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[Preprint]. 2023 Sep 1:2023.03.14.532631. Originally published 2023 Mar 15. [Version 2] doi: 10.1101/2023.03.14.532631

Figure 4. N-WASP-activated Arp2/3 sustains the lifetime and the centripetal movement of inner F-actin foci.

Figure 4.

LifeAct-GFP-expressing B-cells were incubated with or without various inhibitors and imaged live by TIRF during interaction with Fab’-PLB at 37°C. Three kymographs were generated for each cell using time-lapse images and positioned to track as many inner actin foci as possible. (A) A representative kymograph from TIRF time-lapse images of a DMSO-treated B-cell (Left panels). Inner F-actin foci were identified as described in Figure 3, and those that emerged during the 60 sec window after maximal spreading (white rectangles in B) and can be tracked for ≥ 4 sec (dashed lines) in individual kymographs were analyzed. Relative lifetimes of inner F-actin foci were measured using the duration each focus could be detected in a kymograph. The relative distances traveled by the foci were measured using the displacement of each focus in a kymograph. Relative speed was calculated for each inner F-actin focus by dividing its relative distance by its relative lifetime (right panels). (B-D) B-cells were treated with CK-689 or CK-666 (50 μM) from the beginning of the incubation with Fab’-PLB (0 min). Shown are representative kymographs (B), relative lifetimes (C), and relative speed (D) of inner F-actin foci in CK-689- versus CK-666-treated B-cells. (E-G) B-cells were treated with CK-666 at maximal spreading (2 min). Shown are a representative kymograph of a CK-666-treated cell (E), relative lifetimes (F), and relative speeds (G) of inner F-actin foci in 30-sec windows before the inhibition and 10 sec after the inhibition in the same cells (linked by blue lines). (H-J) B-cells were treated with DMSO or Wisko (10 μM) 10 min before and during interaction with Fab’-PLB. Shown is a representative kymograph of a Wisko-treated B-cell (H), relative lifetime (I), and relative speed (J) of inner F-actin foci in DMSO versus Wisko-treated B-cells. (K-M) LifeAct-GFP-expressing WT and WKO B-cells were incubated with Fab’-PLB. Shown are a representative kymograph of a WKO B-cell (K), relative lifetime (L), and relative speed (M) of WT versus WKO B-cells. Data points represent the averaged values from inner F-actin foci in individual cells, with 3 kymographs per cell and ~12 cells per condition per experiment from 3 independent experiments. Scale bar, 2 μm. * p <0.05, ** p <0.01, *** p<0.001, by non-parametric and paired student’s t-test.