Fig. 4

Inhibition of myosin II or formin reduces filopodia adhesion. a–d Growth of filopodia attached to laser-trapped beads upon stage movement in HeLa-JW cells treated as indicated (see Supplementary Movies 11, 13, 14, and 16). Cells were transfected with GFP-myosin X (green) and F-Tractin-tdTomato (red). Position of the center of laser trap is indicated by blue dashed line. a At 944 s filopodia started to retract and at 960 s (dark red arrow) pulled the bead out of the trap. b–f Filopodia in cells treated with: 20 μM of S-nitro-blebbistatin (b); myosin IIA siRNA (c); 40 μM of SMIFH2 (d). The detachment of the filopodium from the bead, detected by returning of the bead to the center of the trap, occurred at 404 s (b), 230 s (c) after starting the stage movement (blue arrows). Upon switching off the trap (at 760, 256 s, respectively) the detached beads disappeared from the field of view (b, c). In (d) the return of bead to the center of trap at 200 s (white arrow) was accompanied by formation of membrane tether and the bead remained within the field of view after switching off the trap at 284 s (Supplementary Movie 16). e Statistics of the outcomes of the pulling experiments. Color pie charts represent the fractions of outcomes for each treatment. Red—bead withdrawal from the trap, blue—filopodia detachment from the bead, white—formation of membrane tether. The numbers of assessed filopodia indicated in each slice. Each dot on the graphs underneath the pie charts represents time interval between start of pulling and corresponding outcome for individual filopodium in cell treated as indicated; color-coded bars represent the mean values. The filopodia were attached to fibronectin-coated or ConA-coated beads as indicated. Concentration of Y-27632 was 30 μM. f Peak values of the forces exerted by filopodia on the fibronectin-coated beads during the stage movement. Mean values (horizontal lines) and s.e.m. (error bars) are shown. Numbers of measured filopodia (n) are indicated. p Values calculated according to unpaired two-tailed t test with Welch’s correction were all less than 0.0001