads point to centrosomes at early metaphase and arrows point to the centrosomes at late metaphase, before anaphase onset. Displacement of the spindle toward the posterior can be observed during metaphase. Displacement began during early metaphase or at the end of prometaphase in all embryos examined in this way (n = 8; see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Bars, 5 μm.
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fig1: Posterior spindle displacement begins at metaphase. (A) Time-lapse images of an early C. elegans embryo expressing both γ-tubulin and histone H2B fused to GFP. (B) Kymograph analysis of spindle behavior from these time-lapse images. In both panels, arrowheads point to centrosomes at early metaphase and arrows point to the centrosomes at late metaphase, before anaphase onset. Displacement of the spindle toward the posterior can be observed during metaphase. Displacement began during early metaphase or at the end of prometaphase in all embryos examined in this way (n = 8; see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Bars, 5 μm.

Mentions: In the one-cell stage C. elegans embryo, the spindle forms at the center of the cell and moves toward the posterior before cytokinesis. Previous experiments assessing the forces acting on the spindle were performed at anaphase B (Grill et al., 2001, 2003). As a baseline for further studies, we first determined the stage of the cell cycle during which posterior spindle displacement occurs by imaging embryos expressing both γ-tubulin and histone H2B fused to GFP (Oegema et al., 2001), which allowed us to simultaneously monitor the behavior of centrosomes and chromosomes, respectively. After the spindle arrived at the center of the embryo, both the centrosomes and chromosomes began to move posterior of the center 60.9 ± 20.8 s before anaphase (n = 7), near the time when sister chromatids were first aligned at the metaphase plate (Fig. 1 and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Chromosome separation occurred after the spindle began moving toward the posterior in the cell, and the posterior spindle pole continued to move posteriorly after entry into anaphase. Therefore, the mitotic spindle begins to move to an asymmetric position during metaphase, before anaphase onset, which is consistent with observations made previously (Oegema et al., 2001). This result suggests that spindle positioning is unlikely to be regulated by anaphase entry.


The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly.

Labbé JC, McCarthy EK, Goldstein B - J. Cell Biol. (2004)

Posterior spindle displacement begins at metaphase. (A) Time-lapse images of an early C. elegans embryo expressing both γ-tubulin and histone H2B fused to GFP. (B) Kymograph analysis of spindle behavior from these time-lapse images. In both panels, arrowheads point to centrosomes at early metaphase and arrows point to the centrosomes at late metaphase, before anaphase onset. Displacement of the spindle toward the posterior can be observed during metaphase. Displacement began during early metaphase or at the end of prometaphase in all embryos examined in this way (n = 8; see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172534&req=5

fig1: Posterior spindle displacement begins at metaphase. (A) Time-lapse images of an early C. elegans embryo expressing both γ-tubulin and histone H2B fused to GFP. (B) Kymograph analysis of spindle behavior from these time-lapse images. In both panels, arrowheads point to centrosomes at early metaphase and arrows point to the centrosomes at late metaphase, before anaphase onset. Displacement of the spindle toward the posterior can be observed during metaphase. Displacement began during early metaphase or at the end of prometaphase in all embryos examined in this way (n = 8; see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Bars, 5 μm.
Mentions: In the one-cell stage C. elegans embryo, the spindle forms at the center of the cell and moves toward the posterior before cytokinesis. Previous experiments assessing the forces acting on the spindle were performed at anaphase B (Grill et al., 2001, 2003). As a baseline for further studies, we first determined the stage of the cell cycle during which posterior spindle displacement occurs by imaging embryos expressing both γ-tubulin and histone H2B fused to GFP (Oegema et al., 2001), which allowed us to simultaneously monitor the behavior of centrosomes and chromosomes, respectively. After the spindle arrived at the center of the embryo, both the centrosomes and chromosomes began to move posterior of the center 60.9 ± 20.8 s before anaphase (n = 7), near the time when sister chromatids were first aligned at the metaphase plate (Fig. 1 and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Chromosome separation occurred after the spindle began moving toward the posterior in the cell, and the posterior spindle pole continued to move posteriorly after entry into anaphase. Therefore, the mitotic spindle begins to move to an asymmetric position during metaphase, before anaphase onset, which is consistent with observations made previously (Oegema et al., 2001). This result suggests that spindle positioning is unlikely to be regulated by anaphase entry.

Bottom Line: The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex.Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans.We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. jc.labbe@umontreal.ca

ABSTRACT
Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

Show MeSH
Related in: MedlinePlus