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.

细胞外环境的机械特性调节轴突生长。延伸轴突尖端的生长锥通过离合器和细胞粘附分子将由肌动球蛋白收缩和 F-肌动蛋白聚合产生的肌动蛋白丝逆行流动的力传递到粘合基质，从而产生轴突向外生长的牵引力。提出肌动蛋白丝流和底物之间的分子离合器有助于细胞机械传感。然而，负责机械敏感生长锥推进的离合器界面的分子身份尚不清楚。我们之前报道了肌动蛋白丝逆行流动和粘合剂基质之间通过离合器分子shootin1a 和细胞粘附分子 L1 之间的机械耦合，为轴突生长和引导产生牵引力。这里，我们表明，在与软脑环境相对应的弹性条件下，培养的小鼠海马神经元在更硬的基质上延伸更长的轴突。我们证明这种依赖于刚度的轴突生长需要由底物上的shootin1a、L1 和层粘连蛋白介导的肌动蛋白粘附耦合。散斑成像分析表明，生长锥膜上的 L1 在两种粘附状态之间切换：L1 被固定并在基板上经历逆行运动。在较硬的基材上，固定阶段的持续时间较长；这伴随着肌动蛋白 - 粘附耦合和施加在基板上的牵引力的增加。这些数据表明，L1 和层粘连蛋白之间的相互作用在较硬的基质上得到增强，从而促进轴突生长的力产生。