Figure 4. Real-Time Observations of Cofilin Severing Actin Filaments and Dissociating Branches by Fluorescence Microscopy.
(A and B) Time series of total internal reflection fluorescence micrographs showing the formation of actin filament branches and dissociation of branches by cofilin. Conditions: 10 mM imidazole (pH 7.0), 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 100 mM DTT, 0.2 mM ATP, 50 mM CaCl2, 15 mM glucose, 20 mg/ml catalase, 100 mg/ml glucose oxidase, and 0.5% methylcellulose (15 cP at 2%).
(A) Time course of actin filament branching and elongation by 2 mM Mg-ATP-actin monomers (40% Oregon green), 20 nM Arp2/3 complex, and 200 nM VCA imaged every 10 s.
(B) Time course after addition of 1 µM cofilin imaged every second.
(C) Time course of the loss of branches after adding a range of concentrations of cofilin.
(D) Dependence of the severing and debranching rates on the cofilin occupancy of the actin filaments during the first 30 s after adding cofilin. Rates are the total number of observed severing and debranching events divided by the time of observation (30 s).