Bundling promotes barbed end attachment to WCA domains in the absence of Arp2/3 complex.

<p>Conditions: 8 µM (30% labeled) actin was polymerized in low Mg²⁺ F buffer. Filaments were incubated with GST-WCA coated microspheres with 1 µM ATP-actin monomers and the indicated final concentration of Mg²⁺, Lys-Lys²⁺, fascin, or K⁺ for 10 minutes. Beads were centrifuged, resuspended in low Mg²⁺ buffer in the absence of methylcellulose, and imaged on poly-lysine coated coverslips. (<b>A</b>) TIRF microscopy images of actin filaments and bundles attached to the bottom of coated microspheres in the indicated concentration of Mg²⁺, Lys-Lys²⁺, fascin, or K⁺. The number of actin filaments and bundles crossing the bead boundary (dashed circle) were counted for each bead. Scale bar is 2 µm. An example measurement is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031385#pone.0031385.s006" target="_blank">Figure S<b>6</b></a>. (<b>B</b>) Count of average number of captured filaments per bead as a function of Mg²⁺ (○), Lys-Lys²⁺ (•), fascin (□), or K⁺ (⋄) concentration. Bundles were counted as two filaments. Error bars show S.D. from at least 60 beads for each condition from three independent experiments. Coincidental filament overlap with control, BSA coated microsphere (⧫) was negligible. (C) Stacked bar chart showing average number of filaments (light gray) or bundles (dark gray) captured by WCA-coated microspheres, with indicated Mg²⁺, Lys-Lys²⁺, fascin concentrations. The proportion of captured bundles increased with increasing polycation or fascin.