To determine if the altered actin patch morphology observed in cap2Δ cells results from ectopic formin recruitment to the patches, we generated a strain that enabled temperature-sensitive inactivation of formin activity, cap2Δ bni1-1 bnr1Δ. This strain was generated by crossing BGY24 (cap2Δ) to BGY715 (bni1-1 bnr1Δ; Sagot et al., 2002 (link)). Two independent isolates of cap2Δ bni1-1 bnr1Δ were used in all experiments. We performed statistical tests (one-way ANOVA), which revealed that the observed changes in F-actin signal (at 25 and 37°C) are consistent between independent isolates and that any differences between the isolates are extremely small and not contributing to the statistical differences we observe between genotypes (as graphed in Fig. S5, A–C ). The data presented are pooled from the two isolates. To visualize F-actin in wild-type and cap2Δ cells before and after formin inactivation, single colonies of wild-type, cap2Δ, bni1-1 bnr1Δ, and cap2Δ bni1-1 bnr1Δ strains were used to inoculate 5 ml yeast extract peptone dextrose cultures and grown at 25°C to OD600 0.3–0.6. Cultures were then split, such that half was shifted to the nonpermissive temperature of 37°C for 30 min before fixing in 3.7% formaldehyde for 40 min (at 37°C). The other half of the culture was maintained at 25°C and then fixed as above (at 25°C). Cells were stained with Alex488-phalloidin as above, mounted, and samples were imaged separately on a spinning-disk confocal and an Airyscan microscope, as described above for fixed cell imaging. As above, in all experiments, a wild-type control strain (marked with RFPmScarlet) was grown at 25°C in parallel, then mixed with experimental cells during fixation and phalloidin stained to control for tube-to-tube variability in staining. The total F-actin signal per cell was measured as described above for fixed-cell imaging. The signal intensity of individual patches was measured in ImageJ from a single Airyscan Z-section by drawing a 5 × 5-pixel box around the central Z-plane of each patch. Signal background (outside of the cell) was subtracted from these measurements and values are reported relative to patch intensities of the internal control strain.
To determine whether formin activity is required for tropomyosin recruitment to patches, we generated a cap2Δ bni1-1 bnr1Δ strain expressing mNeon–Tpm1 and the patch marker Arc15-RFPmScarlet. This strain was constructed by crossing a cap2Δ bni1-1 bnr1Δ strain to a strain expressing mNeon–Tpm1 and Arc15-RFPmScarlet. These strains were then grown in synthetic complete media (2% glucose) and imaged on a spinning-disk confocal microscope as above for live cell imaging. To inactivate formins, cells were shifted to 37°C for 10 min, then imaged as above except at 37°C using a CherryTemp (Cherry Biotech) stage heater.
To determine whether formin activity is required for tropomyosin recruitment to patches, we generated a cap2Δ bni1-1 bnr1Δ strain expressing mNeon–Tpm1 and the patch marker Arc15-RFPmScarlet. This strain was constructed by crossing a cap2Δ bni1-1 bnr1Δ strain to a strain expressing mNeon–Tpm1 and Arc15-RFPmScarlet. These strains were then grown in synthetic complete media (2% glucose) and imaged on a spinning-disk confocal microscope as above for live cell imaging. To inactivate formins, cells were shifted to 37°C for 10 min, then imaged as above except at 37°C using a CherryTemp (Cherry Biotech) stage heater.
