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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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Confirming kinetochore and cohesin inactivation. (A) Chromosome attachment schematic. Correct attachment of a chromosome to the spindle results in biorientation, visualized as two GFP dots separated by ≤1 μm. A single GFP dot is a kinetochore pair separated by <0.3 μm or a chromosome attached to only one or neither spindle pole (red dots). Cohesin-less chromosomes separate prematurely during metaphase. GFP dots at poles are separated by >1 μm. (B) Chromosome biorientation. ndc10-1 and PGAL1-MCD1 strains have few bioriented chromosomes compared with wild type. Error bars are SDs. (C) Chromosome position. Positions of GFP dots were scored to distinguish between unattached (one dot, ndc10-1) and prematurely separated sister chromatids (two dots, PGAL1-MCD1). Error bars are SDs. Scale bar, 3 μm.
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Figure 3: Confirming kinetochore and cohesin inactivation. (A) Chromosome attachment schematic. Correct attachment of a chromosome to the spindle results in biorientation, visualized as two GFP dots separated by ≤1 μm. A single GFP dot is a kinetochore pair separated by <0.3 μm or a chromosome attached to only one or neither spindle pole (red dots). Cohesin-less chromosomes separate prematurely during metaphase. GFP dots at poles are separated by >1 μm. (B) Chromosome biorientation. ndc10-1 and PGAL1-MCD1 strains have few bioriented chromosomes compared with wild type. Error bars are SDs. (C) Chromosome position. Positions of GFP dots were scored to distinguish between unattached (one dot, ndc10-1) and prematurely separated sister chromatids (two dots, PGAL1-MCD1). Error bars are SDs. Scale bar, 3 μm.

Mentions: To confirm that kinetochores were inactivated in ndc10-1 cells and cohesin function was lost in glucose-grown PGAL1-MCD1 cells, we scored the position of sister chromatids. When a chromosome is bioriented, its sister chromatids come under tension as they are pulled toward opposite poles but kept from separating by cohesin (Figure 1A). This conflict visibly separates the sister kinetochores and their associated centromeric DNA from each other, as seen by placing a LacO array near the centromere (200 base pairs downstream of CEN15 (Supplemental Table S1) and labeling it with GFP-LacI (Figure 3A; Goshima and Yanagida, 2000). The presence of two GFP dots separated by 0.2–1 μm indicates a bioriented chromosome, and a single GFP dot indicates a bioriented chromosome whose sister kinetochores have not stretched far enough apart to be resolved by light microscopy or a mono-oriented chromosome (Figure 3A). In wild-type cells with functional kinetochores and cohesin, 49 ± 2.8% of cells with GFP-labeled chromosome XV had two dots, indicating biorientation. In ndc10-1, only 4 ± 3.7% of cells had two GFP dots, and 96% had a single GFP dot, confirming kinetochore inactivation (Figure 3B). Similar to ndc10-1, only 14 ± 2.6% of the glucose-treated PGAL1-MCD1 cells had the two dots separated by the small distances (0.2–1.0 μm) that indicate a bioriented chromosome, but unlike ndc10-1, 77 ± 2.6% of PGAL1-MCD1 cells have two GFP dots localized near either spindle pole body. These dots are >1 μm apart and are located close to the spindle poles, showing that the sister chromatids were prematurely separated in metaphase, as expected if kinetochores were functional but cohesin was not (Figure 2C).


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)

Confirming kinetochore and cohesin inactivation. (A) Chromosome attachment schematic. Correct attachment of a chromosome to the spindle results in biorientation, visualized as two GFP dots separated by ≤1 μm. A single GFP dot is a kinetochore pair separated by <0.3 μm or a chromosome attached to only one or neither spindle pole (red dots). Cohesin-less chromosomes separate prematurely during metaphase. GFP dots at poles are separated by >1 μm. (B) Chromosome biorientation. ndc10-1 and PGAL1-MCD1 strains have few bioriented chromosomes compared with wild type. Error bars are SDs. (C) Chromosome position. Positions of GFP dots were scored to distinguish between unattached (one dot, ndc10-1) and prematurely separated sister chromatids (two dots, PGAL1-MCD1). Error bars are SDs. Scale bar, 3 μm.
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Related In: Results  -  Collection

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Figure 3: Confirming kinetochore and cohesin inactivation. (A) Chromosome attachment schematic. Correct attachment of a chromosome to the spindle results in biorientation, visualized as two GFP dots separated by ≤1 μm. A single GFP dot is a kinetochore pair separated by <0.3 μm or a chromosome attached to only one or neither spindle pole (red dots). Cohesin-less chromosomes separate prematurely during metaphase. GFP dots at poles are separated by >1 μm. (B) Chromosome biorientation. ndc10-1 and PGAL1-MCD1 strains have few bioriented chromosomes compared with wild type. Error bars are SDs. (C) Chromosome position. Positions of GFP dots were scored to distinguish between unattached (one dot, ndc10-1) and prematurely separated sister chromatids (two dots, PGAL1-MCD1). Error bars are SDs. Scale bar, 3 μm.
Mentions: To confirm that kinetochores were inactivated in ndc10-1 cells and cohesin function was lost in glucose-grown PGAL1-MCD1 cells, we scored the position of sister chromatids. When a chromosome is bioriented, its sister chromatids come under tension as they are pulled toward opposite poles but kept from separating by cohesin (Figure 1A). This conflict visibly separates the sister kinetochores and their associated centromeric DNA from each other, as seen by placing a LacO array near the centromere (200 base pairs downstream of CEN15 (Supplemental Table S1) and labeling it with GFP-LacI (Figure 3A; Goshima and Yanagida, 2000). The presence of two GFP dots separated by 0.2–1 μm indicates a bioriented chromosome, and a single GFP dot indicates a bioriented chromosome whose sister kinetochores have not stretched far enough apart to be resolved by light microscopy or a mono-oriented chromosome (Figure 3A). In wild-type cells with functional kinetochores and cohesin, 49 ± 2.8% of cells with GFP-labeled chromosome XV had two dots, indicating biorientation. In ndc10-1, only 4 ± 3.7% of cells had two GFP dots, and 96% had a single GFP dot, confirming kinetochore inactivation (Figure 3B). Similar to ndc10-1, only 14 ± 2.6% of the glucose-treated PGAL1-MCD1 cells had the two dots separated by the small distances (0.2–1.0 μm) that indicate a bioriented chromosome, but unlike ndc10-1, 77 ± 2.6% of PGAL1-MCD1 cells have two GFP dots localized near either spindle pole body. These dots are >1 μm apart and are located close to the spindle poles, showing that the sister chromatids were prematurely separated in metaphase, as expected if kinetochores were functional but cohesin was not (Figure 2C).

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