2. Materials and methods 35
2.6. Yeast cell biology methods 46
2.6.1.1. cdc25-‐22 strains (G2 – M phase block)
Strains with the cdc25-‐22 TS mutation are blocked at the G2 – M phase transition when shifted to their restrictive temperature (they also continue to increase in length during this time). Using a programmable shaker if necessary, a cell culture grown at the permissive temperature is shifted to 36°C for 3 – 3.5 hours to initiate the block. Cells will then enter M phase and begin to form an AMR about 30 – 45 minutes after being returned to the permissive temperature.
2.6.1.2. nda3-‐KM311 strains (Metaphase block)
nda3-‐KM311 is a cold-‐sensitive (CS) mutation in the beta tubulin gene which prevents the formation of microtubules when at the restrictive temperature of 18°C. This precludes the formation of the mitotic spindle in mitotic cells, so that chromosome segregation does not occur. Importantly for this research, nda3-‐KM311 cells form a complete AMR, which does not begin to contract until the cells are returned to their permissive temperature (24°C or higher).
To initiate the block, cells grown at the permissive temperature are shifted to 18°C until > 50% of cells have an AMR, which normally occurs after 6 – 8 hrs. After returning to their permissive temperature, nda3-‐KM311 cells will quickly form a mitotic spindle and undergo karyokinesis within 5 minutes, after which ring constriction will quickly follow.
2.6.1.3. cps1-‐191 strains (Cytokinetic block)
Fission yeast strains with the cps1-‐191 genotype contain a mutation in the bgs1 gene, which is responsible for synthesis of the primary septum during ring constriction. Cells with this mutation cannot form a primary septum when shifted to 36°C, meaning that ring contraction does not
occur. To initiate the block cells are shifted to 36°C for 3 hrs before imaging. When returned to the permissive temperature of 24°C, cells will initiate ring constriction within a few minutes, or alternatively they can be kept at 36°C in order to block cytokinesis indefinitely.
2.6.2. Fission Yeast Spheroplasting
Spheroplasts are cells that have been separated from their cell wall, causing them to adopt a spherical shape. They form normal looking AMRs, which slide along the inner surface of the membrane. Due to the change in cell shape, these rings are normally much larger than those in normal cells, and can be orientated in any direction, and both of these differences can be advantageous in certain situations.
To prepare spheroplasts, fission yeast cells are grown in YEA media up to an OD of approx. 0.5 in 20 mL. Using a cdc25-‐22 strain, and blocking the cells at 36°C for 3 – 3.5 hrs beforehand will result in larger spheroplasts (and larger rings), although this is not necessary. Cells are then spun down in a falcon tube at 1900 RCF for 2 minutes, washed once with E-‐buffer (50 mM Sodium citrate, 100 mM Sodium phosphate), spun down and resuspended in 5 mL E-‐buffer with 1.2 M sorbitol.
To enzymatically digest the cell wall, 0.025g lysing enzyme (Sigma) is added to the cells, which are incubated at 80 rpm for 1 hr 30 mins, with the falcon tube laid flat on its side, at either 24°C or 36°C if using cdc25-‐22 cells. Then, 25 μL of zymolyase (G-‐biosciences, 1.5 units/μL) was added, and the mixture was incubated for a further hour at the same temperature and rpm as before.
After this time, spheroplast formation is monitored via bench top microscopy, imaging the cells without a cover slip. When the cell wall appears sufficiently weakened, usually after ~1 hour, cells were spun down at 450 RCF for 2 mins, washed in E-‐buffer with 0.6 M sorbitol, then spun down and resuspended in 10 mL culturing medium (minimal medium + 0.8 M sorbitol + supplements). 50 μL of 2-‐Deoxy-‐D-‐glucose (Sigma Aldrich) at a concentration of 164.16 mg/mL was added, to
prevent cell wall regrowth, and the mixture was placed at 24°C, 80 rpm, with the falcon tube laid on its side.
After 3 hrs, we began to check the spheroplasts for ring formation using fluorescence microscopy, imaging the spheroplasts on a rectangular coverslip. When a majority of the spheroplasts had formed rings, the spheroplasts were imaged using the cell suspension method, detailed later.
2.6.3. Cell fixation and staining with Calcofluor white and DAPI For cell-‐fixation, 5 mL of mid-‐log phase culture was centrifuged at 900 RCF for 3 minutes, and then washed with 1× phosphate-‐buffered saline (PBS). Cells were resuspended in 0.5 mL 1x PBS + 0.5 mL of 8% paraformaldehyde, and fixed on a shaker at 24°C for 12 minutes. The fixed cells were then washed once with PBS, and resuspended in 100 μL PBS. For permeabilisation, 100 μL of PBS + 1% Triton X-‐100 was added to 100 μL of fixed cells, and after 2 minutes the cells were washed twice with PBS, and resuspended in 25 μL PBS. For staining with calcofluor white (CW) and 4’, 6-‐diamidino-‐2-‐phenylindole (DAPI), 10 μL of the final cell mixture was taken, and mixed with 10 μL of DAPI (2 μg/mL), and 2 μL CW at 500× dilution. For staining with just CW, cells were fixed but not permeabilised. After the final stage of fixation, cells were resuspended in 25 μL of PBS, and then 2 μL CW at 500× dilution was added to 10 μL of cells. For imaging fixed cells, these were placed on bare microscope slides, and then sealed under a coverslip using VALAP (section 2.7.3).
2.7. Microscopy and data analysis