Figure 1. Actin filament and branch geometry in comet tails under TIRF microscopy.

Conditions: A–B, 8.5 µM (8% labeled) actin, 9 µM profilin, 100 nM Arp2/3, CP as indicated; C–E, 2 µM (10% labeled) actin, 3 µM profilin, 20 nM Arp2/3, 40 nM CP; F–H, 8.5 µM (8% labeled) actin, 9 µM profilin, 100 nM Arp2/3, CP as indicated; nanofibers coated with 10 µM GST-WCA from N-WASP, motility buffer, 0.38 mM total ATP. (A–B) Actin architecture in comet tails (T) of moving nanofibers (dashed outline) was visible under TIRF microscopy. In 100 nM CP, comet tails consisted primarily of long filament bundles. Increasing CP to 200 nM generated a branched actin networks with short bundles (Black arrowhead). (C) Lowering profilin-actin, Arp2/3, and CP concentrations showed individual filaments and branches (white arrowheads) in the comet tail (T). Some filaments (white arrows) crossed the nanofiber boundary, while others terminated at the nanofiber (black arrows). Brighter filament bundles (black arrowhead) terminated at the nanofiber. (D) Epi-fluorescence image of panel C. (E) Magnified image of box in B showing bundle (black arrowhead) dissociation. The bundle was formed from daughter filaments from the same mother filament (white arrowheads). (F) In high CP, nanofibers sometimes formed two comet tails. (G) Kymograph of line in F showing tail expansion at the nanofiber surface (dashed outline) under TIRF (left) and DIC (right) microscopy. (H) Nanofibers sometimes formed two comet tails in low CP. Long actin bundles (black arrowheads) appeared within and beyond the comet tails. Scale bars are 1 µm for E and 5 µm for all others. Times are shown in min∶sec.