Figure 2. INF2 binds throughout the filament but severs towards pointed end.

(A)Lifetimes of filament-bound puncta in (A). >15s represents puncta that did not dissociate during the observation period (180 sec). n=74 and 39 puncta (13 and 6 filaments) in the absence and presence of phosphate respectively.

(B)Histogram of severing positions for GFP-INF2-FFC (5–20nM) on DDS/F127 treated slides. 18 filaments (91 severing events) quantified (average length 12.3µm, range 5.83–24.22µm at time = 0). Barbed end fragments were measured as a percentage of total filament length.

(C) Correlation of time of severing with respect to distance from original barbed end (as % of original length). n=80 and 38 severing events (11 and 7 filaments) for GFP-INF2-FFC and A488-cofilin, respectively. Filaments were12.5 and 7.86µm mean length (range 6.49–21.59 and 4.55–11.05µm) prior to INF2-FFC or cofilin addition respectively. Severing events were observed for 57.5–112.3 and 68.6–170.8 s after addition of INF2 or cofilin respectively. Note x-axis scale is different between INF2 and cofilin.

(D)Tree map diagraming severing events on individual filaments with time (sec) on the y-axis. t=0 denotes time of cofilin or INF2-FFC addition. B and P denote barbed and pointed ends. Severed filament fragments are named based on severing history with subscripts denoting lineage (B₁ being the first barbed end fragment, B₁B₂ being the second barbed end fragment originating from the first barbed end fragment, and so forth). Original filament lengths: 11.1 (cofilin) and 13.6 µm (INF2). Time and sizes drawn to scale. Additional examples in Figure S2.