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Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle.

Nannas NJ, O'Toole ET, Winey M, Murray AW - Mol. Biol. Cell (2014)

Bottom Line: The length of the mitotic spindle varies among different cell types.A simple model for spindle length regulation requires balancing two forces: pulling, due to micro-tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles.In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore-microtubule interactions generate an inward force to balance forces that elongate the spindle.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Department, Harvard University, Cambridge, MA 02138 FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.

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Extra kinetochores shorten spindles. (A) Plasmid schematic. The centromeric plasmid assembles a kinetochore (yellow circle) and carries the CUP1 copper resistance gene (green box). Replicated plasmids attach to the spindle via their kinetochores. (B) Driving up plasmid copy number. Plasmid-bearing cells were grown in increasing concentrations of CuSO4 to select for increased plasmid number. (C) Spindle length in strains containing an average of 5, 10, 20, or 30 centromeric plasmids. Spindles are shorter in cells with extra kinetochores and shorten with increasing kinetochore number (p < 0.001, Student's t test). Error bars are SDs in average length. (D) Spindle length scales with attachment number. Each data point is the average spindle length in an independent trial. (E) Extra kinetochores do not alter anaphase dynamics. Wild-type cells (red lines, n = 12) and cells with 10 plasmids (blue lines, n = 12) were synchronized in G1 and released to proceed through mitosis; spindle length was tracked in individual live cells. The time to enter anaphase, maximum length of the anaphase spindle, and rate of anaphase spindle elongation were statistically indistinguishable between wild-type and plasmid-containing cells (p > 0.5 for all parameters, Student's t test). (F) Frames from anaphase movies. Wild type and cells containing 10 plasmids were released from G1 at T = 0:00 (time represented as hours:minutes) and allowed to proceed through mitosis. Frames displayed from the 10-plasmid movie jumps from T = 0:45 to 1:00 to show the second cell entering anaphase. Spindle pole bodies are labeled with mCherry, and spindles are labeled with GFP-tubulin; scale bars, 3 μm. (G, H) Intercentromere separation in the presence of extra kinetochores. (G) Percentage of cells with two close GFP dots signifying a correctly attached chromosome in cells containing a range of plasmids. Error bars are SDs. (H) Mean distance between sister chromatids. Error bars are SDs in average separation.
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Figure 5: Extra kinetochores shorten spindles. (A) Plasmid schematic. The centromeric plasmid assembles a kinetochore (yellow circle) and carries the CUP1 copper resistance gene (green box). Replicated plasmids attach to the spindle via their kinetochores. (B) Driving up plasmid copy number. Plasmid-bearing cells were grown in increasing concentrations of CuSO4 to select for increased plasmid number. (C) Spindle length in strains containing an average of 5, 10, 20, or 30 centromeric plasmids. Spindles are shorter in cells with extra kinetochores and shorten with increasing kinetochore number (p < 0.001, Student's t test). Error bars are SDs in average length. (D) Spindle length scales with attachment number. Each data point is the average spindle length in an independent trial. (E) Extra kinetochores do not alter anaphase dynamics. Wild-type cells (red lines, n = 12) and cells with 10 plasmids (blue lines, n = 12) were synchronized in G1 and released to proceed through mitosis; spindle length was tracked in individual live cells. The time to enter anaphase, maximum length of the anaphase spindle, and rate of anaphase spindle elongation were statistically indistinguishable between wild-type and plasmid-containing cells (p > 0.5 for all parameters, Student's t test). (F) Frames from anaphase movies. Wild type and cells containing 10 plasmids were released from G1 at T = 0:00 (time represented as hours:minutes) and allowed to proceed through mitosis. Frames displayed from the 10-plasmid movie jumps from T = 0:45 to 1:00 to show the second cell entering anaphase. Spindle pole bodies are labeled with mCherry, and spindles are labeled with GFP-tubulin; scale bars, 3 μm. (G, H) Intercentromere separation in the presence of extra kinetochores. (G) Percentage of cells with two close GFP dots signifying a correctly attached chromosome in cells containing a range of plasmids. Error bars are SDs. (H) Mean distance between sister chromatids. Error bars are SDs in average separation.

Mentions: Adding extra kinetochores to wild-type cells should shorten their spindles by creating more attachments between the spindle poles and the chromosomes (Figure 1D). We introduced additional kinetochores in the form of minichromosomes or centromeric plasmids (Figure 5A), which assemble a fully functional kinetochore (Clarke and Carbon, 1980). Multiple centromeric plasmids slow the cell cycle (Futcher and Carbon, 1986), but this delay depends on the spindle checkpoint and is completely eliminated in mad2∆ strains (Wells and Murray, 1996), which lack the checkpoint. We therefore used checkpoint-deficient cells to allow us to add substantial numbers of extra kinetochores without delaying progress through mitosis.


Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle.

Nannas NJ, O'Toole ET, Winey M, Murray AW - Mol. Biol. Cell (2014)

Extra kinetochores shorten spindles. (A) Plasmid schematic. The centromeric plasmid assembles a kinetochore (yellow circle) and carries the CUP1 copper resistance gene (green box). Replicated plasmids attach to the spindle via their kinetochores. (B) Driving up plasmid copy number. Plasmid-bearing cells were grown in increasing concentrations of CuSO4 to select for increased plasmid number. (C) Spindle length in strains containing an average of 5, 10, 20, or 30 centromeric plasmids. Spindles are shorter in cells with extra kinetochores and shorten with increasing kinetochore number (p < 0.001, Student's t test). Error bars are SDs in average length. (D) Spindle length scales with attachment number. Each data point is the average spindle length in an independent trial. (E) Extra kinetochores do not alter anaphase dynamics. Wild-type cells (red lines, n = 12) and cells with 10 plasmids (blue lines, n = 12) were synchronized in G1 and released to proceed through mitosis; spindle length was tracked in individual live cells. The time to enter anaphase, maximum length of the anaphase spindle, and rate of anaphase spindle elongation were statistically indistinguishable between wild-type and plasmid-containing cells (p > 0.5 for all parameters, Student's t test). (F) Frames from anaphase movies. Wild type and cells containing 10 plasmids were released from G1 at T = 0:00 (time represented as hours:minutes) and allowed to proceed through mitosis. Frames displayed from the 10-plasmid movie jumps from T = 0:45 to 1:00 to show the second cell entering anaphase. Spindle pole bodies are labeled with mCherry, and spindles are labeled with GFP-tubulin; scale bars, 3 μm. (G, H) Intercentromere separation in the presence of extra kinetochores. (G) Percentage of cells with two close GFP dots signifying a correctly attached chromosome in cells containing a range of plasmids. Error bars are SDs. (H) Mean distance between sister chromatids. Error bars are SDs in average separation.
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Related In: Results  -  Collection

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Figure 5: Extra kinetochores shorten spindles. (A) Plasmid schematic. The centromeric plasmid assembles a kinetochore (yellow circle) and carries the CUP1 copper resistance gene (green box). Replicated plasmids attach to the spindle via their kinetochores. (B) Driving up plasmid copy number. Plasmid-bearing cells were grown in increasing concentrations of CuSO4 to select for increased plasmid number. (C) Spindle length in strains containing an average of 5, 10, 20, or 30 centromeric plasmids. Spindles are shorter in cells with extra kinetochores and shorten with increasing kinetochore number (p < 0.001, Student's t test). Error bars are SDs in average length. (D) Spindle length scales with attachment number. Each data point is the average spindle length in an independent trial. (E) Extra kinetochores do not alter anaphase dynamics. Wild-type cells (red lines, n = 12) and cells with 10 plasmids (blue lines, n = 12) were synchronized in G1 and released to proceed through mitosis; spindle length was tracked in individual live cells. The time to enter anaphase, maximum length of the anaphase spindle, and rate of anaphase spindle elongation were statistically indistinguishable between wild-type and plasmid-containing cells (p > 0.5 for all parameters, Student's t test). (F) Frames from anaphase movies. Wild type and cells containing 10 plasmids were released from G1 at T = 0:00 (time represented as hours:minutes) and allowed to proceed through mitosis. Frames displayed from the 10-plasmid movie jumps from T = 0:45 to 1:00 to show the second cell entering anaphase. Spindle pole bodies are labeled with mCherry, and spindles are labeled with GFP-tubulin; scale bars, 3 μm. (G, H) Intercentromere separation in the presence of extra kinetochores. (G) Percentage of cells with two close GFP dots signifying a correctly attached chromosome in cells containing a range of plasmids. Error bars are SDs. (H) Mean distance between sister chromatids. Error bars are SDs in average separation.
Mentions: Adding extra kinetochores to wild-type cells should shorten their spindles by creating more attachments between the spindle poles and the chromosomes (Figure 1D). We introduced additional kinetochores in the form of minichromosomes or centromeric plasmids (Figure 5A), which assemble a fully functional kinetochore (Clarke and Carbon, 1980). Multiple centromeric plasmids slow the cell cycle (Futcher and Carbon, 1986), but this delay depends on the spindle checkpoint and is completely eliminated in mad2∆ strains (Wells and Murray, 1996), which lack the checkpoint. We therefore used checkpoint-deficient cells to allow us to add substantial numbers of extra kinetochores without delaying progress through mitosis.

Bottom Line: The length of the mitotic spindle varies among different cell types.A simple model for spindle length regulation requires balancing two forces: pulling, due to micro-tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles.In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore-microtubule interactions generate an inward force to balance forces that elongate the spindle.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Department, Harvard University, Cambridge, MA 02138 FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138.

Show MeSH
Related in: MedlinePlus