Fig. 3

Dynamics of pulling-induced filopodia growth. a Experimental setup used to observe force-induced filopodia growth. Optical tweezers were used to trap fibronectin-coated microbeads attached to filopodia tips of HeLa-JW cells. b Confocal images of a typical cell expressing GFP-myosin X and F-Tractin-tdTomato with an attached bead, taken immediately after starting of stage movement (top) and in the course of sustained growth (bottom). Note that both myosin X and actin remain at the filopodium tip during growth. See also Supplementary Movies 9 and 10. Scale bar, 5 μm. c Top panel: A kymograph showing the dynamics of myosin X and actin in the filopodium shown in (b). This kymograph is composed of two parts following the change of the filopodium direction at 256 s from the beginning of observation. Middle panel: The length of new pulling-induced segment of filopodia is plotted versus time. Bead position in the coordinate system associated with moving microscope stage is indicated. The origin of the coordinate system corresponds to the bead position in the center of the laser trap at the initial time point. The net filopodium length change (ΔL) was calculated as ΔL = S − ΔX, where S is the microscope piezo stage displacement from its initial position, while ΔX is the deviation of the bead from the center of the optical trap. Bottom panel: Forces experienced by the bead. Note the discrete peak force values corresponding to the moments of filopodia growth cessation (seen in the middle panel) as marked with dotted lines. Inset: The distribution of peak force values, based on the pooled measurements of 21 peaks from 6 beads. Graphs were obtained by Origin software package