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Fig. 7. CP inhibition of Twf1 filament binding and severing requires their direct interaction. (A) Coomassie-stained SDS-polyacrylamide gel of purified Twf1-10. Positions of molecular mass markers (kDa) are shown on the left. (B) Time course of actin filament fluorescence (30% pyrene labeled) after dilution at time zero (from 5 µM to 0.1 µM) in the presence of different concentrations of Twf1, Twf1-10 and/or CP at pH 5.0. A.U., arbitrary units. (C) TIRF microscopy of actin filaments incubated with Twf1-10. 1.5 µM monomeric actin (30% Alexa Fluor 488 labeled) was assembled into filaments in a flow cell, then 2 µM Twf1-10 in buffer pH 5.0 was applied at time zero. The time in seconds following addition of proteins is shown in the upper right corner of each panel. (D) Following filament severing by Twf1-10 in C, the immobilized filaments were incubated with fresh 1.5 µM actin monomers (30% Alexa Fluor 488 labeled) at time zero. Yellow arrowheads track the growth of barbed ends in consecutive frames. Blue arrows mark the original positions of barbed ends (at time zero) for frame of reference. The field in D is the same field as that shown in C. (E) TIRF microscopy of filaments incubated with both Twf1-10 and CP. Actin filaments were assembled as in C, then a mixture of 2 µM Twf1-10 and 3 µM CP in buffer pH 5.0 was applied at time zero. (F) Following filament severing in E, the immobilized filaments were incubated with fresh 1.5 µM actin monomers (30% Alexa Fluor 488 labeled) at time zero. Symbols are as in D. The field in F is the same field as that shown in E. Movies of these TIRF microscopy experiments are provided in supplemental materials (Movies 5 and 6, supplementary material).
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