Volume 16, Issue 2, Pages 292-302 (February 2009) Displacement of Formins from Growing Barbed Ends by Bud14 Is Critical for Actin Cable Architecture and Function  Melissa Chesarone, Christopher J. Gould, James B. Moseley, Bruce L. Goode  Developmental Cell  Volume 16, Issue 2, Pages 292-302 (February 2009) DOI: 10.1016/j.devcel.2008.12.001 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Actin Cable Defects in bud14Δ Cells (A) Representative architectural defects in actin cables found in bud14Δ cells. Whereas cables in wild-type cells followed the contour of the cell cortex (large arrow, upper panels), cables in bud14Δ cells were “kinked” or “buckled” (small arrow, lower panels). Cables that changed direction with a >90° turn at the cortex were scored as “bent.” (B) Data were quantified from two experiments. (C) Total number of actin cables visible in bud14Δ versus wild-type cells. Data were averaged from two experiments. (D) Representative images from experiments in which wild-type and bud14Δ cells were treated with 20 μM LatA for 60 s, fixed, and stained with Alexa 488 phalloidin to score the percentage of cells with visible cables. “Enhanced” panels are the same images (LatA-treated) that have been contrast enhanced to highlight the remaining cables. (E) Cells were treated as described above with LatA for different lengths of time before fixation and imaging (0–120 s). Data were quantified from at least two independent experiments (∗∗p < 0.001, ∗p < 0.05). (F) Yeast strains were serially diluted and compared for growth at 25°C and 34°C on YPD plates. (G) GFP-Sec4 localization in strains grown at 25°C. The percentage of cells with a bright spot of GFP-Sec4 at the bud tip was scored (n > 100) and is listed in each panel. (H) DIC microscopy of strains in (F). All error bars represent SD. Developmental Cell 2009 16, 292-302DOI: (10.1016/j.devcel.2008.12.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 BUD14 Functions Upstream of BNR1 and in Parallel to BNI (A) Yeast strains were serially diluted and compared for growth at 25°C and 37°C on YPD plates. (B) DIC microscopy of strains grown at 25°C. (C) The percentage of cells with elongated buds was scored for each strain (n > 200) in two independent experiments. All error bars represent SD. Developmental Cell 2009 16, 292-302DOI: (10.1016/j.devcel.2008.12.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Requirements for GFP-Bud14 Localization (A) Localization of GFP-Bud14. (B) GFP-Bud14 localization after treatment of cells with 50 μM LatA. (C) GFP-Bud14 localization after genetic disruption of actin cables or Myo2 function. Each of the indicated strains carrying GFP-Bud14 was imaged after growth at 25°C or after a 10 min shift to the nonpermissive temperature (37°C). (D) GFP-Bud14 localization in bnr1Δ cells. For all strains, GFP-Bud14 localization patterns were uniform in the populations. Developmental Cell 2009 16, 292-302DOI: (10.1016/j.devcel.2008.12.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Purified Bud14 Directly Inhibits Bnr1 (A) Coommassie-stained gel of purified full-length Bud14. (B and C) Monomeric actin (2 μM, 5% pyrene labeled) was polymerized in the presence of (B) 5 nM Bnr1(FH1-FH2-C) or (C) 30 nM Bni1(FH1-FH2-C) and the indicated concentrations of Bud14. (D) Concentration-dependent effects of Bud14 on Bnr1 or Bni1 activity. The percent activity was determined by dividing the slope of the actin polymerization curve in the presence of a given concentration of Bud14 by the slope of the curve in the absence of Bud14 (considered 100% activity). (E) Visualization of filaments assembled by Bnr1 in the presence and absence of Bud14. Reaction samples were removed (same conditions as described in the legend of [B]) 10 min after initiation; filaments were stabilized with Alexa 488 phalloidin and imaged. (F) Bud14 inhibits Bnr1(FH2). Reaction conditions are as described in the legend of (B), except 20 nM Bnr1(FH2) was used. (G) Concentration-dependent effects of Bud14 on Bnr1(FH2) (determined as in [D]). (H) Association of 500 nM His6-Bnr1(FH1-FH2-C) or 6His-Bnr1(FH2) with GST-Bud14 or GST (control) immobilized on glutathione agarose. Levels of 6His-Bnr1 in the pellet (P) and supernatant (S) fractions compared by immunoblotting with 6His antibodies. Developmental Cell 2009 16, 292-302DOI: (10.1016/j.devcel.2008.12.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Bud14 Displaces Bnr1 from Growing Barbed Ends (A) Effects of Bud14 on Bnr1-capped filament barbed end growth in the presence and absence of capping protein (CP). At time zero, monomeric actin (0.5 μM) was added to mechanically sheared F-actin seeds in the presence of Bnr1(FH1-FH2-C), CP, and/or Bud14 (concentrations are indicated in [B]). (B) Rates of elongation were averaged from multiple experiments. (C) Elongation assays were performed as described in the legend of (A), except instead of premixing Bnr1 with Bud14, Bud14 or control buffer was added to the reactions 65 s after initiation. (D) Rates of elongation were averaged from multiple experiments. All error bars represent SD. Developmental Cell 2009 16, 292-302DOI: (10.1016/j.devcel.2008.12.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 bud14Δ Suppresses the Actin Cable Architecture and Secretory Transport Defects of a bnr1ΔDAD Strain (A) Yeast strains were serially diluted and compared for growth at 25°C, 34°C, and 37°C on YPD plates. (B) Cells were fixed and stained with Alexa 488 phalloidin or transformed with a GFP-Sec4 plasmid to localize secretory vesicles. Images of representative cells are shown. GFP-Sec4 panels include the percentage of cells having >50% GFP-Sec4 fluorescence in the bud (n > 100). (C) Time-lapse movement of GFP-Sec4 particles in live cells. The four categories of movement observed (Class I, II, III, and IV) were scored in each strain (n > 100 cells). Shown to the right of the bar graph are representative cells depicting each category of movement; yellow circles trace the path of movement recorded at 300 ms intervals. See Supplemental Data for movies: wild-type, Movie S1; bud14Δ, Movie S2; bnr1ΔDAD, Movie S3; and bud14Δbnr1ΔDAD, Movie S4. Developmental Cell 2009 16, 292-302DOI: (10.1016/j.devcel.2008.12.001) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Bud14 Domain Analysis (A) Schematic of purified, truncated Bud14 polypeptides. Each Bud14 polypeptide was compared at a range of concentrations for its effects on Bnr1(FH1-FH2-C)-mediated pyrene-actin assembly. Concentrations of Bud14 required for half-maximal Bnr1 inhibition are shown. (B) Dose-dependent effects of two constructs, Bud14-5 and Bud14-3, on Bnr1(FH1-FH2-C) activity. (C) Effects on cell growth after Gal-driven overexpression of full-length Bud14, Bud14-5, and Bud14-9. Cells were grown in selective media, serially diluted on selective plates containing glucose or galactose, and grown at 25°C. (D) Cell morphology and actin organization defects caused by overexpression of Bud14 constructs. After 24 hr of growth in galactose-containing medium, cells were fixed and stained with Alexa 488 phalloidin. DIC panels for Gal-induced cultures include the percentage of cells with an obvious “chained” phenotype and/or with grossly elongated or misshapen buds. Developmental Cell 2009 16, 292-302DOI: (10.1016/j.devcel.2008.12.001) Copyright © 2009 Elsevier Inc. Terms and Conditions