Budding Yeast Chromosome Structure and Dynamics during Mitosis

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.

Condensin Regulates the Stiffness of Vertebrate Centromeres

“…This pericentric LacO array underwent movements resembling those of kinetochore pairs in living SMC2 ON and SMC2 OFF cells (Figure 4c; Supplementary Figure S2, a and c; Supplementary Movies 4 and 5), and on average, the LacI:GFP signals on sister chromatids were further apart in SMC2 OFF :CEN cells fixed at metaphase (Figure 4b). Similar separation of pericentric regions was previously observed in wild-type budding yeast for reporter arrays adjacent to centromeres (Goshima and Yanagida, 2000;He et al, 2000;Pearson et al, 2001), but has not previously been observed in vertebrates.Kymographs revealed the dynamic separation between the LacI:GFP CEN signals (Figure 4c), and their uncoordinated behavior was readily observed in movies of SMC2 OFF : CEN cells (Supplementary Movies 4 and 5). Remarkably, the loss of condensin caused an approximately twofold increase in the maximum separation of the chromosomal region linking the two LacO arrays (Figure 4d).…”

In Vivo Analysis of Chromosome Condensation inSaccharomyces cerevisiae

“…(This was also the case when we analyzed the MMP1-CMS1 region of chromosome XII after cell cycle arrest in the presence of nocodazole; see Supplementary Figure 2, A and C.) Using pds1-⌬db, an inhibitor of sister chromatid separation, we arrested cells carrying single LacO repeats at CEN4, TRP1, or LYS4. As expected, the CEN4 region possessed separated dots, as has been previously observed to reveal tension at the centromere that results from biorientation of the chromosome on the spindle ( Figure 3B; Goshima and Yanagida, 2001;Pearson et al, 2001). However, most of the TRP1 and almost all of the LYS4 cells arrested with a single dot ( Figure 3B and data not shown).…”

“…However, the apparent dependence of decondensation on Mad2 indicates that the spindle checkpoint is required for decondensation. In metaphase-arrested cells, with intact spindles, the dynamic movement of the spindle (Pearson et al, 2001) could account for stretching of the chromatin between the TRP1 and the LYS4 loci, resulting in dot separation. And in anaphase, linear chromosome stretching has been previously described, a result of telomere cohesion that is not resolved until late anaphase (Straight et al, 1997;Pearson et al, 2001).…”

“…In metaphase-arrested cells, with intact spindles, the dynamic movement of the spindle (Pearson et al, 2001) could account for stretching of the chromatin between the TRP1 and the LYS4 loci, resulting in dot separation. And in anaphase, linear chromosome stretching has been previously described, a result of telomere cohesion that is not resolved until late anaphase (Straight et al, 1997;Pearson et al, 2001). We similarly observed separation of the TRP1 and LYS4 loci in some anaphase cells as the sister chromatids moved to opposite poles ( Figure 6D and Supplementary Figure 4, A and B).…”

β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

“…The mitotic spindle, consisting of kinetochore and interpolar microtubules, is typically disassembled within a few minutes of the completion of anaphase Pearson et al, 2001). The kinetochore microtubules depolymerize during anaphase A (Winey et al, 1995), and the interpolar microtubules separate at the midzone and depolymerize toward the spindle poles during telophase .…”