Adhesion-clutch between DCC and netrin-1 mediates netrin-1-induced axonal haptotaxis.

The growth cone, a motile structure located at the tip of growing axons, senses extracellular guidance cues and translates them into directional forces that drive axon outgrowth and guidance. Axon guidance directed by chemical cues on the extracellular adhesive substrate is termed haptotaxis. Recent studies reported that netrin-1 on the substrate functions as a haptotactic axon guidance cue. However, the mechanism mediating netrin-1-induced axonal haptotaxis remains unclear. Here, we demonstrate that substrate-bound netrin-1 induces axonal haptotaxis by facilitating physical interactions between the netrin-1 receptor, DCC, and the adhesive substrates. DCC serves as an adhesion receptor for netrin-1. The clutch-linker molecule shootin1a interacted with DCC, linking it to actin filament retrograde flow at the growth cone. Speckle imaging analyses showed that DCC underwent either grip (stop) or retrograde slip on the adhesive substrate. The grip state was more prevalent on netrin-1-coated substrate compared to the control substrate polylysine, thereby transmitting larger traction force on the netrin-1-coated substrate. Furthermore, disruption of the linkage between actin filament retrograde flow and DCC by shootin1 knockout impaired netrin-1-induced axonal haptotaxis. These results suggest that the directional force for netrin-1-induced haptotaxis is exerted on the substrates through the adhesion-clutch between DCC and netrin-1 which occurs asymmetrically within the growth cone.

Mechanical regulation of synapse formation and plasticity.

Dendritic spines are small protrusions arising from dendrites and constitute the major compartment of excitatory post-synapses. They change in number, shape, and size throughout life; these changes are thought to be associated with formation and reorganization of neuronal networks underlying learning and memory. As spines in the brain are surrounded by the microenvironment including neighboring cells and the extracellular matrix, their protrusion requires generation of force to push against these structures. In turn, neighboring cells receive force from protruding spines. Recent studies have identified BAR-domain proteins as being involved in membrane deformation to initiate spine formation. In addition, forces for dendritic filopodium extension and activity-induced spine expansion are generated through cooperation between actin polymerization and clutch coupling. On the other hand, force from expanding spines affects neurotransmitter release from presynaptic terminals. Here, we review recent advances in our understanding of the physical aspects of synapse formation and plasticity, mainly focusing on spine dynamics.

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

Simultaneous analyses of clutch coupling and actin polymerization in dendritic spines of rodent hippocampal neurons during chemical LTP.

Dendritic spine enlargement by synaptic activation is thought to increase synaptic efficacy underlying learning and memory. This process requires forces generated by actin polymerization and actin-adhesion coupling (clutch coupling). Here, we describe a protocol to monitor actin filament retrograde flow and actin polymerization within spines using a standard epi-fluorescence microscope. In combination with chemical long-term potentiation, this protocol allows us to quantify clutch coupling efficiency and actin polymerization rate, which are essential variables for generating forces for activity-dependent spine enlargement. For complete details on the use and execution of this protocol, please refer to Kastian et al. (2021).

Shootin1a-mediated actin-adhesion coupling generates force to trigger structural plasticity of dendritic spines.

Dendritic spines constitute the major compartments of excitatory post-synapses. They undergo activity-dependent enlargement, which is thought to increase the synaptic efficacy underlying learning and memory. The activity-dependent spine enlargement requires activation of signaling pathways leading to promotion of actin polymerization within the spines. However, the molecular machinery that suffices for that structural plasticity remains unclear. Here, we demonstrate that shootin1a links polymerizing actin filaments in spines with the cell-adhesion molecules N-cadherin and L1-CAM, thereby mechanically coupling the filaments to the extracellular environment. Synaptic activation enhances shootin1a-mediated actin-adhesion coupling in spines. Promotion of actin polymerization is insufficient for the plasticity; the enhanced actin-adhesion coupling is required for polymerizing actin filaments to push against the membrane for spine enlargement. By integrating cell signaling, cell adhesion, and force generation into the current model of actin-based machinery, we propose molecular machinery that is sufficient to trigger the activity-dependent spine structural plasticity.

Forces to Drive Neuronal Migration Steps.

To establish and maintain proper brain architecture and elaborate neural networks, neurons undergo massive migration. As a unique feature of their migration, neurons move in a saltatory manner by repeating two distinct steps: extension of the leading process and translocation of the cell body. Neurons must therefore generate forces to extend the leading process as well as to translocate the cell body. In addition, neurons need to switch these forces alternately in order to orchestrate their saltatory movement. Recent studies with mechanobiological analyses, including traction force microscopy, cell detachment analyses, live-cell imaging, and loss-of-function analyses, have begun to reveal the forces required for these steps and the molecular mechanics underlying them. Spatiotemporally organized forces produced between cells and their extracellular environment, as well as forces produced within cells, play pivotal roles to drive these neuronal migration steps. Traction force produced by the leading process growth cone extends the leading processes. On the other hand, mechanical tension of the leading process, together with reduction in the adhesion force at the rear and the forces to drive nucleokinesis, translocates the cell body. Traction forces are generated by mechanical coupling between actin filament retrograde flow and the extracellular environment through clutch and adhesion molecules. Forces generated by actomyosin and dynein contribute to the nucleokinesis. In addition to the forces generated in cell-intrinsic manners, external forces provided by neighboring migratory cells coordinate cell movement during collective migration. Here, we review our current understanding of the forces that drive neuronal migration steps and describe the molecular machineries that generate these forces for neuronal migration.

Shootin1b Mediates a Mechanical Clutch to Produce Force for Neuronal Migration.

As an essential step for brain morphogenesis, neurons migrate via mechanical interactions with components of their environment such as neighboring cells and the extracellular matrix. However, the molecular mechanism by which neurons exert forces on their environment during migration remains poorly understood. Here, we show that shootin1b is expressed in migrating mouse olfactory interneurons and accumulates at their leading process growth cone. We demonstrate that shootin1b, by binding to cortactin and L1-CAM, couples F-actin retrograde flow and the adhesive substrate as a clutch molecule. Shootin1b-mediated clutch coupling at the growth cone generates traction force on the substrate, thereby promoting leading process extension and subsequent somal translocation of olfactory interneurons. Furthermore, loss of shootin1 causes abnormal positioning of the interneurons and dysgenesis of the olfactory bulb. Our findings indicate that shootin1b plays a key role in force-driven leading process extension, which propels the migration of olfactory interneurons during olfactory bulb formation.

Identification of a shootin1 isoform expressed in peripheral tissues.

Shootin1 is a brain-specific cytoplasmic protein involved in neuronal polarity formation and axon outgrowth. It accumulates at the leading edge of axonal growth cones, where it mediates the mechanical coupling between F-actin retrograde flow and cell adhesions as a clutch molecule, thereby producing force for axon outgrowth. In this study, we report a novel splicing isoform of shootin1 which is expressed not only in the brain but also in peripheral tissues. We have renamed the brain-specific shootin1 as shootin1a and termed the novel isoform as shootin1b. Immunoblot and immunohistochemical analyses with a shootin1b-specific antibody revealed that shootin1b is distributed in various mouse tissues including the lung, liver, stomach, intestines, spleen, pancreas, kidney and skin. Interestingly, shootin1b immunoreactivity was widely detected in epithelial cells that constitute simple and stratified epithelia; in some cells, it colocalized with E-cadherin and cortactin at cell-cell contact sites. Shootin1b also localized in dendritic cells in the spleen. These results suggest that shootin1b may function in various peripheral tissues including epithelial cells.

Shootin1-cortactin interaction mediates signal-force transduction for axon outgrowth.

Motile cells transduce environmental chemical signals into mechanical forces to achieve properly controlled migration. This signal-force transduction is thought to require regulated mechanical coupling between actin filaments (F-actins), which undergo retrograde flow at the cellular leading edge, and cell adhesions via linker "clutch" molecules. However, the molecular machinery mediating this regulatory coupling remains unclear. Here we show that the F-actin binding molecule cortactin directly interacts with a clutch molecule, shootin1, in axonal growth cones, thereby mediating the linkage between F-actin retrograde flow and cell adhesions through L1-CAM. Shootin1-cortactin interaction was enhanced by shootin1 phosphorylation by Pak1, which is activated by the axonal chemoattractant netrin-1. We provide evidence that shootin1-cortactin interaction participates in netrin-1-induced F-actin adhesion coupling and in the promotion of traction forces for axon outgrowth. Under cell signaling, this regulatory F-actin adhesion coupling in growth cones cooperates with actin polymerization for efficient cellular motility.