Figure 4. Nanomolar concentrations of Lifeact-mCherry inhibit filament severing by cofilin.

Conditions: Actin filaments were preformed in the microscopy chamber by incubating 0.5 μM actin monomers (20% Alexa 488-labeled) in microscopy buffer (see Methods) for ~5 min. The buffer containing unpolymerized actin monomers was replaced with the same buffer containing cofilin and Lifeact-mCherry. Images were collected over time by TIRF microscopy. (a) Representative time-series of fluorescent micrographs of preformed actin filaments incubated with 20 nM Sp cofilin or 5 μM Hs cofilin in the absence or presence of 1.5 or 1 μM Lifeact-mCherry. (b, c) Dependence of the rate of actin filament severing on the concentration of Lifeact-mCherry. The units of severing activity are severing events • 10⁻⁴ μm⁻¹ s⁻¹. Data represent one of 3 sets of independent experiments that each produced samples containing hundreds of filaments and severing events. (b) Severing by 20 nM Sp cofilin. Inset shows low Lifeact-mCherry concentrations. (c) Severing by (closed circles) 5 μM, (open circles) 10 μM or (squares) 20 μM Hs cofilin. (d) Thermodynamic scheme of cofilin and Lifeact binding to two contiguous filamentous actin sites. Dashed box outlines interactions between a single Lifeact (L) and cofilin (C) with a filamentous actin subunit (F), resulting in Lifeact-bound actin (L_F), cofilin-bound actin (F_C) and Lifeact- and cofilin-bound actin (L_F_C) (no cooperativity). See methods section for a complete description of the model. Asterisks denote binding constants measured in Cao et al.⁴⁰. Daggers denote binding constants were derived from detailed balance.