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. 2018 Oct 23;7:e41313. doi: 10.7554/eLife.41313

Figure 3. Direct interactions of Twinfilin with Capping Protein attenuate CARMIL-mediated uncapping.

(A) Bulk fluorescence assays comparing the rates of actin assembly in the presence of 25 nM muscle Capping Protein (CPα1β1) and increasing concentrations of mTwf1. To initiate uncapping, 250 nM CBR fragment of CARMIL (see schematic, Figure 2A) was spiked into the reaction at 400 s. Data shown are representative curves from experiments repeated three independent times. (B) Representative time-lapse images from TIRF microscopy assays monitoring the displacement of labeled CP from barbed ends. Filaments were first polymerized and tethered using 1 µM actin (10% OG-labeled, 0.5% biotin–actin), then capped at their barbed ends by flowing in SNAP-649-CP (100% labeled). Next, 50 nM CBR fragment of CARMIL and different concentrations of mTwf1 were flowed in, and CP dissociation was monitored over time. Scale bar, 5 μm. (C) Quantification of the percentage of filaments retaining CP at the barbed ends in the presence of 50 nM CBR fragment of CARMIL and variable concentrations of mTwf1, determined from TIRF reactions as in (B). Control curve, buffer alone (no CBR or mTwf1). n > 45 events measured from at least two independent experiments. (D) Representative time-lapse images from TIRF microscopy assays monitoring CP displacement from barbed ends, analyzed as in (B), except using 1 μM mTwf1-11 instead of mTwf1. n > 45 events measured from at least two independent experiments. (E) Quantification of the percentage of filaments retaining CP at the barbed end in the presence of 50 nM CBR fragment of CARMIL and different concentrations of mTwf1-11, determined from TIRF assays as in (D). n > 45 events measured from at least two independent experiments.

Figure 3.

Figure 3—figure supplement 1. Supporting data for Figure 3 showing that multiple Twinfilin isoforms antagonize CARMIL uncapping of barbed ends.

Figure 3—figure supplement 1.

(A) Representative time-lapse images from TIRF microscopy assays showing that mTwf2a protects Capping Protein (CP) from CARMIL-mediated displacement from barbed ends. Filaments were first polymerized and tethered using 1 µM actin (10% OG-labeled, 0.5% biotin–labeled), then SNAP-649-CP was flowed in to cap barbed ends. Subsequently, 50 nM CBR fragment of CARMIL and 250 nM mTwf2a was flowed in, and uncapping was monitored over time. (B) Quantification of the percentage of filaments retaining SNAP-649-CP at their barbed ends after flow in of 50 nM CBR fragment of CARMIL and variable concentrations of mTwf2a, measured from TIRF reactions as in (A). Control curve, buffer alone (no CBR or mTwf2a). n > 45 events measured from one experiment. (C) Concentration-dependent effects of Twinfilin isoforms (and mutant mTwf1-11) on time to 50% disappearance of CP from barbed ends, measured from data shown in Figure 3C (mTwf1), Figure 3E (mTwf1-11), and Supplemental Figure 3—figure supplement 1 (mTwf2a).