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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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A transition in forces occurs in metaphase-arrested embryos. Quantification of anterior centrosome movement after posterior centrosome irradiation in untreated (light gray bars) and c-LβL–treated, metaphase-arrested wild-type embryos (dark gray bars). Displacement of the anterior centrosome toward the anterior increases as the cell cycle progresses from late prophase/prometaphase to metaphase. Error bars represent SD over, from top to bottom, 19, 5, 13, 8, 8, and 9 embryos, respectively, for each case.
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fig4: A transition in forces occurs in metaphase-arrested embryos. Quantification of anterior centrosome movement after posterior centrosome irradiation in untreated (light gray bars) and c-LβL–treated, metaphase-arrested wild-type embryos (dark gray bars). Displacement of the anterior centrosome toward the anterior increases as the cell cycle progresses from late prophase/prometaphase to metaphase. Error bars represent SD over, from top to bottom, 19, 5, 13, 8, 8, and 9 embryos, respectively, for each case.

Mentions: That forces transition from tethering to pulling in the anterior of the embryo during mitosis prompted us to ask whether or not the forces are regulated by cell cycle transitions. To test this, we irradiated the posterior centrosome in c-LβL–treated embryos at various times during the cell cycle. If forces are not regulated by the cell cycle machinery, then the anterior pulling force observed at the onset of posterior spindle displacement in metaphase-arrested embryos should transition to the stronger force, comparable to the net anterior force observed at metaphase in untreated embryos. We performed laser irradiations of centrosomes in metaphase-arrested embryos and confirmed this hypothesis; when the posterior centrosome was irradiated at the time of metaphase in c-LβL–treated embryos, we observed a net anterior movement of the anterior centrosome similar to the net anterior movement observed at the onset of posterior spindle displacement in untreated embryos (4.2 ± 1.3% EL compared with 4.4 ± 2.3% EL, n = 8 and n = 13, respectively, P = 0.89; Fig. 4). Irradiating the posterior centrosome at the time of late prophase, with or without treatment with the proteasome inhibitor, gave indistinguishable results (Fig. 4), thereby indicating that forces are not grossly misregulated in c-LβL–treated embryos. This result suggests that the pulling forces responsible for moving the spindle posteriorly are not regulated by the proteasome-dependent events at the metaphase–anaphase transition during progression through mitosis in the early embryo.


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)

A transition in forces occurs in metaphase-arrested embryos. Quantification of anterior centrosome movement after posterior centrosome irradiation in untreated (light gray bars) and c-LβL–treated, metaphase-arrested wild-type embryos (dark gray bars). Displacement of the anterior centrosome toward the anterior increases as the cell cycle progresses from late prophase/prometaphase to metaphase. Error bars represent SD over, from top to bottom, 19, 5, 13, 8, 8, and 9 embryos, respectively, for each case.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172534&req=5

fig4: A transition in forces occurs in metaphase-arrested embryos. Quantification of anterior centrosome movement after posterior centrosome irradiation in untreated (light gray bars) and c-LβL–treated, metaphase-arrested wild-type embryos (dark gray bars). Displacement of the anterior centrosome toward the anterior increases as the cell cycle progresses from late prophase/prometaphase to metaphase. Error bars represent SD over, from top to bottom, 19, 5, 13, 8, 8, and 9 embryos, respectively, for each case.
Mentions: That forces transition from tethering to pulling in the anterior of the embryo during mitosis prompted us to ask whether or not the forces are regulated by cell cycle transitions. To test this, we irradiated the posterior centrosome in c-LβL–treated embryos at various times during the cell cycle. If forces are not regulated by the cell cycle machinery, then the anterior pulling force observed at the onset of posterior spindle displacement in metaphase-arrested embryos should transition to the stronger force, comparable to the net anterior force observed at metaphase in untreated embryos. We performed laser irradiations of centrosomes in metaphase-arrested embryos and confirmed this hypothesis; when the posterior centrosome was irradiated at the time of metaphase in c-LβL–treated embryos, we observed a net anterior movement of the anterior centrosome similar to the net anterior movement observed at the onset of posterior spindle displacement in untreated embryos (4.2 ± 1.3% EL compared with 4.4 ± 2.3% EL, n = 8 and n = 13, respectively, P = 0.89; Fig. 4). Irradiating the posterior centrosome at the time of late prophase, with or without treatment with the proteasome inhibitor, gave indistinguishable results (Fig. 4), thereby indicating that forces are not grossly misregulated in c-LβL–treated embryos. This result suggests that the pulling forces responsible for moving the spindle posteriorly are not regulated by the proteasome-dependent events at the metaphase–anaphase transition during progression through mitosis in the early embryo.

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