Volume 28, Issue 4, Pages 555-568 (November 2007) Analysis of the Function of Spire in Actin Assembly and Its Synergy with Formin and Profilin  Montserrat Bosch, Kim Ho Diep Le, Beata Bugyi, John J. Correia, Louis Renault, Marie-France Carlier  Molecular Cell  Volume 28, Issue 4, Pages 555-568 (November 2007) DOI: 10.1016/j.molcel.2007.09.018 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Analysis of NTspire and Its Complex with G-Actin by Size Exclusion Chromatography (A) The following mixtures of NTspire (S) and G-actin (A) were loaded on the column. Black, 5 μM NTspire, no G-actin; green, 8 μM NTspire, 32 μM G-actin; red, 5 μM NTspire, 32 μM G-actin; blue, 5 μM NTspire, 25 μM G-actin, 25 μM profilin (P). Column buffer was supplemented with either no G-actin (black line), 0.1 μM G-actin (green and red lines), or 0.1 μM G-actin and 0.5 μM profilin (blue line). Absorbance scale, right axis for the black curve, left axis for others. (B) SDS-PAGE analysis of actin and NTspire in the eluted Spire-actin complex. Two samples of the eluted complex (frame A) are loaded in lanes 1 and 2 and coelectrophoresed with actin and NTspire standards as indicated. (C) Densitometric analysis of the Coomassie blue-stained gel shown in (B), showing evidence for the 1:4 Spire:actin ratio in samples 1 and 2 (color coded as in [B]). (D) NTspire (10 μM) with various amounts of G-actin (57 [blue], 45 [red], 30 [green], 13 [cyan], and 0 [black] μM) were loaded and eluted (no G-actin in column buffer). SA4 prevails, with excess Spire, at actin:Spire < 4:1. Molecular Cell 2007 28, 555-568DOI: (10.1016/j.molcel.2007.09.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Ultracentrifugation Analysis of NTspire and NTspire-Actin Complex (A) S20,w values (and their standard deviations estimated by DCDT+2 and indicated by error bars) for actin (solid line), pyrene-actine (dotted line), and NTspire (dashed line and solid symbols) are plotted versus total protein concentration (mg/ml). (B) Dilutions of column-purified spire-pyrene-actin SA4 complex (2.1 μM, black; 1.96 μM, red; 1.75 μM, green; 0.875 μM, blue) were run at 50,000 rpm, 3.6°C collecting data at 278 and 344 nm. Data at 344 and 278 nm (data not shown) reveal a tight complex and a zone of excess pyrene-actin. (C and D) AUC data were collected at 278 nm (C) and 344 nm (D) on solutions of NTspire (2 μM) and pyrene-actin at 8 μM (green), 12 μM (red), and 16 μM (black). Direct boundary global fitting of the data returned best-fitted ratios of actin:spire consistent with the loading concentrations, the appearance of excess pyrene-actin in the distributions, and the partial formation of F-actin in the solutions. (E and F) Conditions identical to (C) and (D), respectively, with samples supplemented with 20 μM latrunculin A, thus preventing F-actin formation. Direct boundary global fitting of the data returned best-fitted ratios of actin:spire consistent with the loading concentrations and appearance of excess pyrene-actin in the distribution. Molecular Cell 2007 28, 555-568DOI: (10.1016/j.molcel.2007.09.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Fluorescence Measurements of NTspire-Actin Interaction (A) Pyrenyl-G-actin (1 μM) was titrated by NTspire in F buffer. Note the equivalence point is at 0.25 μM NTspire. (Inset) Titration of NTspire (0.5 μM) by pyrenyl-labeled G-actin (open circles, no NTspire; closed circles, + NTspire). Note the equivalence point is at 2.0 μM actin. (B) NBD-G-actin fluorescence in the absence (closed circles) and in the presence (open circles) of 0.45 μM NTspire. Note the equivalence point at 1.8 μM actin. (C) Tryptophane fluorescence versus G-actin concentration in the absence (closed circles) and in the presence (open circles) of 0.5 μM NTspire. (D) Kinetic analysis of NTspire interaction with G-actin. Pyrenyl-G-actin (2 μM) was mixed with NTspire in a stopped-flow apparatus. Open circles, light line, final extent of fluorescence change. Closed circles, thick line, rate constant. Closed triangle represents the off-rate constant derived from chase experiments (Figure S2B). (Inset) Stopped-flow fluorescence traces recorded upon mixing 2 μM pyrenyl-G-actin with 0 (lower trace) or 1 μM NTspire (upper trace). (E) Anisotropy change of AEDANS-actin (1.14 μM) upon addition of NTspire, in the absence (closed circles) or in the presence (open circles) of 5 μM latrunculin A. (F) AEDANS-actin anisotropy change in the absence (squares) and presence of NTspire at 0.2 μM (closed circles), 0.4 μM (open circles), and 0.6 μM (triangles). Curves are calculated assuming cooperative binding of four actins to NTspire with KS = (0.175 μM)4 for the SA4 complex and values of 0.096 and 0.159 for the anisotropy of free actin and actin in SA4 complex, respectively. Molecular Cell 2007 28, 555-568DOI: (10.1016/j.molcel.2007.09.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 NTspire Sequesters Actin in a Nonpolymerizable SA4 Complex (A) Actin (10% pyrenyl-labeled) was polymerized at 0.4, 0.6, 1, 1.2, 1.3, 1.4, and 1.6 μM (bottom to top curves) in the presence of 0.2 μM NTspire. (Inset) Extent of final change in fluorescence. (B) Actin (2.5 μM, 10% pyrenyl labeled) was polymerized in the presence of NTspire at the following concentrations (nM, in numbered order): 0, 3, 6, 16, 31, 63, 156, 234, 313, 469, 781, 940, and 1094. (Inset) Extent of pyrenyl-F-actin fluorescence change upon assembly of actin (4, 2.5, and 1.5 μM, top to bottom) in the presence of NTspire. (C) Actin (2.5 μM, 10% pyrenyl labeled) was polymerized in the absence (closed cicles) and in the presence of 2.2 μM (open circles) or 4.4 μM (closed triangles) Tβ4 and the indicated amounts of NTspire. (D) Actin (1.2 μM, 10% pyrenyl labeled) was polymerized in the absence (closed circles) or in the presence (open circles) of 5 nM gelsolin and NTspire, as indicated. Molecular Cell 2007 28, 555-568DOI: (10.1016/j.molcel.2007.09.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Binding of NTspire to Barbed Ends Prevents Filament Assembly from Profilin-Actin (A) Effect of profilin on F-actin (1.3 μM) at steady state in the absence (circles) or presence of 14 μM Tβ4 (triangles). Profilin induces a decrease in Tβ4-actin (increase in F-actin) due to its lowering the steady-state concentration of ATP-G-actin. (B) Effect of profilin on F-actin (3 μM) at steady state in the absence of Spire (control, closed circles) and in the presence of Spire at 0.1 μM (open circles), 0.4 μM (closed triangles), and 0.8 μM (open triangles). (C) Kinetics of actin polymerization (2.5 μM) in the presence of 0.47 μM NTspire and profilin at 0, 0.4, 1.4, and 3 μM (top to bottom curves). (D) Spire blocks filament-barbed end growth from profilin-actin. Normalized rate of barbed end elongation from spectrin-actin seeds (0.14 nM) at 2.5 μM G-actin, either 9.6 (closed circles) or 14.4 μM (open circles) profilin, and NTspire as indicated. The lines are calculated best-fit binding curves with Kd = 9.2 nM at 9.6 μM profilin and 8.0 nM at 14.4 μM profilin (see the Experimental Procedures). (Inset) Raw data at NTspire concentrations (in nM) are the following: 0 (black), 6 (blue), 40 (magenta), and 240 (yellow). Bold and dim colors are 9.6 and 14.4 μM profilin, respectively. Molecular Cell 2007 28, 555-568DOI: (10.1016/j.molcel.2007.09.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 NTspire Severs Filaments and Slows Down Depolymerization upon Binding to Barbed Ends (A) Initial rate of dilution-induced depolymerization of noncapped filaments (closed circles) and gelsolin-capped filaments (open circles). Data from two independent experiments are merged in each curve. (B) Spire severs filaments. Fluorescence microscopy images of filaments assembled from 20% rhodamine-F-actin (1 μM), 30 s after addition of NTspire. (C) Average length of filaments severed by NTspire. Scale bars indicate standard error. (D) Time course of fragmentation of filaments (1 μM of 20% rhodamine-F-actin, 0.2 μM NTspire added at time zero). Filaments were diluted and stabilized at the indicated times following NTspire addition. Data points come from two independent experiments. The curve is an exponential calculated with the fragmentation rate constant of 0.03 s−1. Pyrenyl fluorescence measurements demonstrated that less than 1.5% F-actin was depolymerized within 5 min following addition of 0.2 μM NTspire. Molecular Cell 2007 28, 555-568DOI: (10.1016/j.molcel.2007.09.018) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Synergy between Spire, Formin, and Profilin in a Reconstituted Actin-Based Motility Assay of Formin-Coated Beads (A) Time-lapse recordings of mDia1 FH1-FH2-coated beads propelled in the motility assay in the absence (Aa) and in the presence (Ab) of 0.5 μM NTspire. Conditions, 7 μM F-actin, 6.4 μM profilin, and 9.8 μM ADF. Scale bar, 20 μm. (B) NTspire enhances the velocity of formin-coated beads (conditions as in [A]). (Inset) SDS-PAGE pattern of the supernatants of F-actin (7 μM) polymerized with 9.8 μM ADF, 6.4 μM profilin, and NTspire. Scale bars represent standard error. (C–E) Scheme showing how the control of barbed ends by either a capping protein or Spire affects formin-based processes. Histograms (C), steady-state concentrations of free G-actin (red), profilin-actin (blue), and free profilin (pale green), calculated at 5 μM total profilin, in cases where all barbed ends are free (left), gelsolin bound (middle), or Spire bound (right). Black bar, rate of formin-based processive assembly from profilin-actin. When barbed ends in the medium are capped (D), rapid growth of formin-bound filaments is arrested after a few seconds. When barbed ends in the medium are bound to Spire (E), processive assembly is sustained. In this example, formin is present in minute amounts as compared to the pool of filaments in the medium and serves as an indicator but does not contribute to the steady state. The color code is as follows: ATP-actin, red; ADP-Pi-actin, yellow; ADP-actin, pale blue; profilin, dark blue; capping protein, purple; formin, green; and spire, black. Molecular Cell 2007 28, 555-568DOI: (10.1016/j.molcel.2007.09.018) Copyright © 2007 Elsevier Inc. Terms and Conditions