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. 2021 Sep 9;12:5329. doi: 10.1038/s41467-021-25682-5

Fig. 4. The CP β tentacle is essential for reconstituted branched actin network assembly.

Fig. 4

A Scheme describing bead-motility-assay. Dendritic actin networks are assembled from polystyrene microspheres (Ø =3 μm) coated with WAVE1ΔN via biotin-streptavidin linkages. Dense branched actin networks assemble from these beads and incorporation of all constituting components can be visualized via matching fluorescent labels as indicated. B Top: Representative epifluorescence images of dendritic actin networks grown from WAVE1ΔN-coated microspheres using 5 μM profilin–actin complexes (see Methods), 100 nM CP (either wt or mutants as indicated, 20% TMR-labeled), 100 nM Arp2/3 (20%-Alexa647 labeled). Reactions were kinetically arrested after 4 min using phalloidin (15 μM), Latrunculin-B (15 μM) and Alexa488-lifeact (15 nM) to visualize filamentous actin. The experiment was repeated three times with similar results. Bottom: Quantification of the averaged intensity profiles for the indicated dendritic network components. Source data are provided as a Source Data file. C Plots of network growth velocities of dendritic actin networks grown in presence of either wt or mutant CP. Source data are provided as a Source Data file. D Plots of polymerization, capping and nucleation rates of dendritic actin networks grown in presence of either wt or mutant CP (see Methods). Source data are provided as a Source Data file. Quantifications in C and D were done for n = 25 actin networks from N = 3 independent experiments, error indicator = SD of the mean. P-values were derived from one-way ANOVA Tukey tests.