Microscopy of fission yeast cells

For microscopy, fission yeast cells were first inoculated in a YE5S medium for 2 days at 25°C. Then 1 ml of the exponentially growing cell culture, at a density between 5 × 10⁶/ml and 1.0 × 10⁷/ml, was harvested by centrifugation at 4000 rpm for 1 min. It was washed three times with synthetic EMM medium ([Ca²⁺] = 107 µM) and resuspended in 1 ml of EMM before proceeding for microscopy. Then 20 µl of the resuspended cells were spotted in a 10-mm Petri dish with a glass coverslip (#1.5) at the bottom (D35-10-1.5N, Cellvis). The coverslip was precoated with 50 µl of 50 µg/ml lectin (Sigma, L2380) and allowed to dry overnight at 4°C. The cells were allowed to attach to the coverslip for 10 min at room temperature before addition of another 2 ml EMM in the Petri dish.

We employed a spinning-disk confocal microscope for fluorescence microscopy using an Olympus IX71 unit equipped with a CSU-X1 spinning-disk unit (Yokogawa, Japan). The motorized stage (ASI, USA) included a Piezo Z Top plate for acquiring Z-series. The images were captured on an EMCCD camera (IXON-897, Andor) controlled by iQ3.0 (Andor). Solid-state lasers of 488 and 561 nm were used at a power of no more than 2.5 mW. Unless specified, we used a 60× objective lens (Olympus, Plan Apochromat, NA = 1.40). A Z-series of eight slices at a spatial distance of 1 µm was captured at each time point. The microscopy was carried out in a designated room maintained at 22 ± 2°C. To minimize environmental variations, we typically imaged both control and experimental groups in randomized order on the same day.

We employed a CellASIC ONIX2 system controlled by a desktop computer through ONIX software (EMD Millipore) to apply osmotic shock. Using a yeast haploid microfluidics plate (Y04C, EMD Millipore), we pushed the cells into the imaging chamber at a pressure of 34–55 kPa for a minimum of 2 min using EMM media. The trapped cells were equilibrated in EMM for 10 min at a pressure of 10 kPa. The same perfusion pressure was applied afterward for the media exchange (EMM, EMM-calcium and EMM+2 mM EGTA).