Budding yeast chromosome structure and dynamics during mitosis - PubMed (original) (raw)

Budding yeast chromosome structure and dynamics during mitosis

C G Pearson et al. J Cell Biol. 2001.

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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Figures

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.

Figure 2

Centromere proximal spots exhibited dynamic separation and oscillations along the preanaphase mitotic spindle. The lacO marker was integrated ∼1.1 kb from CEN11 with Spc72–GFP-labeled spindle pole bodies. (A) Selected frames of an ∼7.5-min single-plane time-lapse. (B) A kymograph sequence of the entire time-lapse collected at ∼0.9-s intervals. Narrow tick marks indicate corresponding time points to the above selected frames. (C) Graphical plot of the above time course. In both A and B, arrowheads denote the spindle pole bodies, and arrows define the centromere proximal chromosome spots. Elapsed time provided in seconds. Bar, 2 μm.

Figure 3

Centromere dynamics of all chromosomes using the pan-specific centromere marker Cse4p fused to GFP (Cse4–GFP). Dynamic movements between the two clustered groups indicated a similar separation and dynamic movements to those observed for the ∼1.1-kb centromere proximal marker. Spindle pole bodies are not labeled. (A) Selected images of an ∼9-min single-plane time-lapse. (B) Kymograph of the entire time-lapse taken at 5-s intervals. Arrowheads in A and B indicate occurrences of GFP centromere fluorescence disjoining from the clustered group. Narrow tick marks indicate corresponding times to A. Elapsed time is in seconds. Bar, 2 μm.

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.

Figure 5

Centromere proximal (∼1.1 kb) marker movement to the spindle pole body during anaphase A. Graphical plot of a single representative early anaphase onset (15 observed) using Nuf2–GFP to mark the spindle pole bodies and the ∼1.1-kb CEN11 chromosome marker. Centromere movement to the spindle pole body (anaphase A) began at or shortly after the onset of anaphase B spindle pole body separation. (A) Plot of anaphase onset. (B) Plot showing the separation of spindle pole bodies (▪) and CEN spots (▴) and the distance between the spindle pole body and the CEN proximal spot (•). The onset of anaphase A was defined by the decrease in the distance between the spindle pole body and the ∼1.1-kb spots, indicating that chromosomes were moving toward the spindle pole bodies. (C) Plot of each individual CEN proximal spot to spindle pole bodies with linear regression lines representing the general rate of anaphase A movement to the spindle pole body. The average slope was 0.33 ± 0.16 μm/min (Table ).

Figure 6

Upon anaphase onset chromosome spots exhibit rapid separation from their sister chromosome GFP spots at rates much greater than anaphase A and B movements. (A) Representative plot of sister chromatid spot separation for the ∼1.1-kb CEN11 marker upon anaphase onset. (B) Representative plot of sister chromatid separation for the ∼23-kb CEN3 marker upon anaphase onset. Sequences of rapid spot separation were acquired at single-plane, short acquisition intervals (>1 frame/s).

Figure 7

Model of chromosome oscillations, alignment, and segregation.

References

1. Chen X., Yin H., Huffaker T. The yeast spindle pole body component Spc72p interacts with Stu2p and is required for proper microtubule assembly. J. Cell Biol. 1998;141:1169–1179. - PMC - PubMed
1. Chen Y., Baker R.E., Keith K.C., Harris K., Stoler S., Fitzgerald-Hayes M. The N terminus of the centromere H3-like protein Cse4p performs an essential function distinct from that of the histone fold domain. Mol. Cell. Biol. 2000;20:7037–7048. - PMC - PubMed
1. Cui Y., Bustamante C. Pulling a single chromatin fiber reveals the forces that maintain its higher order structure. Proc. Natl. Acad. Sci. USA. 2000;97:127–132. - PMC - PubMed
1. Finch J.T., Lutter L.C., Rhodes D., Brown R.S., Rushton B., Levitt M., Klug A. Structure of nucleosomal core particles of chromatin. Nature. 1977;269:29–36. - PubMed
1. Goshima G., Yanagida M. Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell. 2000;100:619–633. - PubMed

Budding yeast chromosome structure and dynamics during mitosis - PubMed (original) (raw)