Formin-mediated Actin Assembly Regulation

Fig. S1 compares the in vitro effects of yeast Cap1/2 and vertebrate CapZ on formin-mediated (Bni1, Bnr1, and mDia1) actin assembly in bulk assays, and the inhibitory effects of vertebrate CapZ on yeast formin (Bni1 and Bnr1) stimulated elongation of actin filaments in mf-TIRF assays. Fig. S2 shows the in vitro elongation rates of yeast actin filaments visualized with a SiR-actin probe in mf-TIRF assays, and the minimal effects of formins (Bni1 or Bnr1) on off rate of Cap1/2 from the barbed end (related to Fig. 2 in the main text). Also shown is quantification of the high-level (galactose-induced) expression of Cap1/2-GFP^Envy and GFP^Envy-CapZ in cells and evidence that it has no significant effect on Sac6-RFP^mScarlet marked actin patches (related to Fig. 3 in the main text). Fig. S3 shows the improved detection of formins Bni1 and Bnr1 in cells when tagged with a 3xmNeon tag and the quantification of formin (Bni1 and Bnr1) colocalization with Arc15-RFP^mScarlet labeled patches (related to Fig. 5 in the main text). Fig. S4 shows that even in the absence of formin activity in vivo, there is excessive accumulation of F-actin (indicated by phalloidin staining) at cortical actin patches in cap2∆ cells (related to Fig. 6 in the main text). Further, it shows that actin-binding proteins have altered dynamics at cortical patches in cap2∆ cells, and that aberrant decoration of older patches by tropomyosin (Tpm1) in cap2∆ cells occurs independent of formin activity. Fig. S5 shows that upon CK666 treatment the Arp2/3 complex signal at cortical patches is lost in both wild-type and cap2∆ cells, and provides additional examples of F-actin organization in phalloidin stained wild-type and cap2∆ cells (related to Fig. 7 in the main text). It also shows an alignment of the capping protein β-tentacle primary sequences from budding yeast (S. cerevisiae), fission yeast (S. pombe), human (Homo sapiens), and chicken (G. gallus). Video 1 shows dynamic puncta of 3xmNeon-tagged Cap2 visible at the cell cortex that is distinct from actin patches. Video 2 is an example time series of cells expressing Cap2–3xmNeon treated with control carrier, CK666 or LatB (analyzed in Fig. 5). Video 3 shows continuous imaging of example cells analyzed in Fig. S3 expressing formins (Bni1 or Bnr1) tagged with GFP or 3xmNeon, revealing that the 3xmNeon tag is brighter and more photostable. Videos 4 and 5 (related to Fig. 5) show that 3xmNeon-tagged Bni1 (Video 4) and Bnr1 (Video 5) colocalize with actin patches (marked by Arc15-RFP^mScarlet) in cap2Δ but not WT cells. Video 6 shows that Arc15-RFP^mScarlet marked actin patches in cap2∆ cells acquire cable-like characteristics as they age and inappropriately recruit tropomyosin (mNeon–Tpm1; related to Fig. 6). Table S1 contains a list of yeast strains used in this study. Table S2 contains a list of primers used and a description of each primer. Table S3 contains a list of plasmids used and a description of each plasmid.