Figure 5.

Fascin-induced bundling moderately affects lipid-anchored formin detachment rate but not elongation rate. (A) Sketch of a fascin-induced 2-filament bundle elongated by lipid-anchored formins in a microfluidics flow. (B) Typical field of view of filament bundles elongated by FH1-anchored-to-lipids formins at the edge of a lipid square pattern, in the presence of 0.2 μM 15% alexa488-labeled actin, 2 μM profilin, and 100 nM fascin, in a microfluidics flow. (C) (Top) Raw data of a 2-filament bundle imaged with both 15% alexa488-labeled actin and 100% alexa568-labeled fascin; (bottom) Kymograph of a 2-filament bundle where a leading filament moves upstream as its elongation rate is higher than the elongation rate of the trailing filament. 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. (D) Elongation rate distributions of single filaments (n = 18) or 2-filament bundles (n = 18) elongated by FH2-anchored-to-lipids formins (Student’s t test p-value = 0.34). (E) Survival fractions of lipid-anchored formin-bound single filaments or 2-filament bundles for FH1-anchored-to-lipids (n = 15 and 22 filaments, for single filaments and 2-filament bundles respectively) or FH2-anchored-to-lipids formins (n = 35 and 25 filaments, for single filaments and 2-filament bundles respectively) in the presence of 0.2 μM actin, 2 μM profilin, and 100 nM fascin. (F) Normalized individual formin detachment rates for either FH1-anchored-to-lipids or FH2-anchored-to-lipids individual formins elongating 2-filament bundles, relative to the detachment rate of formins elongating single, unbundled filaments (orange bar). For comparison, the average detachment rate of both leading and trailing unanchored formins is shown (light blue bar). (Error bars are standard errors.)