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Budding yeast chromosome structure and dynamics during mitosis.

Pearson CG, Maddox PS, Salmon ED, Bloom K - J. Cell Biol. (2001)

Bottom Line: Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase.The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere.These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. cgpearso@email.unc.edu

ABSTRACT
Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

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Preanaphase separation and positioning of sister centromeres. Centromeres displayed the greatest average separation with progressively decreasing separation of chromosome markers as they were placed distal from the centromere. (A) Pan-specific centromere marker for all centromeres, Cse4–GFP (green) with Spc29–CFP (red) marked spindle pole bodies. Centromeres clustered into discrete groups that were separated toward the spindle pole bodies. (B) LacO GFP marker placed ∼1.1 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (C) LacO GFP marker placed ∼12.7 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (D) LacO GFP marker placed ∼23 kb from CEN3 (arrows) with Tub3–GFP-labeled spindle (arrowhead). (E) Schematic representing the above average preanaphase separations (Table ). SPB, spindle pole body. Bar, 2 μm.
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Figure 1: Preanaphase separation and positioning of sister centromeres. Centromeres displayed the greatest average separation with progressively decreasing separation of chromosome markers as they were placed distal from the centromere. (A) Pan-specific centromere marker for all centromeres, Cse4–GFP (green) with Spc29–CFP (red) marked spindle pole bodies. Centromeres clustered into discrete groups that were separated toward the spindle pole bodies. (B) LacO GFP marker placed ∼1.1 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (C) LacO GFP marker placed ∼12.7 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (D) LacO GFP marker placed ∼23 kb from CEN3 (arrows) with Tub3–GFP-labeled spindle (arrowhead). (E) Schematic representing the above average preanaphase separations (Table ). SPB, spindle pole body. Bar, 2 μm.

Mentions: To test how sister centromeres and centromere proximal chromosome arms respond to forces generated by kinetochores attached to the mitotic spindle, we used fluorescent probes for centromeres, chromosome arms, and spindle pole bodies (Fig. 1). In live large budded cells before anaphase onset, centromeres labeled with Cse4–GFP were often observed as two clusters or groups separated on average by 0.58 ± 0.36 μm (minimum = 0 μm, maximum = 1.84 μm; n = 63) (Fig. 1 A; Table ). In contrast, average separation of the ∼1.1-kb centromere proximal spot was 0.26 ± 0.32 μm (min = 0 μm, max = 1.48 μm; n = 95) (Fig. 1 B; Table ). This is ∼0.32 μm less separated than the average separation of the Cse4–GFP centromere marker (Fig. 1A and Fig. B; Table ). Chromosome arms with lac operator repeats integrated more distal to the centromere were on average separated by 0.07 ± 0.19 μm (min = 0 μm, max = 0.85 μm; n = 94) at loci ∼12.7-kb CEN11 and were observed as a single unseparated spot when integrated ∼23 kb from CEN3 (n = 101) (Fig. 1C and Fig. D; Table ). The centromere separation and outward stretch of the arms near the centromeric region shown in Fig. 1 E is consistent with previous reports that centromeres are on average separated before anaphase onset by poleward forces (Goshima and Yanagida 2000; He et al. 2000; Tanaka et al. 2000).


Budding yeast chromosome structure and dynamics during mitosis.

Pearson CG, Maddox PS, Salmon ED, Bloom K - J. Cell Biol. (2001)

Preanaphase separation and positioning of sister centromeres. Centromeres displayed the greatest average separation with progressively decreasing separation of chromosome markers as they were placed distal from the centromere. (A) Pan-specific centromere marker for all centromeres, Cse4–GFP (green) with Spc29–CFP (red) marked spindle pole bodies. Centromeres clustered into discrete groups that were separated toward the spindle pole bodies. (B) LacO GFP marker placed ∼1.1 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (C) LacO GFP marker placed ∼12.7 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (D) LacO GFP marker placed ∼23 kb from CEN3 (arrows) with Tub3–GFP-labeled spindle (arrowhead). (E) Schematic representing the above average preanaphase separations (Table ). SPB, spindle pole body. Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199205&req=5

Figure 1: Preanaphase separation and positioning of sister centromeres. Centromeres displayed the greatest average separation with progressively decreasing separation of chromosome markers as they were placed distal from the centromere. (A) Pan-specific centromere marker for all centromeres, Cse4–GFP (green) with Spc29–CFP (red) marked spindle pole bodies. Centromeres clustered into discrete groups that were separated toward the spindle pole bodies. (B) LacO GFP marker placed ∼1.1 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (C) LacO GFP marker placed ∼12.7 kb from CEN11 (arrows) with Spc72–GFP-labeled spindle pole bodies (arrowheads). (D) LacO GFP marker placed ∼23 kb from CEN3 (arrows) with Tub3–GFP-labeled spindle (arrowhead). (E) Schematic representing the above average preanaphase separations (Table ). SPB, spindle pole body. Bar, 2 μm.
Mentions: To test how sister centromeres and centromere proximal chromosome arms respond to forces generated by kinetochores attached to the mitotic spindle, we used fluorescent probes for centromeres, chromosome arms, and spindle pole bodies (Fig. 1). In live large budded cells before anaphase onset, centromeres labeled with Cse4–GFP were often observed as two clusters or groups separated on average by 0.58 ± 0.36 μm (minimum = 0 μm, maximum = 1.84 μm; n = 63) (Fig. 1 A; Table ). In contrast, average separation of the ∼1.1-kb centromere proximal spot was 0.26 ± 0.32 μm (min = 0 μm, max = 1.48 μm; n = 95) (Fig. 1 B; Table ). This is ∼0.32 μm less separated than the average separation of the Cse4–GFP centromere marker (Fig. 1A and Fig. B; Table ). Chromosome arms with lac operator repeats integrated more distal to the centromere were on average separated by 0.07 ± 0.19 μm (min = 0 μm, max = 0.85 μm; n = 94) at loci ∼12.7-kb CEN11 and were observed as a single unseparated spot when integrated ∼23 kb from CEN3 (n = 101) (Fig. 1C and Fig. D; Table ). The centromere separation and outward stretch of the arms near the centromeric region shown in Fig. 1 E is consistent with previous reports that centromeres are on average separated before anaphase onset by poleward forces (Goshima and Yanagida 2000; He et al. 2000; Tanaka et al. 2000).

Bottom Line: Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase.The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere.These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. cgpearso@email.unc.edu

ABSTRACT
Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

Show MeSH
Related in: MedlinePlus