Figure 3. Inner F-actin foci are originated from lamellipodia behind the spreading membrane.

Mouse splenic B-cells from LifeAct-GFP transgenic mice were treated with DMSO or Wisko (10 μM), imaged live using TIRF during incubation with Fab’-PLB at 37°C, and analyzed using kymographs generated by NIH ImageJ. (A) One frame of TIRF time-lapse images of LifeAct-GFP in the contact zone of B-cells treated with DMSO or Wisko and a WKO B-cell. Lines indicate eight kymographs that were randomly generated from each cell. (B) Representative kymographs were generated from TIRF time-lapse images of LifeAct-GFP at the red line in (A). Top panel, a contracting cell. Arrows indicate the start of contraction with inner F-actin foci originating from lamellipodia. Bottom panel, a non-contracting cell. Lamellipodia-derived inner F-actin foci were identified by their LifeAct-GFP FI ≥2 fold of their nearby region, migrating out of the lamellipodial F-actin toward the center of the contact zone, and trackable for >8 sec. (C) Percentages (±SEM) of kymographs showing inner F-actin foci originating from lamellipodia per cell that did and did not undergo contraction. Data were generated from 3 independent experiments with ~10 cells per condition per experiment. (D) A histogram of inner F-actin foci emerging (expressed as percentages of the total events, blue line) over time relative to the time of B-cell contraction (defined as 0 sec, indicated by a purple dash line and arrow). Data were generated from 5 independent experiments with ~9 cells per condition per experiment. (E) Percentage (±SEM) of inner F-actin foci originated from lamellipodia observed in 8 randomly positioned kymographs of each DMSO- or Wisko-treated WT or untreated WKO B-cell. Data were generated from 3 independent experiments with ~10 cells per condition per experiment. Scale bars, 2 μm. *** p<0.001, by non-parametric student’s t-test.