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.

Biomechanical microenvironment in peripheral nerve regeneration: from pathophysiological understanding to tissue engineering development

“…Mechanical properties of the extracellular substrate modulate signal transduction for biological activities of the axons, such as electrical conduction and protein transport 31 , 32 . Stiffness-dependent axon outgrowth requires actin-adhesion coupling mediated by laminin and ligand molecules on the substrate 11 , 29 , 30 , which may be triggered and modulated by the mechanical effectors (YAP/TAZ) of Hippo pathway induced laminin receptor expression as described by a recent study 24 . Both endogenous and exogenous mechanical factors are necessary for the well-organized radial sorting of peripheral nerve axons.…”

“…actin and microtubules) play an important role in cell motility, adhesion and axonal transport, contributing to growth cone activities and axon extension 33 - 37 . Growth cones at the tip of extending axons generate traction force for axon outgrowth by transmitting the force of actin filament retrograde flow originating from actomyosin contraction and F-actin polymerization, to adjacent cells or adhesive substrates through membrane clutch and CAMs 29 , 31 , 38 . A cluster of mechanosensitive molecules between the actin filament flow and substrate govern dynamic signal transduction for neural activities 34 , 35 .…”

“…For instance, shootin1 interacting with cortactin or L1 generates traction force for axon outgrowth and guidance, while impairing these interactions, either by shootin1 RNA interference or disturbing the interaction between shootin1 and actin filament flow, will inhibit shootin1-dependent axon outgrowth 30 , 38 . Therefore, the actin retrograde flow-clutch protein-adhesive substrate axis is mainly responsible for endogenous mechanotransduction of neurons in a stiffness-dependent manner because substrate stiffness could directly modulate the interactional efficiency between clutch proteins and adhesion molecules 29 .…”

Substrate stiffness in nerve cells

“…For instance, rat hippocampal neurons have a higher number of neurite branches [5–7] with increasing substrate stiffness in the range of 300 Pa to 7 kPa. For a substrate stiffness range of 7–20 kPa, the number of branches in mice hippocampal neurons increased with increasing substrate stiffness [8], in the substrate stiffness range of 100 Pa to 4 kPa, the neurite length of mice hippocampal neurons extended with increasing substrate stiffness [9]. However, Kostic et al demonstrated that mice hippocampal neurons prefer a soft (500 Pa) to a more rigid substrate (7.5 kPa) [10], which was further confirmed by Chen et al who showed that the development of hippocampal neurons was biased towards a soft substrate (88 kPa) [11].…”

Rlip76: An Unexplored Player in Neurodegeneration and Alzheimer’s Disease?

“…In the case of CDE, the actin cytoskeleton is important for providing the force required to constrict and separate vesicles from the plasma membrane [ 27 ]. In the case of mitochondrial fission, dynamin-related protein 1 (Drp1) oligomers surround and mechanically constrict the mitochondria [ 28 ], while plasma membrane outgrowths such invadopodia and neurites require forces transmitted through actin bundles [ 29 , 30 ]. For the sake of comprehensiveness, it is also worth noting that Rlip is frequently bound to tubulin [ 31 ], and a RALBP1 splice variant called cytocentrin functions in spindle separation during mitosis, a process in which microtubules play a key role [ 32 ].…”