Fascin crosslinking does not appear to slow down Pi release in actin filaments. Actin filaments elongated from spectrin–actin seeds in microfluidics chambers were bundled and aged for 15 min by exposing them to 0.15 µM actin and 200 nM fascin. Actin filaments were then unbundled and they depolymerized as single filaments upon exposure to buffer only. In the absence of fascin in solution, bundled filaments become individual isolated filaments after typically 30 s. The depolymerization rates (measured over 3 min) of individual actin filaments that were initially either isolated filaments or part of two-filament bundles when exposed to fascin, were quantified (N = 3 repeats, with n = 44, 47, and 44 for single filaments, and n = 45, 63, and 42 for two-filament bundles, respectively). Large symbols represent median values. The P value is calculated from the comparison of the paired median values.