Coronin Switches Roles in Actin Disassembly Depending on the Nucleotide State of Actin  Meghal Gandhi, Vérane Achard, Laurent Blanchoin, Bruce L. Goode  Molecular Cell  Volume 34, Issue 3, Pages 364-374 (May 2009) DOI: 10.1016/j.molcel.2009.02.029 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Effects of crn1Δ on Actin Turnover In Vivo (A) Yeast strains compared for growth at 34°C on YPD. (B) Complementation of crn1Δ cof1-22 growth defects by plasmid expression of Crn1 (ΔCC) or Crn1 (FL). Transformants were serially diluted and spotted on selective medium. Complementation was tested at 37°C, because crn1Δ cof1-22 cells do not exhibit temperature-sensitive growth at 34°C on selective medium. (C) Crn1 domains; CC, coiled-coil domain. (D–I) Deletion of CRN1 reduces rates of actin turnover in wild-type (WT) and cof1-22 cells. Strains were treated with indicated amounts of LatA. Samples removed at time points were fixed and stained with Alexa 488-phalloidin. Cells (n > 200) were scored for (D) visible cables or (E and G) patches. Data shown (mean ± SD) were obtained from two experiments. (F) Representative cells from (E). (H) Representative cells from (G). (I) The average F-actin intensity per cell was calculated by using ImageJ software (n > 50 cells for each data point). Molecular Cell 2009 34, 364-374DOI: (10.1016/j.molcel.2009.02.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Effects of Crn1 (FL) on Cof1-Mediated Actin Assembly and Disassembly (A) Quenching of F-actin pyrene fluorescence by Cof1 (125, 400, 600, 875, 1200, 1400, 1625, 1750, and 2000 nM). The circled data point (125 nM) is the concentration of Cof1 used in all disassembly assays in this study. (B) Kinetics of DBP-induced actin disassembly. Reactions contained 2 μM F-actin, ± 125 nM Cof1, ± 600 nM Crn1 (FL). (C) Distribution of actin between pellet and supernatant for the reactions in (B). After 1700 s and 15 hr, samples were centrifuged at 80,000 rpm for 20 min and analyzed on Coomassie-stained gels. (D) Concentration-dependent inhibitory effects of Crn1 (FL) (60, 180, 300, 600, and 900 nM) on Cof1-mediated actin disassembly. (E) Actin assembly assays containing 2 μM actin, 250 nM Cof1, and variable concentrations of Crn1 (FL). (F) Rates of assembly determined from curves in (E). (G) Time-lapse TIRF microscopy of ATP-actin polymerized in the presence of Cof1 and/or Crn1 (FL). Reactions contained 1.5 μM ATP-actin, 4.5 μM profilin, ± 500 nM Cof1, ± 1 μM Crn1 (FL). (H) Filament length (mean ± SEM; n = 22 filaments) was determined in the window of time between 5 and 12 min for the reactions in (G). Molecular Cell 2009 34, 364-374DOI: (10.1016/j.molcel.2009.02.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Effects of the Crn1 Coiled-Coil Domain on Cof1-Mediated ATP-Actin Assembly (A) 2 μM actin was polymerized with 250 nM Cof1 and different concentrations of Crn1 (CC). (B) Rates of assembly determined from curves in (A). (C) Time-lapse TIRF microscopy of 1.5 μM ATP-actin polymerized in the presence of 4.5 μM profilin, ± 500 nM Cof1, ± 4 μM Crn1 (CC). (D) Filament length (mean ± SEM; n = 22 filaments) determined in the window of time between 5 and 12 min for reactions in (C). (E) A Coomassie-stained gel showing binding of Crn1 (CC) to 2 μM F-actin in cosedimentation assays. Molecular Cell 2009 34, 364-374DOI: (10.1016/j.molcel.2009.02.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Effects of Crn1 (ΔCC) on Cof1-Mediated Actin Assembly and Disassembly (A) Kinetics of DBP-induced actin disassembly. Reactions contained 2 μM F-actin, ± 125 nM Cof1, ± 600 nM Crn1 (ΔCC). (B) A Coomassie-stained gel showing the distribution of actin between the high-speed pellet and the supernatant after 1700 s and 15 hr for samples of reactions in (A). (C) Assembly of 2 μM actin, ± 250 nM Cof1, ± 800 nM Crn1 (ΔCC). (D) Effects of Crn1 (ΔCC) (25, 100, 200, 400, 600, and 800 nM) on the rate of 2 μM actin assembly ± 250 nM Cof1. (E) Two-step seeded assembly assay. First reactions (inset) contain 2 μM actin, ± 250 nM Cof1, ± 600 nM Crn1 (ΔCC). Second reactions (shown) contain G-actin and “seeds” from the first reactions. Control reactions (1–4) lack seeds and contain G-actin, ± 25 nM Cof1, ± 60 nM Crn1 (ΔCC). (F) Rates of assembly determined from curves in (E). Data from two experiments (mean ± SD). (G) Visualization of F-actin seeds produced in first reactions in (E). (H) An integrated crn1ΔCC allele complements crn1Δ cof1-22 synthetic growth defects at 34°C. Molecular Cell 2009 34, 364-374DOI: (10.1016/j.molcel.2009.02.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Enhanced Cof1-Mediated Actin Assembly Requires Crn1 (ΔCC) Binding to F-Actin (A) Coomassie-stained gels showing binding of 0.75 μM wild-type and mutant Crn1 (ΔCC) to 3 μM F-actin. (B) Quantification of data from two experiments as in (A) (mean ± SD). (C) Effects of 600 nM wild-type and mutant Crn1 (ΔCC) on assembly of 2 μM actin + 250 nM Cof1. (D) Quantification of data from two experiments as in (C) (mean ± SD) normalized to the effects of wild-type Crn1 (ΔCC) (100% activity). (E) Cosedimentation assay showing that 300 nM Crn1 (ΔCC) increases the association of 100 nM Cof1 with 3 μM F-actin. (Upper panel) Coomassie-stained gel. (Lower panel) Western blot probed with anti-Cof1 antibodies. (F) Quantification of data from four experiments as in (E) (mean ± SD). Molecular Cell 2009 34, 364-374DOI: (10.1016/j.molcel.2009.02.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Effects of Crn1 (FL) and Crn1 (CC) on the Cof1-Mediated Severing of ADP-Actin (A) Assembly of 2 μM ATP-actin, ± 600 nM Crn1 (FL), ± 250 nM Cof1. (B) Assembly of 4 μM ADP-actin, ± 1.2 μM Crn1 (FL), ± 500 nM Cof1. (C) Effects of Crn1 (FL) (75, 125, 600, 1200, 1600, and 2000 nM) on the assembly of 4 μM ADP-G-actin + 500 nM Cof1. (D) Comparison of assembly rates for ATP-actin (as in [A]) and ADP-actin (as in [B]) from two experiments (mean ± SD). (E) Time-lapse TIRF microscopy of 2 μM ADP-actin polymerized with ± 1 μM Cof1 ± 1.2 μM Crn1 (FL). (F) Filament length (mean ± SEM; n = 22 filaments) for reactions in (E) determined in the window of time between the 13 and 20 min time points. (G) Filament length (mean ± SEM; n = 22 filaments) as in (F) for experiment similar to those in (E), but using 1 μM Crn1 (CC) rather than Crn1 (FL). (H) Cosedimentation assays testing competition between 1 μM Crn1 (FL) and Cof1 (0–32 μM) for binding to 2 μM ATP/ADP+Pi-F-actin (black) or ADP-F-actin (red). (I) Same as (H), but using 3 μM Crn1 (CC) instead of Crn1 (FL). Molecular Cell 2009 34, 364-374DOI: (10.1016/j.molcel.2009.02.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Crn1 Excludes GFP-Cof1 from Newly Assembled Regions of Actin Patches (A) Abp1-RFP and GFP-Cof1 localization in sla2Δ CRN1+ and sla2Δ crn1Δ strains; representative images. (B) Quantification of Abp1-RFP and GFP-Cof1 fluorescence intensities as a function of distance from the cell cortex (three measurements averaged per actin patch; n = 10 cells for each strain). (C) Working model for coronin function in actin disassembly with two separate actin-binding domains (β propeller and CC). At newly assembled regions of actin networks rich in ATP/ADP+Pi-actin, the CC-actin interaction blocks cofilin binding and severing. At older regions of networks rich in ADP-actin, the CC-actin interaction no longer blocks cofilin binding, and the coronin β propeller synergizes with cofilin to sever filaments. This model also depicts the CC domain binding to the Arp2/3 complex, which may locally disrupt CC-actin interactions to promote cofilin severing of ATP/ADP+Pi-actin branches. Molecular Cell 2009 34, 364-374DOI: (10.1016/j.molcel.2009.02.029) Copyright © 2009 Elsevier Inc. Terms and Conditions