Limits...
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.

Show MeSH

Related in: MedlinePlus

Posterior spindle displacement occurs in metaphase-arrested embryos. (A) Time-lapse images of untreated wild-type embryos and wild-type embryos treated with c-LβL during pronuclear migration, before meeting. All cellular events appear to occur normally in both cases until anaphase onset. Embryos treated with c-LβL remain arrested in metaphase and fail to undergo cytokinesis. Arrowheads indicate the position of anterior and posterior centrosomes in these time-lapse images. Time is indicated in minutes and the 0 time point is at pronuclear envelope breakdown. Bar, 5 μm. (B) Quantification of the distance between the two spindle poles in untreated (closed circles) and c-LβL–treated (open triangles) wild-type embryos. At the normal time of anaphase onset, the spindle fails to elongate in treated embryos and remains at constant length for at least 30 min. The 0 time point corresponds to pronuclear envelope breakdown. (C) Quantification of the extent of posterior spindle displacement at anaphase onset in untreated and c-LβL–treated, metaphase-arrested wild-type embryos. The spindle initiates posterior movement at the correct time and moves a comparable distance in both cases, and the spindle midpoint was positioned at 56 ± 2% EL in untreated embryos and 56 ± 3% EL in c-LβL–treated embryos. In all panels, n = 5 for untreated embryos and n = 4 for c-LβL–treated embryos. Error bars represent SD over five and four embryos.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172534&req=5

fig2: Posterior spindle displacement occurs in metaphase-arrested embryos. (A) Time-lapse images of untreated wild-type embryos and wild-type embryos treated with c-LβL during pronuclear migration, before meeting. All cellular events appear to occur normally in both cases until anaphase onset. Embryos treated with c-LβL remain arrested in metaphase and fail to undergo cytokinesis. Arrowheads indicate the position of anterior and posterior centrosomes in these time-lapse images. Time is indicated in minutes and the 0 time point is at pronuclear envelope breakdown. Bar, 5 μm. (B) Quantification of the distance between the two spindle poles in untreated (closed circles) and c-LβL–treated (open triangles) wild-type embryos. At the normal time of anaphase onset, the spindle fails to elongate in treated embryos and remains at constant length for at least 30 min. The 0 time point corresponds to pronuclear envelope breakdown. (C) Quantification of the extent of posterior spindle displacement at anaphase onset in untreated and c-LβL–treated, metaphase-arrested wild-type embryos. The spindle initiates posterior movement at the correct time and moves a comparable distance in both cases, and the spindle midpoint was positioned at 56 ± 2% EL in untreated embryos and 56 ± 3% EL in c-LβL–treated embryos. In all panels, n = 5 for untreated embryos and n = 4 for c-LβL–treated embryos. Error bars represent SD over five and four embryos.

Mentions: To determine whether or not anaphase entry is indeed dispensable for posterior spindle displacement, we examined if posterior spindle displacement can occur when cells are arrested in metaphase. In eukaryotic cells, the transition from metaphase to anaphase depends on the activity of the proteasome, which degrades proteins such as securin and B-type cyclins (for reviews see Nasmyth, 2002; Peters, 2002). Spindle positioning was monitored in embryos treated with clasto-lactacystin β-lactone (c-LβL), a potent, irreversible inhibitor of the 26S proteasome (see Materials and methods). The initial timing and progression of the first cell cycle events in c-LβL–treated embryos was normal, and embryos entered mitosis at the normal time (Fig. 2 A). As expected, the treated embryos failed to complete mitosis. Monitoring centrosome separation revealed that these embryos were arrested in metaphase and did not undergo anaphase B (Fig. 2 B). Such c-LβL–treated embryos could remain in this arrested state for extended periods of time (at least 30 min) and remained arrested even when pole separation was allowed to occur (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1).


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 occurs in metaphase-arrested embryos. (A) Time-lapse images of untreated wild-type embryos and wild-type embryos treated with c-LβL during pronuclear migration, before meeting. All cellular events appear to occur normally in both cases until anaphase onset. Embryos treated with c-LβL remain arrested in metaphase and fail to undergo cytokinesis. Arrowheads indicate the position of anterior and posterior centrosomes in these time-lapse images. Time is indicated in minutes and the 0 time point is at pronuclear envelope breakdown. Bar, 5 μm. (B) Quantification of the distance between the two spindle poles in untreated (closed circles) and c-LβL–treated (open triangles) wild-type embryos. At the normal time of anaphase onset, the spindle fails to elongate in treated embryos and remains at constant length for at least 30 min. The 0 time point corresponds to pronuclear envelope breakdown. (C) Quantification of the extent of posterior spindle displacement at anaphase onset in untreated and c-LβL–treated, metaphase-arrested wild-type embryos. The spindle initiates posterior movement at the correct time and moves a comparable distance in both cases, and the spindle midpoint was positioned at 56 ± 2% EL in untreated embryos and 56 ± 3% EL in c-LβL–treated embryos. In all panels, n = 5 for untreated embryos and n = 4 for c-LβL–treated embryos. Error bars represent SD over five and four embryos.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Posterior spindle displacement occurs in metaphase-arrested embryos. (A) Time-lapse images of untreated wild-type embryos and wild-type embryos treated with c-LβL during pronuclear migration, before meeting. All cellular events appear to occur normally in both cases until anaphase onset. Embryos treated with c-LβL remain arrested in metaphase and fail to undergo cytokinesis. Arrowheads indicate the position of anterior and posterior centrosomes in these time-lapse images. Time is indicated in minutes and the 0 time point is at pronuclear envelope breakdown. Bar, 5 μm. (B) Quantification of the distance between the two spindle poles in untreated (closed circles) and c-LβL–treated (open triangles) wild-type embryos. At the normal time of anaphase onset, the spindle fails to elongate in treated embryos and remains at constant length for at least 30 min. The 0 time point corresponds to pronuclear envelope breakdown. (C) Quantification of the extent of posterior spindle displacement at anaphase onset in untreated and c-LβL–treated, metaphase-arrested wild-type embryos. The spindle initiates posterior movement at the correct time and moves a comparable distance in both cases, and the spindle midpoint was positioned at 56 ± 2% EL in untreated embryos and 56 ± 3% EL in c-LβL–treated embryos. In all panels, n = 5 for untreated embryos and n = 4 for c-LβL–treated embryos. Error bars represent SD over five and four embryos.
Mentions: To determine whether or not anaphase entry is indeed dispensable for posterior spindle displacement, we examined if posterior spindle displacement can occur when cells are arrested in metaphase. In eukaryotic cells, the transition from metaphase to anaphase depends on the activity of the proteasome, which degrades proteins such as securin and B-type cyclins (for reviews see Nasmyth, 2002; Peters, 2002). Spindle positioning was monitored in embryos treated with clasto-lactacystin β-lactone (c-LβL), a potent, irreversible inhibitor of the 26S proteasome (see Materials and methods). The initial timing and progression of the first cell cycle events in c-LβL–treated embryos was normal, and embryos entered mitosis at the normal time (Fig. 2 A). As expected, the treated embryos failed to complete mitosis. Monitoring centrosome separation revealed that these embryos were arrested in metaphase and did not undergo anaphase B (Fig. 2 B). Such c-LβL–treated embryos could remain in this arrested state for extended periods of time (at least 30 min) and remained arrested even when pole separation was allowed to occur (see online supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1).

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