Figure 6.
Figure 6. Cortactin is involved in netrin-1–induced F-actin–substrate coupling and promotion of traction force for axon outgrowth. (A) Fluorescent feature images of mRFP-actin at axonal growth cones expressing control miRNA or cortactin miRNA in the presence or absence of 300 ng/ml netrin-1. Kymographs of the fluorescent features of mRFP-actin in filopodia at 5-s intervals are shown (F-actin flows are indicated by broken yellow lines). (B) F-actin retrograde flow rate measured from the kymograph analysis in A. 200 fluorescent features were analyzed. (C) Fluorescence images (left) showing an axonal growth cone of a 2 d in vitro neuron expressing EGFP and cultured on L1-CAM–coated polyacrylamide gel embedded with 200-nm fluorescent beads. The pictures show representative images from time-lapse series taken every 3 s for 150 s before (control) and 60 min after netrin-1 (300 ng/ml) stimulation (see Video 7). The original and displaced positions of the beads in the gel are indicated by green and red colors, respectively. The bead displacements are also indicated by red bars. Broken lines indicate the boundary of the growth cone. The kymographs (middle) along the axis of bead displacement (white broken arrows) at the indicated areas 1 and 2 of the growth cone show movement of beads recorded every 3 s. The bead in area 2 is a reference bead. The right panel shows the stress maps during the initial 30-s observations. (D) Statistical analyses (right) of the magnitude and angle (θ) of the traction forces under axonal growth cones expressing control miRNA or miRNA against cortactin before or after netrin-1 stimulation. RNAi-refractory cortactin was also coexpressed in the indicated experiments. n = 32 growth cones. (E) 3 h after plating, neurons expressing control miRNA or cortactin miRNA #1 were incubated with BSA (control) or 300 ng/ml netrin-1 for 40 h. RNAi-refractory cortactin was also coexpressed in the indicated experiments. Axon length was then analyzed. Data represent means ± SEM; ***, P < 0.01; **, P < 0.02; *, P < 0.05; ns, not significant. Bars: (A) 2 µm; (C) 5 µm. n = 543 neurons.

Cortactin is involved in netrin-1–induced F-actin–substrate coupling and promotion of traction force for axon outgrowth. (A) Fluorescent feature images of mRFP-actin at axonal growth cones expressing control miRNA or cortactin miRNA in the presence or absence of 300 ng/ml netrin-1. Kymographs of the fluorescent features of mRFP-actin in filopodia at 5-s intervals are shown (F-actin flows are indicated by broken yellow lines). (B) F-actin retrograde flow rate measured from the kymograph analysis in A. 200 fluorescent features were analyzed. (C) Fluorescence images (left) showing an axonal growth cone of a 2 d in vitro neuron expressing EGFP and cultured on L1-CAM–coated polyacrylamide gel embedded with 200-nm fluorescent beads. The pictures show representative images from time-lapse series taken every 3 s for 150 s before (control) and 60 min after netrin-1 (300 ng/ml) stimulation (see Video 7). The original and displaced positions of the beads in the gel are indicated by green and red colors, respectively. The bead displacements are also indicated by red bars. Broken lines indicate the boundary of the growth cone. The kymographs (middle) along the axis of bead displacement (white broken arrows) at the indicated areas 1 and 2 of the growth cone show movement of beads recorded every 3 s. The bead in area 2 is a reference bead. The right panel shows the stress maps during the initial 30-s observations. (D) Statistical analyses (right) of the magnitude and angle (θ) of the traction forces under axonal growth cones expressing control miRNA or miRNA against cortactin before or after netrin-1 stimulation. RNAi-refractory cortactin was also coexpressed in the indicated experiments. n = 32 growth cones. (E) 3 h after plating, neurons expressing control miRNA or cortactin miRNA #1 were incubated with BSA (control) or 300 ng/ml netrin-1 for 40 h. RNAi-refractory cortactin was also coexpressed in the indicated experiments. Axon length was then analyzed. Data represent means ± SEM; ***, P < 0.01; **, P < 0.02; *, P < 0.05; ns, not significant. Bars: (A) 2 µm; (C) 5 µm. n = 543 neurons.

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