Supplemental Material

This article contains the following supporting material:

• Figure S1 - Combinations of LacO and TetO arrays integrated at different loci on the right arm of chromosome IV in S. cerevisiae.

  Photomicrographs of strains with integrated LacO arrays visualized by binding of LacR-GFP and integrated TetO arrays visualized by binding of TetR-mRFP. Images are overlays of DIC and eGFP or DIC, eGFP and red fluorescent channels. Integration sites are indicated above each panel. Red signals always correspond to TetO sequences integrated at TRP1. For each panel, the set of four images are representative of (from left to right) G1, S G2/M and telophase cells.

• Figure S2 - A. Photomicrographs of cells containing LacO sequences integrated at MMP1 and CMS1 and expressing LacR-GFP. DIC and green fluorescent channels are over-laid. Top panel shows cells in different cell cycle stages - from the left; G1 (alpha factor induced arrest), small budded S-phase cell, G2/M cell, anaphase/telophase cell. Lower panel shows cells arrested after nocodazole treatment. B,C. Time course experiments examining the timing of condensation of the MMP1-CMS1 region in wild type cells. Cells were first synchronized in G1 with alpha factor then released into rich medium (B) or medium containing nocodazole (C). Cells were scored according to the criteria described in Figure 1. D. Decondensation of the TRP1-LYS4 region occurs before Pds1 degradation in nocodazole arrested cells. The cells contained LacO sequences integrated at TRP1 and LYS4, expressed LacR-GFP and Pds1-GFP. After release from alpha-factor-induced arrest, nocodazole was added, and time points taken after the cells arrested in G2/M. Pds1 decay began around 3.5 hours after release from G1, around the time when cells began to progress beyond the G2/M arrest. E. Decondensation of the TRP1-LYS4 region occurs before Pds1 degradation in apc2-4 cells. This experiment was identical to that described in part (D) except that the strains possessed a temperature sensitive apc2-4 allele, and instead of adding nocodazole upon release from G1 synchrony, the culture was shifted to 30°C, a semi-permissive temperature for apc2-4. F. Cdc28 is required for condensation of the TRP1-LYS4 region. Time course experiment examining the timing of condensation of the TRP1-LYS4 region in cdc28 mutant cells. The cells contained LacO sequences integrated at TRP1 and LYS4, expressed LacR-GFP and possessed a mutant CDC28 allele, cdc28-1N, defective for mitotic functions but capable of performing DNA replication and other pre-mitotic events. Cells were scored according to the criteria in Figure 1, i.e. the number of fluorescent signals per cell was scored; 1 dot = condensed TRP1-LYS4 region, 2 dots = decondensed TRP1-LYS4region, segregated dots = anaphase/telophase.
• Figure S3 - Loss of cohesion during nocodazole arrest.

  A. Loss of cohesion at TRP1 correlates with Pds1 degradation only in some nocodazole arrested cells. The cells contained LacO sequences integrated at TRP1 and expressed LacR-GFP and Pds1-GFP. After release from alpha-factor-induced arrest, nocodazole was added, and time points taken after the cells arrested in G2/M. B. The timing of loss of cohesion at the TRP1 locus was determined in wild type cells, smc2-8 condensin mutant cells, and mcd1-1 cohesin mutant cells. Strains were arrested in G1 and released at a non-permissive temperature for each mutant into medium containing nocodazole. The timing of loss of cohesion was determined (appearance of cells with 2 fluorescent signals) relative to the timing of checkpoint adaptation (based on re-budding).

• Figure S4 - Low resolution time lapse analysis of the TRP1-LYS4 strain.

  Using the TRP1-LYS4 strain, we visualized chromosome movement and compaction in unperturbed cell cycles by time-lapse microscopy. Images could be captured at no less than five minute intervals because more frequent exposure of the cells to ultra-violet light was phototoxic. In strains expressing other GFP-fusion proteins (such as Tub1-GFP), UV-light was far less cytotoxic, suggesting that the TRP1-LYS4 strain in particular is sensitive. We could capture images of cells with five Z-sections (Z-interval 0.25 μm) at exposures of 4 ms, 5 minute intervals. Even at this low time-resolution of time-lapse imaging, we were able to visualize condensation prior to anaphase. An interesting observation made from this imaging was that the chromosomes were highly dynamic from telophase into the next G1. At this stage, the chromosomes repeatedly appeared to condense and decondense. When the cell under observation entered the next cycle and began the process of budding, two clear fluorescent signals were visible that coalesced at a larger bud size. Anaphase normally occurred 5-10 minutes after the two dots coalesced. As the chromosomes segregated to opposite poles, the fluorescent signals at TRP1 and LYS4 sometimes separated from each other, but subsequently coalesced when that chromosome reached the cell pole. A. Four time-lapse series (i-iv) captured as described above using the TRP1-LYS4 strain. Time interval between each frame is 5 minutes (sequences begin with the upper-leftmost panel and end with the lower-rightmost panel). Bar = 5 μm. Each photomicrograph is a merged image from 5 Z-sections of the eGFP channel and a single DIC section at the mid-point of the stack. B. Examples of wild type early anaphase cells (TRP1-LYS4 strain) in which the TRP1-LYS4 region has become stretched during segregation of the chromosomes. From left to right; a cell shown with the eGFP channel only; a merged image of the DIC and eGFP channel; a merged image of the DIC, eGFP and red fluorescent channels.

• Figure S5 - Estimated distances between the TRP1 and LYS4 loci in live cells.

  To estimate the degrees of chromatin compaction in live S. cerevisiae cells at different cell cycle stages, we calculated distances between the TRP1 and LYS4 loci and determined compaction ratios. To obtain accurate measurements of the physical distance between TRP1 and LYS4, we took into account the z-distance between the two loci. The strain with TetO sequences at TRP1 and LacO sequences at LYS4 was used, combined with TetR-RFP and LacO-GFP fusion proteins. Utilizing two different colors allowed us to accurately determine the center of each fluorescent signal. The actual distance between these two regions of chromosome IV was calculated as shown in A, using z-stacks taken at 0.02-0.1 μm intervals, and a Zeiss 100x/1.45n.a oil objective for maximum resolution. The distance “l” is the distance in the z-plane between the centers of the two dots. The distance “b” is the distance between the centers of the two dots in the two dimensional flattened image. “h” is the actual distance between the two dots. Compaction ratios were calculated from the actual observed distance between the dots, based on the formula previously described (Guacci et al. , 1994). B. Example of z-series of images from which compaction ratios were estimated. The top panel is a flattened image of the z-stack. The panels beneath show a portion of the z-stack containing the TRP1 and LYS4 signals. The distance between z-images was 0.1 μm