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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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Anaphase A centromere movement to the spindle pole body and half-spindle shortening began coincident with or shortly after the onset of anaphase B spindle pole body separation. (A) Progression through anaphase was followed using the ∼1.1-kb CEN11 marker and Nuf2–GFP to label the spindle pole bodies. Selected frames from a Z-series time-lapse sequence taken at 45-s intervals. (B) Graphical plot of the above time-lapse (A), using measurements with the Z-distance taken into account. In A and B, arrowheads denote the spindle pole bodies, and arrows define centromere proximal chromosome spots. (C) Centromere movement to the spindle pole body was coincident with the onset of anaphase B spindle pole body separation, as determined using Cse4–GFP to label all centromeres. Spindle pole bodies were labeled using Spc29–CFP, allowing two-color imaging of anaphase onset. Centromere clusters underwent a synchronous separation at anaphase onset. Frames are from a Z-series time-lapse sequence taken at 45-s intervals. (D) Graphical plot of the above time-lapse (C), with the Z-distance taken into account. (E) Half-spindle microtubule or shortening to the spindle pole body began near the onset of anaphase B spindle pole body separation. Using GFP–Tub1, we show that, upon elongation of the spindle, the fluorescent tufts emanating from each spindle pole body, presumed to be kinetochore microtubules, shortened toward the spindle pole bodies. Plots also indicate that there were dynamic movements in the lengths of half-spindle microtubules emanating from the spindle pole body. When the half-spindle had shortened to within the resolution limit of the light microscope (∼250–300 nm), integrated fluorescent intensities, standardized to the last distance measurement (6.26 min), were plotted to show that fluorescence intensities decreased, indicating that microtubule polymer further decreased from the optical resolution limited tufts (▴). Images were from a single plane acquisition time-lapse sequence at 5-s intervals. Arrows indicate the spindle pole bodies and plus ends of half-spindles. (F) Graphical plot of the above time-lapse showing plots of each spindle pole body (▪) relative to the ends of each fluorescent tuft (▴; 0–6.3 min). At 6.26–7.26 min, the fluorescent intensity of each tuft was standardized to the last distance measurement at 6.26 min and plotted relative to their respective spindle pole body (•, 6.26–7.26 min). Elapsed time for A–E is in minutes. Bars, 2 μm.
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Figure 4: Anaphase A centromere movement to the spindle pole body and half-spindle shortening began coincident with or shortly after the onset of anaphase B spindle pole body separation. (A) Progression through anaphase was followed using the ∼1.1-kb CEN11 marker and Nuf2–GFP to label the spindle pole bodies. Selected frames from a Z-series time-lapse sequence taken at 45-s intervals. (B) Graphical plot of the above time-lapse (A), using measurements with the Z-distance taken into account. In A and B, arrowheads denote the spindle pole bodies, and arrows define centromere proximal chromosome spots. (C) Centromere movement to the spindle pole body was coincident with the onset of anaphase B spindle pole body separation, as determined using Cse4–GFP to label all centromeres. Spindle pole bodies were labeled using Spc29–CFP, allowing two-color imaging of anaphase onset. Centromere clusters underwent a synchronous separation at anaphase onset. Frames are from a Z-series time-lapse sequence taken at 45-s intervals. (D) Graphical plot of the above time-lapse (C), with the Z-distance taken into account. (E) Half-spindle microtubule or shortening to the spindle pole body began near the onset of anaphase B spindle pole body separation. Using GFP–Tub1, we show that, upon elongation of the spindle, the fluorescent tufts emanating from each spindle pole body, presumed to be kinetochore microtubules, shortened toward the spindle pole bodies. Plots also indicate that there were dynamic movements in the lengths of half-spindle microtubules emanating from the spindle pole body. When the half-spindle had shortened to within the resolution limit of the light microscope (∼250–300 nm), integrated fluorescent intensities, standardized to the last distance measurement (6.26 min), were plotted to show that fluorescence intensities decreased, indicating that microtubule polymer further decreased from the optical resolution limited tufts (▴). Images were from a single plane acquisition time-lapse sequence at 5-s intervals. Arrows indicate the spindle pole bodies and plus ends of half-spindles. (F) Graphical plot of the above time-lapse showing plots of each spindle pole body (▪) relative to the ends of each fluorescent tuft (▴; 0–6.3 min). At 6.26–7.26 min, the fluorescent intensity of each tuft was standardized to the last distance measurement at 6.26 min and plotted relative to their respective spindle pole body (•, 6.26–7.26 min). Elapsed time for A–E is in minutes. Bars, 2 μm.

Mentions: The ∼1.1-kb CEN11 lacO marker was integrated into strains containing spindle pole bodies marked with Nuf2–GFP or Spc72–GFP. Z-series time-lapses were acquired at 15–45 s. We observed the ∼1.1-kb CEN11 proximal spots to move toward the GFP-marked spindle pole bodies coincident with or within 3 min after anaphase onset, as defined by the start of anaphase B spindle elongation (Fig. 4A and Fig. B and Fig. 5). Fig. 5 B (arrow) shows the separation of the spindle pole bodies to begin at the 0.0-min time point. Movement of the CEN proximal spots toward the spindle pole body ensues at 1.0 min (Fig. 5 B, arrow). This delay results in a transient increase in the distance between the centromere proximal spot and its spindle pole body. The centromere proximal spot began anaphase A chromosome movement toward the spindle pole body at an average spindle length of 2.87 ± 0.51 μm (Table IV; n = 15). Regression lines were drawn through each plot of centromere proximal chromosome spot movement to the spindle pole body (Fig. 5 C). The average rate observed was 0.33 ± 0.16 μm/min (Fig. 5 C; Table IV; n = 23). The average distance moved was 0.98 ± 0.28 μm (Table III; n = 23).


Budding yeast chromosome structure and dynamics during mitosis.

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

Anaphase A centromere movement to the spindle pole body and half-spindle shortening began coincident with or shortly after the onset of anaphase B spindle pole body separation. (A) Progression through anaphase was followed using the ∼1.1-kb CEN11 marker and Nuf2–GFP to label the spindle pole bodies. Selected frames from a Z-series time-lapse sequence taken at 45-s intervals. (B) Graphical plot of the above time-lapse (A), using measurements with the Z-distance taken into account. In A and B, arrowheads denote the spindle pole bodies, and arrows define centromere proximal chromosome spots. (C) Centromere movement to the spindle pole body was coincident with the onset of anaphase B spindle pole body separation, as determined using Cse4–GFP to label all centromeres. Spindle pole bodies were labeled using Spc29–CFP, allowing two-color imaging of anaphase onset. Centromere clusters underwent a synchronous separation at anaphase onset. Frames are from a Z-series time-lapse sequence taken at 45-s intervals. (D) Graphical plot of the above time-lapse (C), with the Z-distance taken into account. (E) Half-spindle microtubule or shortening to the spindle pole body began near the onset of anaphase B spindle pole body separation. Using GFP–Tub1, we show that, upon elongation of the spindle, the fluorescent tufts emanating from each spindle pole body, presumed to be kinetochore microtubules, shortened toward the spindle pole bodies. Plots also indicate that there were dynamic movements in the lengths of half-spindle microtubules emanating from the spindle pole body. When the half-spindle had shortened to within the resolution limit of the light microscope (∼250–300 nm), integrated fluorescent intensities, standardized to the last distance measurement (6.26 min), were plotted to show that fluorescence intensities decreased, indicating that microtubule polymer further decreased from the optical resolution limited tufts (▴). Images were from a single plane acquisition time-lapse sequence at 5-s intervals. Arrows indicate the spindle pole bodies and plus ends of half-spindles. (F) Graphical plot of the above time-lapse showing plots of each spindle pole body (▪) relative to the ends of each fluorescent tuft (▴; 0–6.3 min). At 6.26–7.26 min, the fluorescent intensity of each tuft was standardized to the last distance measurement at 6.26 min and plotted relative to their respective spindle pole body (•, 6.26–7.26 min). Elapsed time for A–E is in minutes. Bars, 2 μm.
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Figure 4: Anaphase A centromere movement to the spindle pole body and half-spindle shortening began coincident with or shortly after the onset of anaphase B spindle pole body separation. (A) Progression through anaphase was followed using the ∼1.1-kb CEN11 marker and Nuf2–GFP to label the spindle pole bodies. Selected frames from a Z-series time-lapse sequence taken at 45-s intervals. (B) Graphical plot of the above time-lapse (A), using measurements with the Z-distance taken into account. In A and B, arrowheads denote the spindle pole bodies, and arrows define centromere proximal chromosome spots. (C) Centromere movement to the spindle pole body was coincident with the onset of anaphase B spindle pole body separation, as determined using Cse4–GFP to label all centromeres. Spindle pole bodies were labeled using Spc29–CFP, allowing two-color imaging of anaphase onset. Centromere clusters underwent a synchronous separation at anaphase onset. Frames are from a Z-series time-lapse sequence taken at 45-s intervals. (D) Graphical plot of the above time-lapse (C), with the Z-distance taken into account. (E) Half-spindle microtubule or shortening to the spindle pole body began near the onset of anaphase B spindle pole body separation. Using GFP–Tub1, we show that, upon elongation of the spindle, the fluorescent tufts emanating from each spindle pole body, presumed to be kinetochore microtubules, shortened toward the spindle pole bodies. Plots also indicate that there were dynamic movements in the lengths of half-spindle microtubules emanating from the spindle pole body. When the half-spindle had shortened to within the resolution limit of the light microscope (∼250–300 nm), integrated fluorescent intensities, standardized to the last distance measurement (6.26 min), were plotted to show that fluorescence intensities decreased, indicating that microtubule polymer further decreased from the optical resolution limited tufts (▴). Images were from a single plane acquisition time-lapse sequence at 5-s intervals. Arrows indicate the spindle pole bodies and plus ends of half-spindles. (F) Graphical plot of the above time-lapse showing plots of each spindle pole body (▪) relative to the ends of each fluorescent tuft (▴; 0–6.3 min). At 6.26–7.26 min, the fluorescent intensity of each tuft was standardized to the last distance measurement at 6.26 min and plotted relative to their respective spindle pole body (•, 6.26–7.26 min). Elapsed time for A–E is in minutes. Bars, 2 μm.
Mentions: The ∼1.1-kb CEN11 lacO marker was integrated into strains containing spindle pole bodies marked with Nuf2–GFP or Spc72–GFP. Z-series time-lapses were acquired at 15–45 s. We observed the ∼1.1-kb CEN11 proximal spots to move toward the GFP-marked spindle pole bodies coincident with or within 3 min after anaphase onset, as defined by the start of anaphase B spindle elongation (Fig. 4A and Fig. B and Fig. 5). Fig. 5 B (arrow) shows the separation of the spindle pole bodies to begin at the 0.0-min time point. Movement of the CEN proximal spots toward the spindle pole body ensues at 1.0 min (Fig. 5 B, arrow). This delay results in a transient increase in the distance between the centromere proximal spot and its spindle pole body. The centromere proximal spot began anaphase A chromosome movement toward the spindle pole body at an average spindle length of 2.87 ± 0.51 μm (Table IV; n = 15). Regression lines were drawn through each plot of centromere proximal chromosome spot movement to the spindle pole body (Fig. 5 C). The average rate observed was 0.33 ± 0.16 μm/min (Fig. 5 C; Table IV; n = 23). The average distance moved was 0.98 ± 0.28 μm (Table III; n = 23).

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