Figure 4.

Single surface attachment of the filaments reduces single formin elongation and processivity. (A) (top) Sketch of a glass-anchored formin elongating a single filament whose rotation is blocked by surface attachment upon binding of a NEM–myosin to the filament side. (bottom) Kymograph showing an actin filament elongated by a FH1-anchored-to-glass formin, whose elongation is slowed down upon filament binding to a surface-bound NEM–myosin, until the filament unbinds from the surface-anchored formin (arrow). Filament unbinding from the formin-decorated surface is interpreted as a formin-barbed end detachment event and used to quantify formin off rates k_off. (B) Distribution of elongation rates of individual actin filaments, elongated by FH1-anchored-to-glass formins, bound or not to NEM-myosins attached to the glass surface (n = 48 for both conditions; Student’s t test p-value <0.001). (C) Survival fractions of formin-bound single filaments attached to the surface via a NEM–myosin or not, for FH1-anchored-to-glass (n = 78 for NEM–myosin-bound and 35 for single filaments) or FH2-anchored-to-glass formins (n = 53 for NEM–myosin-bound and 76 for single filaments) in the presence of 0.2 μM actin and 2 μM profilin. (D) Normalized formin detachment rates for either FH1-anchored-to-glass or FH2-anchored-to-glass individual formins, whose elongating filaments are rotationally blocked by a surface-attached NEM-myosin, relative to glass-anchored formins elongating rotationally unconstrained single filaments.