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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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Chromosome segregation occurs without anaphase A and significant poleward flux. (A) Spindle-centered kymograph of an embryo expressing both γ-tubulin and histone H2B fused to GFP. This kymograph was produced similarly to the one in Fig. 1 B, except that posterior movement of the spindle was eliminated: the spindle in each frame of time-lapse images was rotationally aligned and recentered on the midpoint between the centrosomes to allow the observation of symmetries in the spindle. Frames were acquired at 7-s intervals. (B and C) Time-lapse images of embryos expressing β-tubulin::GFP in which a short region of anterior (left) or posterior (right) spindle microtubules were photobleached during prometaphase (B) or anaphase (C) onset. The bottom panels follow FRAP as well as movement of the photobleached region (indicated by gray arrowheads). Frames were acquired at 7-s intervals. For photobleaches of the spindle during prometaphase, kymographs were aligned to the location of chromosomes in the center of the spindle. For photobleaches of the anterior or posterior half of the spindle at anaphase onset, kymographs were aligned to the center of the posterior or anterior centrosome, respectively. (D) Quantification of FRAP during prometaphase (gray triangles) and anaphase (open squares). To correct for fluorophore bleaching and embryo to embryo variations, fluorescence intensity in the photobleached region is expressed as a ratio of bleached over unbleached midzone microtubules in the same embryo. FRAP occurs faster during prometaphase (t1/2 = 10.6 s; polynomial equation: y = −3E − 07 x4 + 4E − 05x3 − 0.0021x2 + 0.0559x + 0.1571; R2 = 0.997) compared with anaphase (t1/2 = 17.7 s; polynomial equation: y = 4E − 08x4 + 2E − 06x3 − 0.0005x2 + 0.0303x + 0.1223; R2 = 0.989). Time points were acquired at 7-s intervals. Error bars represent SD over six embryos. (E) Quantification of the distance variation between chromosomes to photobleached region (black triangles), chromosomes and spindle poles (gray squares), and spindle pole to photobleached region (open circles) during anaphase. The distance remains constant between these three positions throughout anaphase. Time points were acquired at 7-s intervals. Error bars represent SD over six embryos.
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fig5: Chromosome segregation occurs without anaphase A and significant poleward flux. (A) Spindle-centered kymograph of an embryo expressing both γ-tubulin and histone H2B fused to GFP. This kymograph was produced similarly to the one in Fig. 1 B, except that posterior movement of the spindle was eliminated: the spindle in each frame of time-lapse images was rotationally aligned and recentered on the midpoint between the centrosomes to allow the observation of symmetries in the spindle. Frames were acquired at 7-s intervals. (B and C) Time-lapse images of embryos expressing β-tubulin::GFP in which a short region of anterior (left) or posterior (right) spindle microtubules were photobleached during prometaphase (B) or anaphase (C) onset. The bottom panels follow FRAP as well as movement of the photobleached region (indicated by gray arrowheads). Frames were acquired at 7-s intervals. For photobleaches of the spindle during prometaphase, kymographs were aligned to the location of chromosomes in the center of the spindle. For photobleaches of the anterior or posterior half of the spindle at anaphase onset, kymographs were aligned to the center of the posterior or anterior centrosome, respectively. (D) Quantification of FRAP during prometaphase (gray triangles) and anaphase (open squares). To correct for fluorophore bleaching and embryo to embryo variations, fluorescence intensity in the photobleached region is expressed as a ratio of bleached over unbleached midzone microtubules in the same embryo. FRAP occurs faster during prometaphase (t1/2 = 10.6 s; polynomial equation: y = −3E − 07 x4 + 4E − 05x3 − 0.0021x2 + 0.0559x + 0.1571; R2 = 0.997) compared with anaphase (t1/2 = 17.7 s; polynomial equation: y = 4E − 08x4 + 2E − 06x3 − 0.0005x2 + 0.0303x + 0.1223; R2 = 0.989). Time points were acquired at 7-s intervals. Error bars represent SD over six embryos. (E) Quantification of the distance variation between chromosomes to photobleached region (black triangles), chromosomes and spindle poles (gray squares), and spindle pole to photobleached region (open circles) during anaphase. The distance remains constant between these three positions throughout anaphase. Time points were acquired at 7-s intervals. Error bars represent SD over six embryos.

Mentions: In vertebrate and Drosophila spindles, microtubule poleward flux is a significant component of chromosome segregation (Desai et al., 1998; Maddox et al., 2002, 2003). One striking observation made previously in C. elegans embryos is that the mitotic spindle does not undergo anaphase A during chromosome segregation (Oegema et al., 2001; Fig. 5, A and E). Furthermore, despite an asymmetry in pulling forces on each side of the spindle, we have found that chromosome segregation and centrosome separation in each spindle half appear symmetric (Fig. 5 A), suggesting that the forces within the mitotic spindle itself may also be symmetric. Together with the finding that the spindle midzone limits the rate of anaphase pole separation (Grill et al., 2001), these observations suggested that the forces responsible for mediating pulling on the asters during posterior spindle displacement may also drive the segregation of chromosomes at anaphase B. However, poleward flux has been shown to generate tension at the kinetochore of mitotic spindles in Xenopus extracts, through constant microtubule plus end net polymerization and minus end depolymerization (Desai et al., 1998; Maddox et al., 2003). Therefore, one possibility remained that spindle microtubules might be under tension during chromosome segregation through poleward flux, despite the apparent absence of anaphase A.


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)

Chromosome segregation occurs without anaphase A and significant poleward flux. (A) Spindle-centered kymograph of an embryo expressing both γ-tubulin and histone H2B fused to GFP. This kymograph was produced similarly to the one in Fig. 1 B, except that posterior movement of the spindle was eliminated: the spindle in each frame of time-lapse images was rotationally aligned and recentered on the midpoint between the centrosomes to allow the observation of symmetries in the spindle. Frames were acquired at 7-s intervals. (B and C) Time-lapse images of embryos expressing β-tubulin::GFP in which a short region of anterior (left) or posterior (right) spindle microtubules were photobleached during prometaphase (B) or anaphase (C) onset. The bottom panels follow FRAP as well as movement of the photobleached region (indicated by gray arrowheads). Frames were acquired at 7-s intervals. For photobleaches of the spindle during prometaphase, kymographs were aligned to the location of chromosomes in the center of the spindle. For photobleaches of the anterior or posterior half of the spindle at anaphase onset, kymographs were aligned to the center of the posterior or anterior centrosome, respectively. (D) Quantification of FRAP during prometaphase (gray triangles) and anaphase (open squares). To correct for fluorophore bleaching and embryo to embryo variations, fluorescence intensity in the photobleached region is expressed as a ratio of bleached over unbleached midzone microtubules in the same embryo. FRAP occurs faster during prometaphase (t1/2 = 10.6 s; polynomial equation: y = −3E − 07 x4 + 4E − 05x3 − 0.0021x2 + 0.0559x + 0.1571; R2 = 0.997) compared with anaphase (t1/2 = 17.7 s; polynomial equation: y = 4E − 08x4 + 2E − 06x3 − 0.0005x2 + 0.0303x + 0.1223; R2 = 0.989). Time points were acquired at 7-s intervals. Error bars represent SD over six embryos. (E) Quantification of the distance variation between chromosomes to photobleached region (black triangles), chromosomes and spindle poles (gray squares), and spindle pole to photobleached region (open circles) during anaphase. The distance remains constant between these three positions throughout anaphase. Time points were acquired at 7-s intervals. Error bars represent SD over six embryos.
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Related In: Results  -  Collection

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fig5: Chromosome segregation occurs without anaphase A and significant poleward flux. (A) Spindle-centered kymograph of an embryo expressing both γ-tubulin and histone H2B fused to GFP. This kymograph was produced similarly to the one in Fig. 1 B, except that posterior movement of the spindle was eliminated: the spindle in each frame of time-lapse images was rotationally aligned and recentered on the midpoint between the centrosomes to allow the observation of symmetries in the spindle. Frames were acquired at 7-s intervals. (B and C) Time-lapse images of embryos expressing β-tubulin::GFP in which a short region of anterior (left) or posterior (right) spindle microtubules were photobleached during prometaphase (B) or anaphase (C) onset. The bottom panels follow FRAP as well as movement of the photobleached region (indicated by gray arrowheads). Frames were acquired at 7-s intervals. For photobleaches of the spindle during prometaphase, kymographs were aligned to the location of chromosomes in the center of the spindle. For photobleaches of the anterior or posterior half of the spindle at anaphase onset, kymographs were aligned to the center of the posterior or anterior centrosome, respectively. (D) Quantification of FRAP during prometaphase (gray triangles) and anaphase (open squares). To correct for fluorophore bleaching and embryo to embryo variations, fluorescence intensity in the photobleached region is expressed as a ratio of bleached over unbleached midzone microtubules in the same embryo. FRAP occurs faster during prometaphase (t1/2 = 10.6 s; polynomial equation: y = −3E − 07 x4 + 4E − 05x3 − 0.0021x2 + 0.0559x + 0.1571; R2 = 0.997) compared with anaphase (t1/2 = 17.7 s; polynomial equation: y = 4E − 08x4 + 2E − 06x3 − 0.0005x2 + 0.0303x + 0.1223; R2 = 0.989). Time points were acquired at 7-s intervals. Error bars represent SD over six embryos. (E) Quantification of the distance variation between chromosomes to photobleached region (black triangles), chromosomes and spindle poles (gray squares), and spindle pole to photobleached region (open circles) during anaphase. The distance remains constant between these three positions throughout anaphase. Time points were acquired at 7-s intervals. Error bars represent SD over six embryos.
Mentions: In vertebrate and Drosophila spindles, microtubule poleward flux is a significant component of chromosome segregation (Desai et al., 1998; Maddox et al., 2002, 2003). One striking observation made previously in C. elegans embryos is that the mitotic spindle does not undergo anaphase A during chromosome segregation (Oegema et al., 2001; Fig. 5, A and E). Furthermore, despite an asymmetry in pulling forces on each side of the spindle, we have found that chromosome segregation and centrosome separation in each spindle half appear symmetric (Fig. 5 A), suggesting that the forces within the mitotic spindle itself may also be symmetric. Together with the finding that the spindle midzone limits the rate of anaphase pole separation (Grill et al., 2001), these observations suggested that the forces responsible for mediating pulling on the asters during posterior spindle displacement may also drive the segregation of chromosomes at anaphase B. However, poleward flux has been shown to generate tension at the kinetochore of mitotic spindles in Xenopus extracts, through constant microtubule plus end net polymerization and minus end depolymerization (Desai et al., 1998; Maddox et al., 2003). Therefore, one possibility remained that spindle microtubules might be under tension during chromosome segregation through poleward flux, despite the apparent absence of anaphase A.

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