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. 2020 Nov 23;220(1):e202006022. doi: 10.1083/jcb.202006022

Figure 1.

Figure 1.

mTwf1 slows the depolymerization of aged (ADP) actin filament barbed ends. (a) Schematics of conventional TIRF and mf-TIRF experiments to monitor barbed end depolymerization of ADP-actin filaments. In conventional TIRF (left), filaments are polymerized using 2 µM G-actin (15% Alexa-488 labeled and 0.5% biotinylated) and 3 µM profilin. In mf-TIRF assays (right), filaments with free barbed ends are polymerized by exposing coverslip-anchored spectrin-actin seeds to 2 µM G-actin (15% Alexa-488 labeled) and 5 µM profilin. In both setups, filaments are aged to produce ADP F-actin by incubation for 15 min in TIRF buffer or mf-TIRF buffer containing 0.1 µM G-actin (critical concentration at the barbed end, to prevent change in filament length). Then 5 µM mTwf1 or control buffer is flowed in, and barbed end depolymerization is monitored over time. Note that G-actin and profilin are no longer present when depolymerization is monitored. BE, barbed end; PE, pointed end. (b) Representative time-lapse images from conventional TIRF assays showing the effects of 5 µM mTwf1 on barbed end depolymerization. Blue arrowheads, barbed ends. Yellow arrowheads, pointed ends. (c) Comparison of single filament rates (mean ± SD) for barbed end depolymerization in the presence and absence of 5 µM mTwf1 and in conventional TIRF and mf-TIRF assays. *, Statistical comparison by two-sample t test between indicated conditions (P < 0.05). Number of filament ends analyzed for each condition (left to right): 58, 38, 40, and 78. Conventional TIRF experiments were performed two times and mf-TIRF experiments three times and yielded similar results. Data shown are from one experiment each. (d) Representative field of view from mf-TIRF assay, showing filaments aligned under flow in the microfluidic chamber with anchored pointed ends (left) and free barbed ends (right). (e) Representative kymograph of an Alexa-488–labeled actin filament (green) depolymerizing in control buffer from mf-TIRF assay. (f) Same as e but in the presence of 5 µM mTwf1. White dotted lines indicate the slope of the depolymerizing barbed ends.