(A) Cartoon illustrating his10Cherry-SCARAPWCA, his10GFP-Lpd850−1250aa, and his10GFP tethered to a lipid coated beads (LCBs) containing DOGS-NTA(Ni) lipid (blue head groups). Actin network assembly on the bead surface is initiated by adding monomeric actin, profilin 1, Arp2/3, capping protein, and buffer containing KCl. (B, C) Membrane tethered his10-GFP-Lpd850−1250aa slows actin network assembly on LCBs. (B) Representative actin comet tails assembled in the presence of 7.5 µM actin (5% Alexa488-Actin), 3 µM hProfilin 1, 100 nM Arp2/3, 100 nM capping protein, and buffer containing 150 mM NaCl. LCBs (2.3 µm, 4% DOGS-NTA(Ni): 96% DOPC) were charged with 75 nM his10-Cherry-SCARAPWCA, plus 25 nM his10-GFP-Lpd850−1250aa or 25 nM his10-GFP (i.e., 75% his10-Cherry, 25% his10-GFP). Actin network assembly and disassembly was stopped at the indicated time points by combining the bead motility assay, 1:1, with 37.5 µM Latrunculin B-phalloidin mixture. Scale bar, 5 µm. (C) Representative actin comet tails assembled as in (B) for 5 min before transitioning from actin motility mix with 7.5 µM actin (5% Alexa488 labeled, GREEN) into an identical mix, but containing 7.5 µM actin (5% Cy3-Actin, RED). The length of Cy3-actin incorporated into the comet tail was measured to determine the growth velocity of multiple tails (n ≥ 50 tails). Error (±) represents the standard deviation of the mean (p-value = 3 × 10−29; two-tailed t-test for data sets with equal variance). Scale bar, 10 µm. (D) Homogenous distribution of his10-Cherry-SCARAPWCA and his10GFP-Lpd850−1250aa before initiating actin network assembly. Scale bar, 5 µm. (E, F) Spatial distribution of his10Cherry-SCARAPWCA, his10GFP-Lpd850−1250aa, and his10-GFP during steady state actin tail growth and recycling (30 min time point). Actin networks were assembled in the presence of 7.5 µM actin (5% Alexa488), 3 µM hProfilin 1, 100 nM Arp2/3, 100 nM Mm capping protein, and 3 µM hCofilin. (E) his10Cherry-SCARAPWCA and his10GFP-Lpd850−1250aa concentrate on the barbed end dense side of the actin comet tail. (F) his10-Cherry-SCARAPWCA concentrates on the barbed end dense side of the actin comet tail, while his10-GFP is excluded from the barbed end attachment zone. Line scans across LCBs are shown to the right. Scale bar, 5 µm.