Fig. 5. Loss of the CP β tentacle tethers NPFs to capped filament ends via their WH2 actin binding site.

A Scheme of the FRET setup. Surface-bound WAVEΔN molecules are donor-(Alexa488-) labeled upstream of their WH2 site. NPF WH2 can either interact with quencher- (Atto540Q-) labeled actin monomers resulting in a decrease of donor fluorescence or unlabeled terminal protomers of filament barbed ends, resulting in no change in fluorescence. Terminal protomers are unlabeled, since quencher-labeled monomers are introduced only upon network arrest. Loss of the β tentacle vacates the WH2 binding site at the terminal protomer of capped ends, tipping the balance in favor of end-binding. B Left: Representative epifluorescence images of Alexa488-WAVEΔN-coated microspheres (green) and dendritic actin networks (visualized by Cy5-UTRN₂₆₁, blue) 3 min after arrest before (top) and after (bottom) quenching with Atto540Q-labeled actin monomers. Protein concentrations were as in Fig. 4 in the presence of wt or mutant capping proteins, as indicated. Right: Average intensities (continuous lines at the center of the error band, dashed lines error = SD) of the NPF-fluorescent signal intensity on the microsphere surface in the presence or absence of Atto540Q-labeled actin monomers (n = 25 beads per condition). Source data are provided as a Source Data file.