Immunofluorescence microscopy and F-actin staining

Spheroplasts were first permeabilized with isolation buffer containing 0.5% NP-40, and washed twice with reactivation buffer. Permeabilized spheroplasts were then fixed with 3.7% formaldehyde for 12 min at room temperature and washed twice with reactivation buffer. A rabbit primary antibody recognizing S. pombe tropomyosin Cdc8p (Balasubramanian et al., 1992) and a mouse CF633 anti-GFP (Sigma; SAB4600146) were added at 1:400 and 1:200, respectively and incubated overnight at 4°C. The samples were then washed twice with reactivation buffer, followed by treatment with the Donkey AlexaFluor 555 anti-Rabbit (Abcam; ab150074) at 1:400 for 2 hr at room temperature. After two washes with reactivation buffer, the samples were mounted on a slide and visualized using the spinning-disk confocal microscopy (Figure 2A and E).

To visualize actin structures in Figure 1C, Figure 1—figure supplement 1H and I, Figures 3C,D,E, and Figure 4—figure supplement 1A, spheroplasts were permeabilized and fixed as described above, and treated with CF633-phalloidin (Biotium; #00046; dissolved in water) at 1:20 for 10 min. Samples were washed twice with reactivation buffer before visualization.

In Figure 1—figure supplement 1A and B, cells and permeabilized spheroplasts were fixed with 3.7% formaldehyde for 12 min at room temperature. The fixed cells were permeabilized with PBS containing 1% Triton X-100 for 1 min, washed twice with PBS, and then treated with Rhodamine-conjugated phalloidin (Life Technologies; R415) at 1:20. Similarly, fixed and permeabilized spheroplasts were treated with Rhodamine-conjugated phalloidin at 1:20 to visualize actin structures.