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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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Chromosomes restrain spindle elongation. (A) Spindle length measurement. Cells were released from G1 into a metaphase arrest. Spindle length (yellow bar), labeled spindle pole bodies (Spc42-mCherry), and GFP CEN15 are indicated. Scale bar, 2 μm. Temperature-sensitive strains and their control were grown and G1 arrested at 23°C and then arrested in metaphase at 37°C; other strains were grown at 30°C. (B) Spindles elongate without kinetochores or cohesin. Fluorescence images of wild-type, ndc10-1, and PGAL1-MCD1 spindles. PGAL1-MCD1 cells were grown in glucose (Glu) to repress cohesin. Spindle pole bodies (red, Spc42-mCherry) and CEN15 (green, GFP-LacI bound to LacO array) are visible. Scale bar, 3 μm. (C) Effect of kinetochores, cohesin, or motors on spindle length. Spindle length in ndc10-1 strains and glucose-grown PGAL1-MCD1 strains is compared with wild-type cells grown at both temperatures and galactose-grown PGAL1-MCD1 strains. Elongation was statistically significant (p < 0.001, Student's t test). Deleting Kip1, a kinesin, shortens the spindle, whereas eliminating both Kip1 and Ndc10 allows the spindle to approach wild-type length. Spindle length was measured as the 3D distance between spindle pole bodies (n >120 cells). Error bars are SDs in average spindle length. (D) Distribution of spindle lengths. Wild type has a tight distribution compared with ndc10-1 and glucose-grown PGAL1-MCD1.
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Figure 2: Chromosomes restrain spindle elongation. (A) Spindle length measurement. Cells were released from G1 into a metaphase arrest. Spindle length (yellow bar), labeled spindle pole bodies (Spc42-mCherry), and GFP CEN15 are indicated. Scale bar, 2 μm. Temperature-sensitive strains and their control were grown and G1 arrested at 23°C and then arrested in metaphase at 37°C; other strains were grown at 30°C. (B) Spindles elongate without kinetochores or cohesin. Fluorescence images of wild-type, ndc10-1, and PGAL1-MCD1 spindles. PGAL1-MCD1 cells were grown in glucose (Glu) to repress cohesin. Spindle pole bodies (red, Spc42-mCherry) and CEN15 (green, GFP-LacI bound to LacO array) are visible. Scale bar, 3 μm. (C) Effect of kinetochores, cohesin, or motors on spindle length. Spindle length in ndc10-1 strains and glucose-grown PGAL1-MCD1 strains is compared with wild-type cells grown at both temperatures and galactose-grown PGAL1-MCD1 strains. Elongation was statistically significant (p < 0.001, Student's t test). Deleting Kip1, a kinesin, shortens the spindle, whereas eliminating both Kip1 and Ndc10 allows the spindle to approach wild-type length. Spindle length was measured as the 3D distance between spindle pole bodies (n >120 cells). Error bars are SDs in average spindle length. (D) Distribution of spindle lengths. Wild type has a tight distribution compared with ndc10-1 and glucose-grown PGAL1-MCD1.

Mentions: Does kinetochore inactivation lead to spindle elongation? We used the ndc10-1 mutant to produce cells with no chromosomal attachments and two manipulations to control the progress of the cell cycle: treating cells with a mating pheromone, α-factor, to arrest them in G1, and removing Cdc20, an activator of the anaphase-promoting complex (APC), to arrest cells in metaphase (Hartwell et al., 1973; Hartwell and Smith, 1985). Wild-type and ndc10-1 cells were grown to log phase at 23°C, synchronized in G1 with α-factor, and released into a Cdc20 depletion–induced metaphase arrest at 37°C (Figure 2A). Spindle pole bodies were labeled by fusing a red fluorescent protein (RFP) variant to a spindle pole body component (SPC42-mCherry), and chromosome XV was labeled with green fluorescent protein (GFP) by inserting a Lac operator (LacO) array ∼200 base pairs downstream of CEN15 (see Supplemental Table S1 for exact position; Shonn et al., 2000) and expressing a GFP-Lac repressor fusion (GFP-LACI; Straight et al., 1996). Spindle length was measured as the three-dimensional (3D) distance between the two spindle poles and reported as the average length of measured spindles; error bars are SDs of average spindle length over all trials. Figure 2B shows examples of fluorescent spindles.


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)

Chromosomes restrain spindle elongation. (A) Spindle length measurement. Cells were released from G1 into a metaphase arrest. Spindle length (yellow bar), labeled spindle pole bodies (Spc42-mCherry), and GFP CEN15 are indicated. Scale bar, 2 μm. Temperature-sensitive strains and their control were grown and G1 arrested at 23°C and then arrested in metaphase at 37°C; other strains were grown at 30°C. (B) Spindles elongate without kinetochores or cohesin. Fluorescence images of wild-type, ndc10-1, and PGAL1-MCD1 spindles. PGAL1-MCD1 cells were grown in glucose (Glu) to repress cohesin. Spindle pole bodies (red, Spc42-mCherry) and CEN15 (green, GFP-LacI bound to LacO array) are visible. Scale bar, 3 μm. (C) Effect of kinetochores, cohesin, or motors on spindle length. Spindle length in ndc10-1 strains and glucose-grown PGAL1-MCD1 strains is compared with wild-type cells grown at both temperatures and galactose-grown PGAL1-MCD1 strains. Elongation was statistically significant (p < 0.001, Student's t test). Deleting Kip1, a kinesin, shortens the spindle, whereas eliminating both Kip1 and Ndc10 allows the spindle to approach wild-type length. Spindle length was measured as the 3D distance between spindle pole bodies (n >120 cells). Error bars are SDs in average spindle length. (D) Distribution of spindle lengths. Wild type has a tight distribution compared with ndc10-1 and glucose-grown PGAL1-MCD1.
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Related In: Results  -  Collection

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Figure 2: Chromosomes restrain spindle elongation. (A) Spindle length measurement. Cells were released from G1 into a metaphase arrest. Spindle length (yellow bar), labeled spindle pole bodies (Spc42-mCherry), and GFP CEN15 are indicated. Scale bar, 2 μm. Temperature-sensitive strains and their control were grown and G1 arrested at 23°C and then arrested in metaphase at 37°C; other strains were grown at 30°C. (B) Spindles elongate without kinetochores or cohesin. Fluorescence images of wild-type, ndc10-1, and PGAL1-MCD1 spindles. PGAL1-MCD1 cells were grown in glucose (Glu) to repress cohesin. Spindle pole bodies (red, Spc42-mCherry) and CEN15 (green, GFP-LacI bound to LacO array) are visible. Scale bar, 3 μm. (C) Effect of kinetochores, cohesin, or motors on spindle length. Spindle length in ndc10-1 strains and glucose-grown PGAL1-MCD1 strains is compared with wild-type cells grown at both temperatures and galactose-grown PGAL1-MCD1 strains. Elongation was statistically significant (p < 0.001, Student's t test). Deleting Kip1, a kinesin, shortens the spindle, whereas eliminating both Kip1 and Ndc10 allows the spindle to approach wild-type length. Spindle length was measured as the 3D distance between spindle pole bodies (n >120 cells). Error bars are SDs in average spindle length. (D) Distribution of spindle lengths. Wild type has a tight distribution compared with ndc10-1 and glucose-grown PGAL1-MCD1.
Mentions: Does kinetochore inactivation lead to spindle elongation? We used the ndc10-1 mutant to produce cells with no chromosomal attachments and two manipulations to control the progress of the cell cycle: treating cells with a mating pheromone, α-factor, to arrest them in G1, and removing Cdc20, an activator of the anaphase-promoting complex (APC), to arrest cells in metaphase (Hartwell et al., 1973; Hartwell and Smith, 1985). Wild-type and ndc10-1 cells were grown to log phase at 23°C, synchronized in G1 with α-factor, and released into a Cdc20 depletion–induced metaphase arrest at 37°C (Figure 2A). Spindle pole bodies were labeled by fusing a red fluorescent protein (RFP) variant to a spindle pole body component (SPC42-mCherry), and chromosome XV was labeled with green fluorescent protein (GFP) by inserting a Lac operator (LacO) array ∼200 base pairs downstream of CEN15 (see Supplemental Table S1 for exact position; Shonn et al., 2000) and expressing a GFP-Lac repressor fusion (GFP-LACI; Straight et al., 1996). Spindle length was measured as the three-dimensional (3D) distance between the two spindle poles and reported as the average length of measured spindles; error bars are SDs of average spindle length over all trials. Figure 2B shows examples of fluorescent spindles.

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