Volume 11, Issue 8, Pages 620-625 (April 2001) The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension  Maryse Bailly, Ilia Ichetovkin, Wayne Grant, Noureddine Zebda, Laura M Machesky, Jeffrey E Segall, John Condeelis  Current Biology  Volume 11, Issue 8, Pages 620-625 (April 2001) DOI: 10.1016/S0960-9822(01)00152-X

Figure 1 The effect of AE360 anti-p34 antibody on the nucleation and side binding functions of the Arp2/3 complex. (a) Immunolocalization of the p34 subunit of the Arp2/3 complex using AE360 antibodies. The scale bar indicates 10 μm. Inset, an immunoblot showing the immunoprecipitation of the p34 subunit of the Arp2/3 complex from whole cell lysates using AE360 antibodies. Lane 1, AE360 antibodies; lane 2, control beads without IgG; the black mark indicates 36.5 kDa molecular weight marker. (b) Nucleation assay in vitro: 3 μM actin (5% pyrene-labeled), 10 nM Arp2/3, and 10 nM GST-VCA (squares, 40:1 ratio of antibodies to Arp2/3) or 2 μM actin, 2 nM Arp2/3, 2 nM GST-VCA (circles, 200:1 ratio of antibodies to Arp2/3) were incubated in the presence of either 400 nM AE360 anti-p34 antibodies (closed symbols) or an equal volume of TBS buffer (open symbols) at 22°C in polymerization buffer (2 mM Tris [pH 8], 0.2 mM CaCl2, 0.02% NaN3, 0.2 mM ATP, 0.5 mM DTT, 1 mM EGTA, 2 mM MgCl2, and 50 mM KCl). (c) The Arp2/3 complex-actin cosedimentation assays: 4 μM F-actin, 0.25 μM Arp2/3 complex, 0.25 μM antibodies as specified, in 5 mM imidazole (pH 7.0), 50 mM KCl, 1 mM MgCl2, 0.2 mM ATP, and 1 mM EGTA. The graph shows the percentage of the Arp2/3 complex that is bound to F-actin after 30 min of incubation at 22°C in the presence of the respective antibodies. (d) The effect of AE360 antibodies on the lag phase in nucleation by the Arp2/3 complex. The Arp2/3 complex (27 nM) and GST-VCA (100 nM) were added to polymerizing actin (4 μM, 5% pyrene-labeled) at 0 s (squares), 400 s (circles), and 700 s (triangles) after initiation of polymerization in the presence of 500 nM nonimmune IgG (open symbols) or AE360 antibodies (closed symbols) Current Biology 2001 11, 620-625DOI: (10.1016/S0960-9822(01)00152-X)

Figure 2 The effect of AE360 anti-p34 antibodies on the branching activity of the Arp2/3 complex as measured in electron and light microscope assays. (a–d) Observations of the actin filaments in the electron microscope. Conditions: 4 μM actin, 4 μM phalloidin, 425 nM GST-VCA, and 300 nM antibodies. Actin filaments polymerized for 30 min in the presence of (a) nonimmune IgG, (b) 75 nM Arp2/3 complex and nonimmune IgG, and (c) 75 nM Arp2/3 complex and anti-p34 antibodies. The arrows point at typical branching. The scale bar indicates 0.2 μm. (d) Quantitation of the branching (number of branches per μm of filament): actin + IgG (n = 1472), actin + Arp2/3 + IgG (n = 2181), actin + Arp2/3 + anti-p34 (n = 5038); significant difference (p = 1.3x10−6). (e–h) Direct observation of actin filaments in the light microscope. Conditions: 2 μM actin, 200 nM phalloidin, 40 nM GST-VCA, and 40 nM Arp2/3. Actin filaments polymerized for 20 min in the presence of (e) TBS buffer blank, (f) 400 nM anti-p34 antibodies, and (g) 800 nM anti-p34 antibodies. The scale bar indicates 5 μm. (h) Quantitation of the total filament number and number of branched filaments (per field, mean ± SEM.). Additional control chambers were perfused with actin in the presence of 1200 nM nonimmune IgG to ensure that IgG has no nonspecific effect on actin binding to nitrocellulose (data not shown) Current Biology 2001 11, 620-625DOI: (10.1016/S0960-9822(01)00152-X)

Figure 3 Inhibition of lamellipod extension after microinjection of AE360 anti-p34 antibodies. Left panel; (a,c) morphology of control noninjected cells, and (b,d) cells microinjected with AE360 anti-p34 antibodies immediately (a, b) before and (c,d) 3 min after EGF stimulation. Lamellipod extension is shown on control cells (arrowheads). Right panel; quantitation of the protrusive activity after EGF stimulation in cells 30–45 min after antibody microinjection, measured as described previously [3, 21]. Triangles, AE360 anti-p34 antibodies (n = 14); squares, nonimmune IgGs (n = 75); and diamonds, control mock-injected cells (n = 17). SEM < 5% Current Biology 2001 11, 620-625DOI: (10.1016/S0960-9822(01)00152-X)

Figure 4 Barbed end number and distribution in vivo after microinjection of AE360 anti-p34 antibodies. Biotin-labeled actin (0.45 μM) was incorporated into permeabilized cells to visualize free barbed ends [3, 23] for actin polymerization after stimulation, and incorporation at the leading edge was quantified 30 s and 60 s after EGF stimulation by measuring the fluorescence intensity within 1.1 μm at the leading edge [3]. (a) exogenous-labeled actin incorporation at the leading edge of cells microinjected with AE360 anti-p34 antibodies after 1 min of EGF stimulation (the arrow points to the microinjected cell). The scale bar indicates 20 μm. (b) The relative number of barbed ends at the leading edge. White columns, control cells in the field (n = 8–30); and gray columns, cells microinjected with anti-p34 antibodies (n = 10–27). Mean ± SEM Current Biology 2001 11, 620-625DOI: (10.1016/S0960-9822(01)00152-X)