Observation of adhesion breaking of an axonal growth cone by femtosecond laser-induced impulsive force.

(A) Schematics of the spatial relation between the femtosecond laser pulse and targeted axonal growth cone of a neuron on a glass substrate. The laser focal point was sequentially moved closer to the growth cone, as indicated by an arrow in the upper cartoon. The effect of the impulsive force to the growth cone was indicated in the lower cartoon, which is enlarged view of the tip of the axon in the upper cartoon. (B) A representative result of adhesion breaking of a growth cone observed by a differential interference contrast (DIC) imaging. The left panel is DIC image before the laser irradiation at the final point. The middle panel indicates the laser irradiation process, in which the laser focal point (cross marks) was sequentially moved along an arrow in the image. The right panel is a merged images of the growth cones before and after the final laser irradiation, which are colorized in green and magenta, respectively. Cross marks are laser focal points. Scale bar, 10 µm.

Quantitative evaluation of breaking force for growth cone adhesion by using femtosecond laser impulsive force.

(A) Pulse energy L dependence of threshold distance R to break the growth cone adhesion on a glass substrate coated with a 10 µg/ml laminin solution, corresponding to an A of 0.45 (see Eq. [4]). n = 36. (B) Box-and-whisker plot of adhesion breaking threshold. Dots in the graph is the threshold calculated independently by substituting data from panel A into Eq. [2]. The mean value and standard deviation are 4.35 and 1.08 kPa, respectively. (C) Images of fluorescent dye-conjugated laminin on the substrate. Concentrations of the laminin solution used for the coating are indicated at the top. Scale bar, 10 µm. (D) Fluorescence intensity as a function of the laminin concentration. The fluorescence intensities were measured on substrates coated with laminin solutions with concentrations of 0.01, 0.1, 1, 10, 50, and 100 µg/ml. The fitting curve was calculated by Eq. [3], where Imax = 25.5 and k = 16.6. n = 50 for each concentration. Data are means ± SDs from three independent experiments.

Adhesion breaking of growth cone on a laminin-coated substrate.

(A) Means and SDs of the adhesion breaking threshold. Dots in the graph is the threshold calculated independently by substituting data from panel A into Eq. [2]. The laminin concentration is indicated at the top. Red and blue dots indicate control neurons and L1CAM knockdown neurons, respectively. (B) Box-and-whisker plots of adhesion breaking threshold. Control neurons: n = 41, 35, 36, 12, and 25 signals for A = 0.01, 0.06, 0.45, 0.78, and 0.99, respectively. L1CAM knockdown neurons: n = 18, 44, 34, 20, and 19 signals for A = 0.01, 0.06, 0.45, 0.78, and 0.99, respectively. **p < 0.01 (two-tailed Student’s t test), n.s., not significant.

Molecular dynamics of F-actin and L1CAM in the axonal growth cone detected by fluorescence speckle microscopy.

(A) Fluorescence speckle images of the HaloTag-actin in a filopodium extended from an axonal growth cone. The coverage of laminin (A) is indicated at the top. Kymographs (right) depict HaloTag-actin behavior in boxed region in the image on the left. Slope of the yellow dashed line corresponds to retrograde flow speed of the F-actin. Time interval between frames, 5 s. Scale bar, 5 μm. (B) Retrograde flow speed of F-actin. n = 125, 160, and 130 signals for A = 0.06, 0.45, and 0.99, respectively. (C) Fluorescence speckle images of L1CAM-HaloTag in a filopodium. Kymographs (right) depict L1CAM-HaloTag behavior in a boxed region in the image on the left. Dashed pink and blue lines connect L1CAM in grip and slip states, respectively. Time interval between frames, 5 s. Scale bar, 5 μm. (D) Ratios of the grip and slip states of L1CAM-HaloTag in filopodia obtained from the kymograph analyses. n = 261, 450, and 197 signals for A = 0.06, 0.45, and 0.99, respectively. (E) Flow speed of L1CAM-HaloTag in the slip state. The speed corresponds to slopes of dashed blue lines in panel C. (F) Duration time of L1-HaloTag grip. White, red, and blue bars represent data for A = 0.06, 0.45, and 0.99, respectively. Data are means ± SDs; **p < 0.01.

Axon elongation on laminin-coated substrate.

(A) Confocal images of neurons visualized with a GFP antibody. The coverage of laminin (A) is indicated on the left. Scale bar; 100 µm. (B) Box-and-whisker plots of axon length. Control neurons (red): n = 91, 28, 79, 60, and 71 signals for A = 0.01, 0.06, 0.45, 0.78, and 0.99, respectively; L1CAM knockdown neurons (blue): n = 43, 49, 73, 103, and 48 signals for A = 0.01, 0.06, 0.45, 0.78, and 0.99, respectively. *p < 0.05, **p < 0.01. n.s., not significant.

Measurement of traction force on a laminin-coated substrate.

(A) DIC (left panels) and fluorescence images (right panels) of an axonal growth cone cultured on polyacrylamide gels with embedded 200 nm fluorescent beads. Dashed lines indicate the boundary of the growth cone. The blue color in the dashed line is due to EGFP which is expressed in the neuron to track the growth cone position. The original and displaced bead positions in the gel are indicated by green and red colors in fluorescence images, respectively. The yellow color overlaps the green and red colors, i.e. the bead is rarely displaced. Laminin coverage A is shown at the top of the panel column. Scale bar; 10 µm. (B) Magnitude of the traction forces under axonal growth cones. Data represent mean ± SEM (error bars).