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. Author manuscript; available in PMC: 2017 Oct 24.
Published in final edited form as: Curr Biol. 2016 Sep 22;26(20):2697–2706. doi: 10.1016/j.cub.2016.07.080

Figure 2. Fascin and α-actinin compete and segregate to different F-actin networks reconstituted on motile beads.

Figure 2

A) Low speed (10,000 × g) sedimentation of F-actin assembled from 3.0 μM Mg-ATP actin monomers with 600 nM fascin and a range of α-actinin concentrations. Coomassie-stained SDS-PAGE gel (top), and graph (bottom) of the amount of actin (A), fascin (F) or α-actinin (α) in pellets. Error bars indicate SEM; n≥2.

(B–G) Addition of GST-pWA-coated beads to polymerization reactions containing 4 μM Mg-ATP actin (1% Oregon green-actin), 5 μM profilin, 100 nM Arp2/3 complex, 20 nM capping protein (CP), 150 nM Cy5-fascin (blue), with and without 50 nM TMR-α-actinin (red).

(B) Cartoon of comet tail and protrusive networks formed on a motile bead.

(C and D) Fluorescent confocal micrographs of beads in the presence of (C) Cy5-fascin or (D) Cy5-fascin and TMR-α-actinin. Shown are representative single planes taken from the center of a confocal z-stack with brightness enhanced in the inset for visualization of protrusions. Scale bar=2 μm.

(E and F) Comparison of the ratio of Cy5-fascin to Oregon green-actin fluorescence in (E) comet tails or (F) protrusions in the absence and presence of α-actinin. Error bars indicate SE; n=2 experiments with >20 beads each.

(G) Ratio of Cy5-Fascin to TMR-α-actinin fluorescence in protrusions and comet tails.