Figure 1.
Collisions between a growth cone and a trapped bead.
(a–c) A growth cone displacing a bead from the optical trap. The red cross indicates the bead's equilibrium position inside the optical trap. Scale bar, 2 µm. (d) Example of a force component obtained with QPD when the bead was distant from the growth cone (upper trace) and when the bead was in contact with the growth cone (lower trace). Red lines are drawn 5 σ from the 0 mark. σ, s.d. of force fluctuations. When the QPD trace crossed the red lines for at least 100 ms and a lamellipodium or filopodium was seen hitting the bead, a reliable collision was detected. (e) Example of Fx and Fy during repetitive collisions between a moving lamellipodium and a trapped bead. Trap stiffness was 0.05 pN nm−1. (f) Comparison of Fx and Fy determined with a QPD (black traces) and video tracking (yellow traces).
Figure 2.
(a–b) Lateral collision between a filopodium and a trapped bead. Trap stiffness was 0.008 pN nm−1. The red cross indicates the bead's equilibrium position inside the optical trap. (c) Fx and Fy from the QPD during the lateral collision shown in (a–b). (d–e) Collision between a protruding filopodium and a trapped bead. (f) Fx and Fy from the QPD during the filopodial protrusion shown in (d–e). Trap stiffness was 0.008 pN nm−1. (g–h) Histograms of forces measured during lateral collisions and protrusions. Data were collected from 75 experiments, each lasting 2 min. Scale bar, 2 µm.
Figure 3.
Effect of trap stiffness on force exerted.
(a–b) Fx from the QPD during collisions between the same filopodium and the same bead trapped with a stiffness of 0.006 and 0.010 pN nm−1. Traces were filtered at 50 Hz and sub-sampled. (c–d) Scatter plot of force duration for collisions between filopodia and beads trapped with a stiffness of 0.006 and 0.010 pN nm−1. Data collected from 15 experiments at each stiffness.
Figure 4.
Force exerted by lamellipodia.
(a–b) A lamellipodium growing and pushing a trapped bead. The red cross indicates the equilibrium position inside the optical trap. Scale bar, 2 µm. (c) Fneu in the x,y plane obtained from a QPD recording. Trap stiffness was 0.009 pN nm−1. (d) The force exerted by a lamellipodium showing step-like jumps. Red lines, drawn by eye, indicate presumed discrete levels. The QPD recording was sub-sampled and filtered at 50 Hz. After low-pass filtering, the value of σ was reduced to 0.05 pN. Trap stiffness was 0.01 pN nm−1. (e) Histogram of forces measured during collisions between lamellipodia and trapped beads. Data reflect 65 experiments, each lasting 2 min. (f) Scatter plot of force duration for the collisions shown in (e).
Figure 5.
Force field exerted by lamellipodia.
(a) A lamellipodium colliding with three trapped beads. (b) Direction and amplitude (in arbitrary units, a.u.) of forces exerted on the three beads. Superposition of bead displacements was obtained by video tracking at 5 Hz from a 4-min recording. Scale bar, 2 µm.
Figure 6.
Effect of inhibitors on force exertion.
A growth cone before (a) and after (b–c) application of 100 nM latrunculin A. No motion was observed after 3.5 min of exposure. (d) Scatterplot of force duration for collisions after application of 50 nM (black symbols) and 100 nM (red symbols) latrunculin A. A growth cone is shown before (e) and after (f–g) application of 50 nM nocodazole. The growth cone retracted, but filopodia continued to move for at least 30 min after drug exposure. (h) Scatterplot of force duration for collisions after application of 50 nM nocodazole. A growth cone is shown before (i) and after (j–k) application of 4 µM ML-7. Filopodia quickly retracted but then regrew and moved for at least 20 min after drug application. (l) Scatterplot of force duration for collisions after application of 4 µM ML-7. Scale bars, 2 µm. Drugs were added at time 0.