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Individual pericentromeres display coordinated motion and stretching in the yeast spindle.

Stephens AD, Snider CE, Haase J, Haggerty RA, Vasquez PA, Forest MG, Bloom K - J. Cell Biol. (2013)

Bottom Line: By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase.The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes.Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8.

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Affiliation: Department of Biology, 2 Department of Mathematics, and 3 Department Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

ABSTRACT
The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.

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4C reveals interactions between pericentromeres of different chromosomes. (A) Primers were used to assay interactions, via 3C technique, between the pericentromeres and arms of chromosome III and V (see Materials and methods). The interaction index is the ratio of the pericentromere (P) to the arm (A; control for random interactions) PCR product was normalized to 1. Pericentromere interaction index is shown for WT, cin8Δ, brn1-9, mcm21Δ, and GalCEN3 (conditionally off centromere). Values listed in Table 1. Error bars represent standard deviations. Asterisks denote significant difference from WT (P < 0.01). Mutants with a different number of asterisks are significantly different, whereas those with the same numbers are similar. (B) Diagram depicting the half-spindle and a results summary for WT and mutants. kMTs emanate from the spindle pole each bound to a different chromosome at the kinetochore/centromere, whereas interpericentromere interaction is facilitated by a cross-linker. Results of 4C suggest cohesin and condensin act as a cross-linker between different pericentromeres. Plus signs, statistically similar to WT; minus, decreased significantly relative to WT; double minus, decreased significantly relative to single minus; N/A, not applicable.
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fig4: 4C reveals interactions between pericentromeres of different chromosomes. (A) Primers were used to assay interactions, via 3C technique, between the pericentromeres and arms of chromosome III and V (see Materials and methods). The interaction index is the ratio of the pericentromere (P) to the arm (A; control for random interactions) PCR product was normalized to 1. Pericentromere interaction index is shown for WT, cin8Δ, brn1-9, mcm21Δ, and GalCEN3 (conditionally off centromere). Values listed in Table 1. Error bars represent standard deviations. Asterisks denote significant difference from WT (P < 0.01). Mutants with a different number of asterisks are significantly different, whereas those with the same numbers are similar. (B) Diagram depicting the half-spindle and a results summary for WT and mutants. kMTs emanate from the spindle pole each bound to a different chromosome at the kinetochore/centromere, whereas interpericentromere interaction is facilitated by a cross-linker. Results of 4C suggest cohesin and condensin act as a cross-linker between different pericentromeres. Plus signs, statistically similar to WT; minus, decreased significantly relative to WT; double minus, decreased significantly relative to single minus; N/A, not applicable.

Mentions: To determine whether pericentromeres are in physical proximity in metaphase, we adapted the 3C (chromosome conformation capture) technique to probe the interaction between two loci on different chromosomes. Inverse primer pairs were used to map the interaction of chromosome 3 and 5 at arm and pericentromere loci (see Materials and methods). Arm loci were used as a control for random interactions between chromosomes 3 and 5. We found that WT pericentromeres interact 1.75 ± 0.05–fold more than random arm interaction (pericentromere/arm, normalized to arm 1.00, n = 10; Fig. 4 A). This recapitulates initial findings of interpericentromere interactions via 3C techniques (cross-linking frequency 1.5; Dekker et al., 2002).


Individual pericentromeres display coordinated motion and stretching in the yeast spindle.

Stephens AD, Snider CE, Haase J, Haggerty RA, Vasquez PA, Forest MG, Bloom K - J. Cell Biol. (2013)

4C reveals interactions between pericentromeres of different chromosomes. (A) Primers were used to assay interactions, via 3C technique, between the pericentromeres and arms of chromosome III and V (see Materials and methods). The interaction index is the ratio of the pericentromere (P) to the arm (A; control for random interactions) PCR product was normalized to 1. Pericentromere interaction index is shown for WT, cin8Δ, brn1-9, mcm21Δ, and GalCEN3 (conditionally off centromere). Values listed in Table 1. Error bars represent standard deviations. Asterisks denote significant difference from WT (P < 0.01). Mutants with a different number of asterisks are significantly different, whereas those with the same numbers are similar. (B) Diagram depicting the half-spindle and a results summary for WT and mutants. kMTs emanate from the spindle pole each bound to a different chromosome at the kinetochore/centromere, whereas interpericentromere interaction is facilitated by a cross-linker. Results of 4C suggest cohesin and condensin act as a cross-linker between different pericentromeres. Plus signs, statistically similar to WT; minus, decreased significantly relative to WT; double minus, decreased significantly relative to single minus; N/A, not applicable.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824013&req=5

fig4: 4C reveals interactions between pericentromeres of different chromosomes. (A) Primers were used to assay interactions, via 3C technique, between the pericentromeres and arms of chromosome III and V (see Materials and methods). The interaction index is the ratio of the pericentromere (P) to the arm (A; control for random interactions) PCR product was normalized to 1. Pericentromere interaction index is shown for WT, cin8Δ, brn1-9, mcm21Δ, and GalCEN3 (conditionally off centromere). Values listed in Table 1. Error bars represent standard deviations. Asterisks denote significant difference from WT (P < 0.01). Mutants with a different number of asterisks are significantly different, whereas those with the same numbers are similar. (B) Diagram depicting the half-spindle and a results summary for WT and mutants. kMTs emanate from the spindle pole each bound to a different chromosome at the kinetochore/centromere, whereas interpericentromere interaction is facilitated by a cross-linker. Results of 4C suggest cohesin and condensin act as a cross-linker between different pericentromeres. Plus signs, statistically similar to WT; minus, decreased significantly relative to WT; double minus, decreased significantly relative to single minus; N/A, not applicable.
Mentions: To determine whether pericentromeres are in physical proximity in metaphase, we adapted the 3C (chromosome conformation capture) technique to probe the interaction between two loci on different chromosomes. Inverse primer pairs were used to map the interaction of chromosome 3 and 5 at arm and pericentromere loci (see Materials and methods). Arm loci were used as a control for random interactions between chromosomes 3 and 5. We found that WT pericentromeres interact 1.75 ± 0.05–fold more than random arm interaction (pericentromere/arm, normalized to arm 1.00, n = 10; Fig. 4 A). This recapitulates initial findings of interpericentromere interactions via 3C techniques (cross-linking frequency 1.5; Dekker et al., 2002).

Bottom Line: By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase.The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes.Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, 2 Department of Mathematics, and 3 Department Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

ABSTRACT
The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.

Show MeSH
Related in: MedlinePlus