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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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PAR proteins regulate pulling forces throughout the cell cycle. Map (left) and quantification (right) of centrosome displacement after OICD in (A) par-2(lw32) and (B) par-3(it71) mutant embryos. Displacement was determined for anterior centrosomes after posterior centrosome irradiation (gray diamonds and gray bars) and for posterior centrosomes after anterior centrosome irradiation (black squares and black bars). (C) Quantification of anterior (gray bars) and posterior (black bars) centrosome displacement at the time of late prophase/prometaphase in wild-type, par-2(lw32), and par-3(it71) embryos. Error bars represent SD over, from top to bottom, 19, 16, 21, 25, 21, and 21 embryos, respectively, for each case. The overall displacement in par-2 mutant embryos (3.0 ± 2.1% EL, n = 46) was different from wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 0.007) as well as from wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 1.3E−5) centrosomes, and the same was observed when comparing displacement in par-3 mutant embryos (3.4 ± 1.5% EL, n = 42) with that of wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 1.4E−5) and wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 5.3E−6) centrosomes.
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fig6: PAR proteins regulate pulling forces throughout the cell cycle. Map (left) and quantification (right) of centrosome displacement after OICD in (A) par-2(lw32) and (B) par-3(it71) mutant embryos. Displacement was determined for anterior centrosomes after posterior centrosome irradiation (gray diamonds and gray bars) and for posterior centrosomes after anterior centrosome irradiation (black squares and black bars). (C) Quantification of anterior (gray bars) and posterior (black bars) centrosome displacement at the time of late prophase/prometaphase in wild-type, par-2(lw32), and par-3(it71) embryos. Error bars represent SD over, from top to bottom, 19, 16, 21, 25, 21, and 21 embryos, respectively, for each case. The overall displacement in par-2 mutant embryos (3.0 ± 2.1% EL, n = 46) was different from wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 0.007) as well as from wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 1.3E−5) centrosomes, and the same was observed when comparing displacement in par-3 mutant embryos (3.4 ± 1.5% EL, n = 42) with that of wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 1.4E−5) and wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 5.3E−6) centrosomes.

Mentions: We performed OICD in par-2(lw32) or par-3(it71) mutant embryos and found that the pulling forces that are acting on anterior and posterior asters during late prophase/prometaphase are more similar to each other than in wild-type embryos (Fig. 6). The estimated forces on anterior and posterior asters in both par-2 and par-3 mutants during late prophase and prometaphase were roughly halfway between those estimated on anterior and posterior asters in wild-type embryos; the overall displacement in par-2 mutant embryos was different from wild-type anterior as well as from wild-type posterior centrosomes, and the same was observed when comparing displacement in par-3 mutant embryos with that of wild-type anterior and wild-type posterior centrosomes (Fig. 6 legend, numbers). However, the difference in overall displacement between par-2 and par-3 mutant embryos was not statistically significant (3.0 ± 2.1% EL compared with 3.4 ± 1.5% EL, n = 46 and n = 42, respectively, P = 0.35), suggesting that although these genes regulate pulling forces, the cortex in par-2 and par-3 mutant embryos cannot simply be regarded as posteriorized or anteriorized with respect to pulling forces before the time of anaphase (see Discussion). It should be noted that par-2 embryos have a partial rotation defect during centration; we irradiated centrosomes only in embryos that had undergone at minimum of 45° rotation before having reached half the distance to the center. Because it is possible that these embryos represent a special subset of par-2 embryos in which PAR-2 has partial function, the results of ablations at this stage might not reveal forces in the complete absence of PAR-2. Even with this caveat, based on stages that follow this, we can conclude that the cortical proteins that regulate spindle positioning also function to regulate the forces that we observed.


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)

PAR proteins regulate pulling forces throughout the cell cycle. Map (left) and quantification (right) of centrosome displacement after OICD in (A) par-2(lw32) and (B) par-3(it71) mutant embryos. Displacement was determined for anterior centrosomes after posterior centrosome irradiation (gray diamonds and gray bars) and for posterior centrosomes after anterior centrosome irradiation (black squares and black bars). (C) Quantification of anterior (gray bars) and posterior (black bars) centrosome displacement at the time of late prophase/prometaphase in wild-type, par-2(lw32), and par-3(it71) embryos. Error bars represent SD over, from top to bottom, 19, 16, 21, 25, 21, and 21 embryos, respectively, for each case. The overall displacement in par-2 mutant embryos (3.0 ± 2.1% EL, n = 46) was different from wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 0.007) as well as from wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 1.3E−5) centrosomes, and the same was observed when comparing displacement in par-3 mutant embryos (3.4 ± 1.5% EL, n = 42) with that of wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 1.4E−5) and wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 5.3E−6) centrosomes.
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Related In: Results  -  Collection

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fig6: PAR proteins regulate pulling forces throughout the cell cycle. Map (left) and quantification (right) of centrosome displacement after OICD in (A) par-2(lw32) and (B) par-3(it71) mutant embryos. Displacement was determined for anterior centrosomes after posterior centrosome irradiation (gray diamonds and gray bars) and for posterior centrosomes after anterior centrosome irradiation (black squares and black bars). (C) Quantification of anterior (gray bars) and posterior (black bars) centrosome displacement at the time of late prophase/prometaphase in wild-type, par-2(lw32), and par-3(it71) embryos. Error bars represent SD over, from top to bottom, 19, 16, 21, 25, 21, and 21 embryos, respectively, for each case. The overall displacement in par-2 mutant embryos (3.0 ± 2.1% EL, n = 46) was different from wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 0.007) as well as from wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 1.3E−5) centrosomes, and the same was observed when comparing displacement in par-3 mutant embryos (3.4 ± 1.5% EL, n = 42) with that of wild-type anterior (1.5 ± 1.1% EL, n = 19, P = 1.4E−5) and wild-type posterior (5.9 ± 2.4% EL, n = 16, P = 5.3E−6) centrosomes.
Mentions: We performed OICD in par-2(lw32) or par-3(it71) mutant embryos and found that the pulling forces that are acting on anterior and posterior asters during late prophase/prometaphase are more similar to each other than in wild-type embryos (Fig. 6). The estimated forces on anterior and posterior asters in both par-2 and par-3 mutants during late prophase and prometaphase were roughly halfway between those estimated on anterior and posterior asters in wild-type embryos; the overall displacement in par-2 mutant embryos was different from wild-type anterior as well as from wild-type posterior centrosomes, and the same was observed when comparing displacement in par-3 mutant embryos with that of wild-type anterior and wild-type posterior centrosomes (Fig. 6 legend, numbers). However, the difference in overall displacement between par-2 and par-3 mutant embryos was not statistically significant (3.0 ± 2.1% EL compared with 3.4 ± 1.5% EL, n = 46 and n = 42, respectively, P = 0.35), suggesting that although these genes regulate pulling forces, the cortex in par-2 and par-3 mutant embryos cannot simply be regarded as posteriorized or anteriorized with respect to pulling forces before the time of anaphase (see Discussion). It should be noted that par-2 embryos have a partial rotation defect during centration; we irradiated centrosomes only in embryos that had undergone at minimum of 45° rotation before having reached half the distance to the center. Because it is possible that these embryos represent a special subset of par-2 embryos in which PAR-2 has partial function, the results of ablations at this stage might not reveal forces in the complete absence of PAR-2. Even with this caveat, based on stages that follow this, we can conclude that the cortical proteins that regulate spindle positioning also function to regulate the forces that we observed.

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