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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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Related in: MedlinePlus

Laser-mediated disruption of microtubule organization allows the estimation of pulling forces throughout the cell cycle. (A) Two-dimensional reconstruction of multiple confocal sections of embryos fixed and stained for α-tubulin. The centrosomes indicated by arrowheads in the right panels were irradiated before fixation. Laser irradiation specifically disrupts microtubule organization at the targeted centrosome, leaving the unirradiated centrosome largely intact. Bar, 5 μm. (B) Map (left) and quantification (right) of centrosome displacement after OICD. 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). In the right panel, displacements were averaged for various phases of the cell cycle. The timing of cell cycle events was determined according to previously reported values and matches observations made by DIC optics (Labbé et al., 2003). Error bars represent SD over, from top to bottom, 6, 4, 19, 16, 13, and 8 embryos, respectively, for each case. (C) Posterior centrosome movement quantified following no irradiation, laser irradiation of a region between the anterior centrosome and the anterior cortex, or laser irradiation of the whole anterior centrosome. A schematic of the procedure for each condition is shown on the left, with the region irradiated marked with an asterisk. Error bars represent SD over, from top to bottom, 8, 8, and 16 embryos, respectively, for each case. (D) Conceptual model depicting the various types of forces that act on the centrosomes throughout the first mitosis of the early C. elegans embryo. In this model, tethering forces are represented as lines, whereas pulling forces are depicted as springs. At late prophase/prometaphase, the pulling force present in the posterior of the embryo is counteracted by the tethering force in the anterior, thereby preventing posterior spindle displacement. During metaphase, the tethering force in the anterior changes to a pulling force.
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fig3: Laser-mediated disruption of microtubule organization allows the estimation of pulling forces throughout the cell cycle. (A) Two-dimensional reconstruction of multiple confocal sections of embryos fixed and stained for α-tubulin. The centrosomes indicated by arrowheads in the right panels were irradiated before fixation. Laser irradiation specifically disrupts microtubule organization at the targeted centrosome, leaving the unirradiated centrosome largely intact. Bar, 5 μm. (B) Map (left) and quantification (right) of centrosome displacement after OICD. 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). In the right panel, displacements were averaged for various phases of the cell cycle. The timing of cell cycle events was determined according to previously reported values and matches observations made by DIC optics (Labbé et al., 2003). Error bars represent SD over, from top to bottom, 6, 4, 19, 16, 13, and 8 embryos, respectively, for each case. (C) Posterior centrosome movement quantified following no irradiation, laser irradiation of a region between the anterior centrosome and the anterior cortex, or laser irradiation of the whole anterior centrosome. A schematic of the procedure for each condition is shown on the left, with the region irradiated marked with an asterisk. Error bars represent SD over, from top to bottom, 8, 8, and 16 embryos, respectively, for each case. (D) Conceptual model depicting the various types of forces that act on the centrosomes throughout the first mitosis of the early C. elegans embryo. In this model, tethering forces are represented as lines, whereas pulling forces are depicted as springs. At late prophase/prometaphase, the pulling force present in the posterior of the embryo is counteracted by the tethering force in the anterior, thereby preventing posterior spindle displacement. During metaphase, the tethering force in the anterior changes to a pulling force.

Mentions: To characterize the forces that drive posterior spindle displacement, we examined in detail the regulation of spindle-positioning pulling forces through the first cell cycle of the C. elegans embryo. Previous experiments have used spindle severing to reveal that spindle poles are pulled apart during anaphase B (Grill et al., 2001), indicating that pulling forces are acting on spindle poles during this stage of the cell cycle. To examine the forces when posterior spindle displacement begins and also throughout the cell cycle, we used an approach termed optically induced centrosome disruption (OICD; Grill et al., 2003), in which we laser irradiated either the anterior or posterior centrosome during various phases of the cell cycle and monitored the resulting movement of the nonirradiated centrosome (see Materials and methods). Our OICD experiments conducted during anaphase succeeded in replicating previous spindle-cutting results (Grill et al., 2001). We found that the OICD procedure was efficient in disrupting one of the two asters without detectably affecting the organization of the nonirradiated one (Fig. 3 A and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). After irradiation of one of the two centrosomes, the spindle was left with only one of the asters maintaining extensive connection with the cell cortex. Quantification of centrosome movement was used to estimate the net relative vectorial force applied on this aster (Fig. S3 and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Using this approach, we generated a map of relative pulling forces acting on each aster throughout the cell cycle (Fig. 3 B). Because the timing of rotation is variable during centration in wild-type embryos, centrosome irradiations that were performed during rotation were performed in the embryos that had undergone a minimum of 45° rotation before having reached half the distance to the center of the 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)

Laser-mediated disruption of microtubule organization allows the estimation of pulling forces throughout the cell cycle. (A) Two-dimensional reconstruction of multiple confocal sections of embryos fixed and stained for α-tubulin. The centrosomes indicated by arrowheads in the right panels were irradiated before fixation. Laser irradiation specifically disrupts microtubule organization at the targeted centrosome, leaving the unirradiated centrosome largely intact. Bar, 5 μm. (B) Map (left) and quantification (right) of centrosome displacement after OICD. 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). In the right panel, displacements were averaged for various phases of the cell cycle. The timing of cell cycle events was determined according to previously reported values and matches observations made by DIC optics (Labbé et al., 2003). Error bars represent SD over, from top to bottom, 6, 4, 19, 16, 13, and 8 embryos, respectively, for each case. (C) Posterior centrosome movement quantified following no irradiation, laser irradiation of a region between the anterior centrosome and the anterior cortex, or laser irradiation of the whole anterior centrosome. A schematic of the procedure for each condition is shown on the left, with the region irradiated marked with an asterisk. Error bars represent SD over, from top to bottom, 8, 8, and 16 embryos, respectively, for each case. (D) Conceptual model depicting the various types of forces that act on the centrosomes throughout the first mitosis of the early C. elegans embryo. In this model, tethering forces are represented as lines, whereas pulling forces are depicted as springs. At late prophase/prometaphase, the pulling force present in the posterior of the embryo is counteracted by the tethering force in the anterior, thereby preventing posterior spindle displacement. During metaphase, the tethering force in the anterior changes to a pulling force.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Laser-mediated disruption of microtubule organization allows the estimation of pulling forces throughout the cell cycle. (A) Two-dimensional reconstruction of multiple confocal sections of embryos fixed and stained for α-tubulin. The centrosomes indicated by arrowheads in the right panels were irradiated before fixation. Laser irradiation specifically disrupts microtubule organization at the targeted centrosome, leaving the unirradiated centrosome largely intact. Bar, 5 μm. (B) Map (left) and quantification (right) of centrosome displacement after OICD. 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). In the right panel, displacements were averaged for various phases of the cell cycle. The timing of cell cycle events was determined according to previously reported values and matches observations made by DIC optics (Labbé et al., 2003). Error bars represent SD over, from top to bottom, 6, 4, 19, 16, 13, and 8 embryos, respectively, for each case. (C) Posterior centrosome movement quantified following no irradiation, laser irradiation of a region between the anterior centrosome and the anterior cortex, or laser irradiation of the whole anterior centrosome. A schematic of the procedure for each condition is shown on the left, with the region irradiated marked with an asterisk. Error bars represent SD over, from top to bottom, 8, 8, and 16 embryos, respectively, for each case. (D) Conceptual model depicting the various types of forces that act on the centrosomes throughout the first mitosis of the early C. elegans embryo. In this model, tethering forces are represented as lines, whereas pulling forces are depicted as springs. At late prophase/prometaphase, the pulling force present in the posterior of the embryo is counteracted by the tethering force in the anterior, thereby preventing posterior spindle displacement. During metaphase, the tethering force in the anterior changes to a pulling force.
Mentions: To characterize the forces that drive posterior spindle displacement, we examined in detail the regulation of spindle-positioning pulling forces through the first cell cycle of the C. elegans embryo. Previous experiments have used spindle severing to reveal that spindle poles are pulled apart during anaphase B (Grill et al., 2001), indicating that pulling forces are acting on spindle poles during this stage of the cell cycle. To examine the forces when posterior spindle displacement begins and also throughout the cell cycle, we used an approach termed optically induced centrosome disruption (OICD; Grill et al., 2003), in which we laser irradiated either the anterior or posterior centrosome during various phases of the cell cycle and monitored the resulting movement of the nonirradiated centrosome (see Materials and methods). Our OICD experiments conducted during anaphase succeeded in replicating previous spindle-cutting results (Grill et al., 2001). We found that the OICD procedure was efficient in disrupting one of the two asters without detectably affecting the organization of the nonirradiated one (Fig. 3 A and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). After irradiation of one of the two centrosomes, the spindle was left with only one of the asters maintaining extensive connection with the cell cortex. Quantification of centrosome movement was used to estimate the net relative vectorial force applied on this aster (Fig. S3 and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200406008/DC1). Using this approach, we generated a map of relative pulling forces acting on each aster throughout the cell cycle (Fig. 3 B). Because the timing of rotation is variable during centration in wild-type embryos, centrosome irradiations that were performed during rotation were performed in the embryos that had undergone a minimum of 45° rotation before having reached half the distance to the center of the 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