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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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Synthetic kinetochores shorten ndc10-1 spindles. (A) Natural and synthetic kinetochores. The budding yeast kinetochore contains many proteins and binds the 125–base pair centromeric DNA. The CBF3 complex (Ndc10, Ctf13, Cep3, and Skp1) binds centromere DNA. The synthetic kinetochore depends on binding of an Ask1-LacI fusion to a LacO array, recruiting other kinetochore components independently of CBF3. (B) Prediction. Adding synthetic kinetochores (sKT) to ndc10-1 cells will shorten their spindles. (C) Synthetic kinetochores shorten ndc10-1 spindles. Each additional synthetic kinetochore shortened spindles significantly (p < 0.04, Student's t test). Error bars are SDs in average spindle length. (D) Shorter spindles require Ask1-LacI. Cells expressing PGAL1-ASK1-LacI were grown in raffinose, synchronized in G1, and released into metaphase arrest in glucose (repressing) or galactose (inducing). Wild-type cells had similar-length spindles in glucose and galactose (p = 0.36, Student's t test), but repressing Ask1-LacI lengthened spindles in ndc10-1 cells carrying a synthetic kinetochore (p = 0.02, Student's t test). Error bars are SDs in average spindle length.
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Figure 4: Synthetic kinetochores shorten ndc10-1 spindles. (A) Natural and synthetic kinetochores. The budding yeast kinetochore contains many proteins and binds the 125–base pair centromeric DNA. The CBF3 complex (Ndc10, Ctf13, Cep3, and Skp1) binds centromere DNA. The synthetic kinetochore depends on binding of an Ask1-LacI fusion to a LacO array, recruiting other kinetochore components independently of CBF3. (B) Prediction. Adding synthetic kinetochores (sKT) to ndc10-1 cells will shorten their spindles. (C) Synthetic kinetochores shorten ndc10-1 spindles. Each additional synthetic kinetochore shortened spindles significantly (p < 0.04, Student's t test). Error bars are SDs in average spindle length. (D) Shorter spindles require Ask1-LacI. Cells expressing PGAL1-ASK1-LacI were grown in raffinose, synchronized in G1, and released into metaphase arrest in glucose (repressing) or galactose (inducing). Wild-type cells had similar-length spindles in glucose and galactose (p = 0.36, Student's t test), but repressing Ask1-LacI lengthened spindles in ndc10-1 cells carrying a synthetic kinetochore (p = 0.02, Student's t test). Error bars are SDs in average spindle length.

Mentions: Synthetic kinetochores can replace natural kinetochores by biorienting and segregating chromosomes (Kiermaier et al., 2009; Lacefield et al., 2009). Recruiting a fusion of the Lac repressor and Ask1, a component of the microtubule-binding Dam1 complex, to a LacO array creates a synthetic kinetochore that bypasses the need for CBF3 and thus does not require Ndc10 or Ctf13 (Figure 4A; Lacefield et al., 2009). We introduced the synthetic kinetochore (sKT) to ndc10-1 cells by integrating LacO arrays on a one, two, or three chromosomes, thus reattaching them to the mitotic spindle (Figure 4B).


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)

Synthetic kinetochores shorten ndc10-1 spindles. (A) Natural and synthetic kinetochores. The budding yeast kinetochore contains many proteins and binds the 125–base pair centromeric DNA. The CBF3 complex (Ndc10, Ctf13, Cep3, and Skp1) binds centromere DNA. The synthetic kinetochore depends on binding of an Ask1-LacI fusion to a LacO array, recruiting other kinetochore components independently of CBF3. (B) Prediction. Adding synthetic kinetochores (sKT) to ndc10-1 cells will shorten their spindles. (C) Synthetic kinetochores shorten ndc10-1 spindles. Each additional synthetic kinetochore shortened spindles significantly (p < 0.04, Student's t test). Error bars are SDs in average spindle length. (D) Shorter spindles require Ask1-LacI. Cells expressing PGAL1-ASK1-LacI were grown in raffinose, synchronized in G1, and released into metaphase arrest in glucose (repressing) or galactose (inducing). Wild-type cells had similar-length spindles in glucose and galactose (p = 0.36, Student's t test), but repressing Ask1-LacI lengthened spindles in ndc10-1 cells carrying a synthetic kinetochore (p = 0.02, Student's t test). Error bars are SDs in average spindle length.
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Related In: Results  -  Collection

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Figure 4: Synthetic kinetochores shorten ndc10-1 spindles. (A) Natural and synthetic kinetochores. The budding yeast kinetochore contains many proteins and binds the 125–base pair centromeric DNA. The CBF3 complex (Ndc10, Ctf13, Cep3, and Skp1) binds centromere DNA. The synthetic kinetochore depends on binding of an Ask1-LacI fusion to a LacO array, recruiting other kinetochore components independently of CBF3. (B) Prediction. Adding synthetic kinetochores (sKT) to ndc10-1 cells will shorten their spindles. (C) Synthetic kinetochores shorten ndc10-1 spindles. Each additional synthetic kinetochore shortened spindles significantly (p < 0.04, Student's t test). Error bars are SDs in average spindle length. (D) Shorter spindles require Ask1-LacI. Cells expressing PGAL1-ASK1-LacI were grown in raffinose, synchronized in G1, and released into metaphase arrest in glucose (repressing) or galactose (inducing). Wild-type cells had similar-length spindles in glucose and galactose (p = 0.36, Student's t test), but repressing Ask1-LacI lengthened spindles in ndc10-1 cells carrying a synthetic kinetochore (p = 0.02, Student's t test). Error bars are SDs in average spindle length.
Mentions: Synthetic kinetochores can replace natural kinetochores by biorienting and segregating chromosomes (Kiermaier et al., 2009; Lacefield et al., 2009). Recruiting a fusion of the Lac repressor and Ask1, a component of the microtubule-binding Dam1 complex, to a LacO array creates a synthetic kinetochore that bypasses the need for CBF3 and thus does not require Ndc10 or Ctf13 (Figure 4A; Lacefield et al., 2009). We introduced the synthetic kinetochore (sKT) to ndc10-1 cells by integrating LacO arrays on a one, two, or three chromosomes, thus reattaching them to the mitotic spindle (Figure 4B).

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