Figure S2.
In vitro effects of formins on yeast Cap1/2 barbed end association, and in vivo effects of Cap1/2 and CapZ overexpression on cortical actin patches. (A and B) Kymographs of yeast actin filaments growing in a microfluidics chamber with 1 μM yeast actin and 2 μM yeast profilin imaged by TIRF and visualized with 100-s pulses of 15% Alexa Flour 488 labeled muscle actin (A) or alternating 100-s pulses of 15% Alexa Flour 488 labeled muscle actin followed by 100 nM SiR-actin (B). (C) Yeast actin elongation rates measured from dark filaments growing for 100 s in the presence and absence of SiR-actin using the Alexa Flour 488 labeled actin to track the increase in filament length during the 100 s pulse of dark actin (n = 100 unlabeled filaments and 100 SiR-actin labeled filaments). Statistical significance calculated by two-sided Student’s t test (****, P ≤ 0.0001). (D) To test if yeast formins can displace Cap1/2, yeast actin filaments (visualized with 15% Alexa Flour 488 labeled muscle actin) were capped with Cap1/2 and then exposed to 1,000 nM Bni1, 1,000 nM Bnr1, or a buffer control for 50 s in the absence of actin. Then, at time 0 s, filaments were returned to a reaction mixture containing 1 μM yeast actin and 2 μM yeast profilin (Pfy1). Filaments were monitored for transitions between being capped and growing, marking a Cap1/2 disassociation event. The percent of capped filaments over time was fit with a one-phase exponential decay curve to calculate Cap1/2 off-rate (k−). The off-rates were 1.0 (0.94–1.1), 1.1 (0.99–1.2), 1.1 (1.0–1.2) s−1 × 10−2 (95% CI) for buffer control, 1,000 nM Bni1, and 1,000 nM Bnr1, respectively. All filaments that returned to growing after being capped grew at the free barbed end rate (n = 87 Bni1-exposed filaments, 49 Bnr1-exposed filaments, and 338 buffer control–exposed filaments). (E) In a second setup, yeast actin filaments assembled in a reaction mixture containing 1 μM yeast actin and 2 μM yeast profilin (Pfy1) were capped by flowing in the same reaction mixture with 100 nM Cap1/2 for 100 s. At time 0, actin and Cap1/2 were flowed out and 2,500 nM Bni1, 2,000 nM Bnr1, or a buffer control was introduced to the chamber. Capped filaments were monitored for Cap1/2 disassociation events when filaments began to depolymerize in the absence of free G-actin. The percent of capped filaments over time was fit with a one phase exponential decay curve to calculate Cap1/2 off-rate (k−; n = 46 Bni1-exposed filaments, 37 Bnr1-exposed filaments, and 117 buffer control exposed filaments). The off-rates were 1.1 (0.96–1.2), 0.95 (0.83–1.1), 1.4 (1.1–1.2) s−1 × 10−2 (95% CI) for buffer control, 2,500 nM Bni1, and 2,000 nM Bnr1, respectively. (F–I) Actin patch assembly is largely unaffected by overexpression of Cap1/2 or CapZ. (F) Average intensity projections of spinning-disk confocal images of cells expressing Cap2-GFPEnvy from the native locus or expressing GAL Cap1/2-GFPEnvy or GAL GFPEnvy-CapZ. All cells were grown in media with 2% galactose to induce overexpression. Images are colored with the Fire LUT to highlight differences in GFPEnvy intensity. Signal intensity in cells expressing Cap2-GFPEnvy from the native locus is also shown multiplied by 10. (G) Quantification of the mean GFPEnvy intensity relative to native Cap2-GFPEnvy (n = 92 Cap2-GFPEnvy cells, 160 GAL Cap1/2-GFPEnvy cells, and 81 GAL GFPEnvy-CapZ cells). (H) To determine if CP overexpression alters actin patch dynamics, an F-actin marker for patch networks (Sac6-RFPmScarlet) was introduced into the Cap2-GFPEnvy, GAL Cap1/2-GFPEnvy, and GAL GFPEnvy-CapZ backgrounds. Overexpression was induced with 2% galactose, and cells were imaged by time-lapse spinning-disk confocal microscopy. Shown are average intensity projections (three Z-sections) from a time series. (I) Quantification of the peak Sac6-RFPmScarlet signal during patch lifetime in Cap2-GFPEnvy, GAL Cap1/2-GFPEnvy, and GAL GFPEnvy-CapZ cells (n = 39 Cap2-GFPEnvy patches, 20 GAL Cap1/2-Cap2-GFPEnvy patches, and 32 GAL Cap2-GFPEnvy-CapZ patches). In all graphs, a line is drawn at the mean, and error bars represent the SD. Statistical significance in all panels calculated by one-way ANOVA (n.s., no significance; P > 0.05; ****, P ≤ 0.0001). Scale bar = 2 μm.

In vitro effects of formins on yeast Cap1/2 barbed end association, and in vivo effects of Cap1/2 and CapZ overexpression on cortical actin patches. (A and B) Kymographs of yeast actin filaments growing in a microfluidics chamber with 1 μM yeast actin and 2 μM yeast profilin imaged by TIRF and visualized with 100-s pulses of 15% Alexa Flour 488 labeled muscle actin (A) or alternating 100-s pulses of 15% Alexa Flour 488 labeled muscle actin followed by 100 nM SiR-actin (B). (C) Yeast actin elongation rates measured from dark filaments growing for 100 s in the presence and absence of SiR-actin using the Alexa Flour 488 labeled actin to track the increase in filament length during the 100 s pulse of dark actin (n = 100 unlabeled filaments and 100 SiR-actin labeled filaments). Statistical significance calculated by two-sided Student’s t test (****, P ≤ 0.0001). (D) To test if yeast formins can displace Cap1/2, yeast actin filaments (visualized with 15% Alexa Flour 488 labeled muscle actin) were capped with Cap1/2 and then exposed to 1,000 nM Bni1, 1,000 nM Bnr1, or a buffer control for 50 s in the absence of actin. Then, at time 0 s, filaments were returned to a reaction mixture containing 1 μM yeast actin and 2 μM yeast profilin (Pfy1). Filaments were monitored for transitions between being capped and growing, marking a Cap1/2 disassociation event. The percent of capped filaments over time was fit with a one-phase exponential decay curve to calculate Cap1/2 off-rate (k−). The off-rates were 1.0 (0.94–1.1), 1.1 (0.99–1.2), 1.1 (1.0–1.2) s−1 × 10−2 (95% CI) for buffer control, 1,000 nM Bni1, and 1,000 nM Bnr1, respectively. All filaments that returned to growing after being capped grew at the free barbed end rate (n = 87 Bni1-exposed filaments, 49 Bnr1-exposed filaments, and 338 buffer control–exposed filaments). (E) In a second setup, yeast actin filaments assembled in a reaction mixture containing 1 μM yeast actin and 2 μM yeast profilin (Pfy1) were capped by flowing in the same reaction mixture with 100 nM Cap1/2 for 100 s. At time 0, actin and Cap1/2 were flowed out and 2,500 nM Bni1, 2,000 nM Bnr1, or a buffer control was introduced to the chamber. Capped filaments were monitored for Cap1/2 disassociation events when filaments began to depolymerize in the absence of free G-actin. The percent of capped filaments over time was fit with a one phase exponential decay curve to calculate Cap1/2 off-rate (k−; n = 46 Bni1-exposed filaments, 37 Bnr1-exposed filaments, and 117 buffer control exposed filaments). The off-rates were 1.1 (0.96–1.2), 0.95 (0.83–1.1), 1.4 (1.1–1.2) s−1 × 10−2 (95% CI) for buffer control, 2,500 nM Bni1, and 2,000 nM Bnr1, respectively. (F–I) Actin patch assembly is largely unaffected by overexpression of Cap1/2 or CapZ. (F) Average intensity projections of spinning-disk confocal images of cells expressing Cap2-GFPEnvy from the native locus or expressing GAL Cap1/2-GFPEnvy or GAL GFPEnvy-CapZ. All cells were grown in media with 2% galactose to induce overexpression. Images are colored with the Fire LUT to highlight differences in GFPEnvy intensity. Signal intensity in cells expressing Cap2-GFPEnvy from the native locus is also shown multiplied by 10. (G) Quantification of the mean GFPEnvy intensity relative to native Cap2-GFPEnvy (n = 92 Cap2-GFPEnvy cells, 160 GAL Cap1/2-GFPEnvy cells, and 81 GAL GFPEnvy-CapZ cells). (H) To determine if CP overexpression alters actin patch dynamics, an F-actin marker for patch networks (Sac6-RFPmScarlet) was introduced into the Cap2-GFPEnvy, GAL Cap1/2-GFPEnvy, and GAL GFPEnvy-CapZ backgrounds. Overexpression was induced with 2% galactose, and cells were imaged by time-lapse spinning-disk confocal microscopy. Shown are average intensity projections (three Z-sections) from a time series. (I) Quantification of the peak Sac6-RFPmScarlet signal during patch lifetime in Cap2-GFPEnvy, GAL Cap1/2-GFPEnvy, and GAL GFPEnvy-CapZ cells (n = 39 Cap2-GFPEnvy patches, 20 GAL Cap1/2-Cap2-GFPEnvy patches, and 32 GAL Cap2-GFPEnvy-CapZ patches). In all graphs, a line is drawn at the mean, and error bars represent the SD. Statistical significance in all panels calculated by one-way ANOVA (n.s., no significance; P > 0.05; ****, P ≤ 0.0001). Scale bar = 2 μm.

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