Constraining two-filament bundles in twist highly enhances their fragmentation. (A) Top: Schematics illustrating the numerical simulations of two 5-µm long actin filaments (yellow) interconnected by fascin (dark gray), where cofilin clusters (light blue) can form and sever filaments (dark blue). Bottom: Kymographs of two interconnected simulated filaments, showing cofilin cluster initiation (arrow) and severing (thunderbolt) events on each filament. The kymographs stop when the severing events on the two filaments result in the fragmentation of the bundle. Numerical values of reaction rates are summarized in Table S1. (B and C) Fraction of unsevered 5-µm segments that are (B) twist-unconstrained or (C) twist-constrained by being doubly attached to the glass surface, for single actin filaments (light blue, n = 53, 34 for filaments unconstrained and constrained in twist respectively) or two-filament bundles (dark blue, n = 47, 16 for bundles unconstrained and constrained in twist, respectively) upon exposure to 200 nM cofilin and 200 nM fascin, as a function of time. 95% confidence intervals are shown as shaded surfaces. Dashed lines correspond to the results obtained from numerically simulated segments (n = 200 for twist-unconstrained, 50 for twist-constrained segments), using experimentally determined rates and considering no inter-filament cooperativity in twist-constrained bundles (see main text). (D) Schematics of the interfilament cooperative twisting model. For a fascin-induced two-filament bundle, a first cofilin cluster on one of the filaments is initiated and starts growing, preventing fascin from binding locally. Local over-twisting caused by cofilin decoration is transmitted to the adjacent filament in the region devoid of fascin. This favors the binding of cofilin on the undecorated filament: the initiation rate of the second cofilin cluster is 48-fold higher than for the first cofilin cluster.