Adhesion-clutch drives three-dimensional axon outgrowth

Axon outgrowth requires forces generated by the growth cone. A key model explaining this force generation is the adhesion-clutch mechanism, through which the backward force of treadmilling actin filaments is transmitted to the extracellular environment via adhesion and clutch molecules. However, this mechanism has not been validated in three-dimensional (3D) environments. Additionally, a recent study reported that inhibiting actin dynamics or the cell adhesion molecule integrin did not affect axon outgrowth in a 3D collagen gel, challenging the adhesion-clutch paradigm. Here, we show that the adhesion molecule N-cadherin and the clutch molecule shootin1a form a non-integrin adhesion-clutch in a 3D environment containing an appropriate adhesive substrate, N-cadherin. We detected forces produced by growth cones when N-cadherin was present. Furthermore, inhibition of N-cadherin, shootin1a or actin dynamics inhibited 3D axon outgrowth. Our findings demonstrate that the adhesion-clutch is a critical machinery for 3D neural network formation under the regulation of specific adhesions.轴突生长依赖于生长锥（growth cone）所产生的作用力。解释该作用力产生过程的核心模型为黏附离合器（adhesion-clutch）机制：肌动蛋白丝通过踏车运动产生的向后作用力，经由黏附分子与离合器分子传递至细胞外环境。然而，该机制尚未在三维（3D）环境中得到验证。此外，近期一项研究表明，在三维胶原凝胶环境中，抑制肌动蛋白动力学或细胞黏附分子整合素（integrin）并不会影响轴突生长，这对黏附离合器机制的范式提出了挑战。本研究证实，在以N-钙黏蛋白（N-cadherin）作为适宜黏附底物的三维环境中，黏附分子N-钙黏蛋白与离合器分子shootin1a可形成非整合素型黏附离合器。我们检测到，当存在N-钙黏蛋白时，生长锥可产生作用力。此外，抑制N-钙黏蛋白、shootin1a或肌动蛋白动力学均可阻碍三维环境中的轴突生长。本研究结果证实，黏附离合器机制是在特定黏附分子调控下，三维神经网络形成的关键核心机制。