Volume 43, Issue 3, Pages 464-477 (August 2011) Cordon-Bleu Uses WH2 Domains as Multifunctional Dynamizers of Actin Filament Assembly  Clotilde Husson, Louis Renault, Dominique Didry, Dominique Pantaloni, Marie-France Carlier  Molecular Cell  Volume 43, Issue 3, Pages 464-477 (August 2011) DOI: 10.1016/j.molcel.2011.07.010 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Cobl Displays Multifunctional Regulation of Actin Assembly (A) Structural organization of Cobl and the constructs used in this study. K, N-terminal lysine-rich region (1142–1161). A, B, and C represent the three WH2 domains of Cobl. (B–F) Polymerization curves of actin (2 μM, 10% pyrenyl labeled) in the presence of Cobl-K-A, Cobl-K-AB, Cobl-K-ABC, Cobl-AB, and Cobl-ABC as indicated. See also Figures S1–S4. Molecular Cell 2011 43, 464-477DOI: (10.1016/j.molcel.2011.07.010) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Binding of Cobl-K-A, Cobl-K-AB, and Cobl-K-ABC to ATP-G-Actin (A) AEDANS-G-actin (0.5 μM) was titrated by Cobl-K-AB in G-buffer. Inset: Titration of Cobl-K-AB (1 μM) by AEDANS-G-actin (open symbols). Closed symbols, actin alone. Equivalence points in both titrations are consistent with a 1:1 complex. (B) AEDANS-actin anisotropy change in the presence of Cobl K-ABC at 1.0 μM (closed squares) and 1.4 μM (open squares). Data are consistent with a 1:1 complex (anisotropy declines following addition of one actin to one Cobl-K-ABC molecule). Curves are calculated using the following anisotropy values: r(actin) = 0.104, r(Cobl-K-ABC-actin) = 0.135, and KA = 0.15 μM for the equilibrium disociation constant. (C) Analysis of Cobl-actin complexes on size-exclusion chromatography and SDS-PAGE. Either actin alone (20 μM) or mixtures of Cobl-K-A or Cobl-K-AB (20 μM) or Cobl-AB (3.4 μM) and actin at indicated molar ratios were loaded on the column. Cobl-K-AB itself eluted with an apparent Stokes radius of 27 Å (data not shown). (D) SDS-PAGE analysis of the eluted complex of actin and Cobl-K-AB. Different volumes of the peak fraction were co-electrophoresed with actin and Cobl-K-AB standards. The actin:Cobl molar ratios in the load were 1:1 (a and b) and 2:1 (c and d). (E) The initial rate of barbed-end growth was measured at different G-actin concentrations in the absence (closed circles, red line) and presence of 25 μM Cobl-K-AB (open circles, blue line). See also Figure S5. Molecular Cell 2011 43, 464-477DOI: (10.1016/j.molcel.2011.07.010) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Cobl-K-AB Sequesters ADP-G-Actin with High Affinity (A) ATP-G-actin 1:1 complex (2 μM, 10% pyrenyl-labeled) was polymerized in presence of either 5 μM or 200 μM ATP and Cobl-K-AB as indicated. (B) ATP-G-actin 1:1 complex, at the indicated concentrations, was polymerized with 5 μM ATP and 2.3 μM Cobl-K-AB. Inset: Change in pyrenyl-actin fluorescence, derived from data in main frame (plus an additional curve at 12.3 μM actin), at the intermediary plateau (open circles) and final plateau (closed squares), representing the steady state in ATP and the equilibrium state in ADP, respectively. Since the critical concentration for assembly of ADP-actin is 1.6 μM, the presence of 3.7 to 3.9 μM unassembled ADP-actin indicates that 2.1 to 2.3 μM actin is in complex with Cobl-K-AB at a total concentration of 2.3 μM, hence KAD = 1.6x 0.1/2.2 = < 0.07 μM. See also Figure S6. Molecular Cell 2011 43, 464-477DOI: (10.1016/j.molcel.2011.07.010) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Cobl Severs Filaments and Enhances the Rate of Depolymerization (A) F-actin (2.5 μM, 50% pyrenyl-labeled) was depolymerized upon 50-fold dilution in the presence of Cobl-K-AB. (B) Histogram of the fold increase in depolymerization rate in the presence of Cobl at 0.5 μM (gray bars). Hatched bars represent the maximal depolymerization rate at high concentration of Cobl-K-A and Cobl-AB. No maximal value is observed with Cobl-K-AB and Cobl-K-ABC, consistent with a severing activity. (C) Fluorescence microscopy evidence for severing of filaments by Cobl and permissive reannealing. Top: Severing and reannealing of Alexa 488- and Alexa 594-labeled F-actin (1 μM) mixed vol:vol at time zero with 0.5 μM Cobl-K-AB. Bottom: F-actin (1 μM) mixed with 0.5 μM Cobl-K-ABC, Cobl-AB, Cobl-ABC, and Cobl-K-A and observed 1 min after addition of Cobl. Molecular Cell 2011 43, 464-477DOI: (10.1016/j.molcel.2011.07.010) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Cobl Does Not Inhibit Filament Assembly from Profilin-Actin (A) Steady-state measurements of F-actin assembled in the presence of increasing amounts of profilin, in the absence (open circles) or presence of Cobl-K-AB (open squares) or Cobl-AB (closed squares). (B) Spontaneous assembly of actin (1.5 μM, 5% pyrenyl-labeled) in the presence of 4 μM profilin and Cobl-K-AB (red curves) or Cobl-K-ABC (blue curves). Nucleating activity of Cobl is low due to the very low level of free G-actin in the presence of profilin. Molecular Cell 2011 43, 464-477DOI: (10.1016/j.molcel.2011.07.010) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Model for the Multifunctional Regulation of Actin Assembly by Cobl (A) Scheme for the multifunctionality of Cobl in actin assembly. First, spontaneous nucleation of actin is facilitated by Cobl. Nucleation of actin alone occurs via subsequent energetically unfavorable formation of a longitudinal dimer and trimer (steps 1 and 2), followed by barbed-end growth, ATP hydrolysis (red to blue transition in the actin cleft (step 4), and release of ADP-actin (step 6). The formation of the Cobl-actin complex CA facilitates electrostatic interaction with another G-actin molecule, promoting formation of prenuclei CA2 and CA3 complexes (steps 14 and 15), which feeds the production of nuclei A3 in higher amounts (step 16). Second, the CA complex participates in barbed-end assembly (step 17); C enhances disassembly of ADP-actin (steps 19 and 20). Third, severing of filaments by Cobl occurs via transient association of Cobl to the side of filaments, structural deformation upon insertion of Cobl, severing and stripping off an ADP-subunit (step 21), followed by spontaneous reannealing (step 10). (B) Theoretical polymerization curves generated using the scheme illustrated in (A) with experimentally determined values of rate and equilibrium parameters (Table S1). The fit to experimental curves recorded at 2 μM actin in absence (curve 0) and presence of Cobl-K-ABC (curves numbered 1 to 4 are data from Figure 1D at 0.05, 0.1, 0.5, and 2 μM Cobl-K-ABC, respectively) is generally satisfactory. The mass amount of F-actin is proportional to the fluorescence change. (C) Computed time course of concentrations of nuclei A3 (green curves) and filament ends F (blue curves) corresponding to the polymerization curves shown in (B). See also Table S1. Molecular Cell 2011 43, 464-477DOI: (10.1016/j.molcel.2011.07.010) Copyright © 2011 Elsevier Inc. Terms and Conditions