Mechanosensitive axon outgrowth mediated by L1-laminin clutch interface

Abstract

Mechanical properties of the extracellular environment modulate axon outgrowth. Growth cones at the tip of extending axons generate traction force for axon outgrowth by transmitting the force of actin filament retrograde flow, produced by actomyosin contraction and F-actin polymerization, to adhesive substrates through clutch and cell adhesion molecules. A molecular clutch between the actin filament flow and substrate is proposed to contribute to cellular mechanosensing. However, the molecular identity of the clutch interface responsible for mechanosensitive growth cone advance is unknown. We previously reported that mechanical coupling between actin filament retrograde flow and adhesive substrates through the clutch molecule shootin1a and the cell adhesion molecule L1 generates traction force for axon outgrowth and guidance. Here, we show that cultured mouse hippocampal neurons extend longer axons on stiffer substrates under elastic conditions that correspond to the soft brain environments. We demonstrate that this stiffness-dependent axon outgrowth requires actin-adhesion coupling mediated by shootin1a, L1, and laminin on the substrate. Speckle imaging analyses showed that L1 at the growth cone membrane switches between two adhesive states: L1 that is immobilized and that undergoes retrograde movement on the substrate. The duration of the immobilized phase was longer on stiffer substrates; this was accompanied by increases in actin-adhesion coupling and in the traction force exerted on the substrate. These data suggest that the interaction between L1 and laminin is enhanced on stiffer substrates, thereby promoting force generation for axon outgrowth.

Figure 1

Hippocampal neurons extend longer axons on stiffer adhesive substrates. (A) Hippocampal neurons cultured on laminin-coated 3.5, 8, and 16% polyacrylamide gels and glasses for 48 h and stained with anti-βIII-tubulin antibody. Arrowheads indicate axonal tips. (B) Quantification of axon length of neurons on laminin-coated gels and glasses in (A) (3.5%, n = 202 neurons; 8%, n = 205 neurons; 16%, n = 199 neurons; glass, n = 203 neurons) and quantification of axon length of neurons on PDL-coated gels and glasses in (C) (3.5%, n = 152 neurons; 8%, n = 157 neurons; 16%, n = 151 neurons; glass, n = 153 neurons). (C) Hippocampal neurons cultured on PDL-coated 3.5, 8, and 16% polyacrylamide gels and glasses for 48 h and stained with anti-βIII-tubulin antibody. Arrowheads indicate axonal tips. Scale bars, 50 μm. Data in (B) represent means ± SE; ^∗∗∗p < 0.01; n.s., nonsignificant (red asterisks represent statistical analyses of neurons on laminin, and blue n.s. represents those on PDL). Axon lengths among laminin-coated substrates or PDL-coated substrates were analyzed by ANOVA with Tukey’s post hoc test. Axon lengths between laminin-coated and PDL-coated substrates were examined by Student’s t-test. To see this figure in color, go online.

Figure 2

Mechanosensitive axon outgrowth requires shootin1a-mediated actin-adhesion coupling. (A) Diagram showing the growth cone clutch machinery for axon outgrowth, involving F-actin, myosin II, cortactin, shootin1a, L1, and laminin (31). (B) Images of WT and shootin1 KO hippocampal neurons cultured on 16% polyacrylamide gels for 48 h and stained with anti-βIII-tubulin antibody. Arrowheads indicate axonal tips. (C) Statistical analysis of axon length of WT and shootin1 KO hippocampal neurons cultured on laminin-coated 3.5, 8, and 16% polyacrylamide gels for 48 h. (WT 3.5%, n = 238 neurons; 8%, n = 205 neurons; 16%, n = 208 neurons; KO 3.5%, n = 206 neurons; 8%, n = 203 neurons; 16%, n = 208 neurons.) (D) Hippocampal neurons overexpressing myc-GST (control) or myc-shootin1a-DN were cultured on 16% polyacrylamide gels for 48 h and stained with anti-myc antibody. Arrowheads indicate axonal tips. (E) Statistical analysis of axon length of neurons overexpressing myc-GST (control) and neurons overexpressing myc-shootin1a-DN cultured on laminin-coated 3.5, 8, and 16% polyacrylamide gels. (myc-GST 3.5%, n = 153 neurons; 8%, n = 147 neurons; 16%, n = 149 neurons; myc-shootin1a-DN 3.5%, n = 132 neurons; 8%, n = 150 neurons; 16%, n = 149 neurons.) Scale bars, 50 μm. Data in (C) and (E) represent means ± SE; ^∗p < 0.05; ^∗∗∗p < 0.01; n.s., nonsignificant (blue n.s. in C represents statistical analyses of shootin1 KO neurons, whereas that in E represents statistical analyses of neurons overexpressing myc-shootin1a-DN). Axon lengths between WT and KO neurons or between neurons overexpressing myc-GST and myc-shootin1a-DN were examined by Student’s t-test. Axon lengths among different substrates were analyzed by ANOVA with Tukey’s post hoc test. To see this figure in color, go online.

Figure 3

Increase in substrate stiffness promotes actin-adhesion coupling and traction force on the substrate. (A) A fluorescent feature image (left) of HaloTag-actin in axonal growth cones of neurons cultured on laminin-coated 8% polyacrylamide gel (left) (see Video S1) and time-lapse montages (right) of fluorescent features of HaloTag-actin in filopodia of axonal growth cones on laminin-coated 3.5, 8, and 16% gels at 5-s intervals (right, F-actin flows are indicated by dashed yellow lines). (B) F-actin retrograde flow speeds obtained from the time-lapse montage analyses in (A) (3.5%, n = 86 signals; 8%, n = 78 signals; 16%, n = 80 signals). (C and D) Brightfield (left panels) and fluorescence (middle panels) images showing axonal growth cones of hippocampal neurons cultured for 1 day on laminin-coated 3.5% (C) or 8% (D) polyacrylamide gel with embedded 200-nm fluorescent beads (see Video S2). The pictures show representative images from time-lapse series taken every 3 s for 147 s. The original and displaced positions of the beads in the gel are indicated by green and red colors, respectively. Dashed lines indicate the boundary of the growth cones. The time-lapse montages (right panels) along the axis of bead displacement (dashed arrows) in the indicated areas 1 and 2 of the growth cone show movement of beads recorded every 3 s. The beads in area 2 are reference beads. (E) Magnitude of averaged stress of axonal growth cones on laminin-coated 3.5 and 8% polyacrylamide gels (3.5%, n = 15 growth cones; 8%, n = 14 growth cones). Scale bars, 5 μm (in the time-lapse montage of A, 2 μm). Data in (B) and (E) represent means ± SE; ^∗∗∗p < 0.01. F-actin retrograde flow speeds were analyzed by ANOVA with Tukey’s post hoc test. Magnitudes of averaged stress were examined by Student’s t-test. To see this figure in color, go online.

Figure 4

Increase in substrate stiffness prolongs the duration of the L1 grip phase. (A) A fluorescent feature image (left) of L1-HaloTag in axonal growth cone cultured on laminin-coated 8% polyacrylamide gel (see Video S3). Time-lapse montages (right) of retrograde flow of L1-HaloTag fluorescent features on laminin-coated 3.5, 8, and 16% gels at 5-s intervals (grip and slip phases are shown by dashed pink and blue lines, respectively), are given. (B) Ratio of grip/slip phases of L1 in growth cones on laminin-coated polyacrylamide gels (3.5%, n = 8 growth cones; 8%, n = 8 growth cones; 16%, n = 8 growth cones). (C) Mean retrograde flow speed of L1-HaloTag in axonal growth cones on laminin-coated polyacrylamide gels (3.5%, n = 188 signals; 8%, n = 232 signals; 16%, n = 205 signals). (D) Histogram showing the distribution of duration time of L1-HaloTag grip phase. (E) Duration time of L1-HaloTag grip phase on laminin-coated polyacrylamide gels (3.5%, n = 232 grip phases; 8%, n = 226 grip phases; 16%, n = 244 grip phases). Scale bars, 5 μm (in the time-lapse montage of A, 2 μm). Data in (B), (C), and (E) represent means ± SE; ^∗p < 0.05; ^∗∗∗p < 0.01; n.s., nonsignificant. Data were analyzed by ANOVA with Tukey’s post hoc test. To see this figure in color, go online.

Figure 5

Stiffness-dependent increase in the duration of the L1 grip phase requires laminin on the substrate. (A) A fluorescent feature image (left) of L1-HaloTag in an axonal growth cone cultured on PDL-coated 8% polyacrylamide gel. Time-lapse montages (right) of retrograde flow of L1-HaloTag fluorescent features on PDL-coated 3.5, 8, and 16% gels at 5-s intervals (grip and slip phases are shown by dashed pink and blue lines, respectively) are given. (B) Ratio of grip/slip phases of L1 in growth cones on PDL-coated polyacrylamide gels (3.5%, n = 6 growth cones; 8%, n = 6 growth cones; 16%, n = 6 growth cones). (C) Mean retrograde flow speed of L1-HaloTag in axonal growth cones on PDL-coated polyacrylamide gels (3.5%, n = 84 signals; 8%, n = 120 signals; 16%, n = 100 signals). (D) Histogram showing the distribution of duration time of L1-HaloTag grip phase. (E) Duration time of L1-HaloTag grip phase on PDL-coated polyacrylamide gels (3.5%, n = 31 grip phases; 8%, n = 48 grip phases; 16%, n = 33 grip phases). Scale bars, 5 μm (in the time-lapse montage of A, 2 μm). Data in (B), (C), and (E) represent means ± SE; ^∗p < 0.05; ^∗∗∗p < 0.01; n.s., nonsignificant. Data were analyzed by ANOVA with Tukey’s post hoc test. To see this figure in color, go online.

Figure 6

A model for mechanosensitive axon outgrowth mediated by catch-bond-like L1-laminin clutch interface. (A) The force generated by actomyosin contraction and F-actin polymerization is transmitted through F-actin retrograde flow (green arrow), cortactin, and shootin1a to L1, thereby pulling the bond between L1 and laminin (blue arrows). When this force exceeds a threshold, the bond breaks and L1 slips retrogradely on the substrate (slip phase). (B) An increase in the substrate rigidity promotes the force-loading rate exerted through the molecular clutch (blue arrows). This, in turn, induces catch-bond-like behavior of the L1-laminin clutch interface, in which the average bond lifetime between L1 and laminin increases with tensile force, thereby promoting the L1 grip phase, actin-adhesion coupling, traction force (red arrow), and axon outgrowth (white arrow). To see this figure in color, go online.