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.

Bayesian traction force estimation using cell boundary-dependent force priors.

Understanding the principles of cell migration necessitates measurements of the forces generated by cells. In traction force microscopy (TFM), fluorescent beads are placed on a substrate's surface and the substrate strain caused by the cell traction force is observed as displacement of the beads. Mathematical analysis can estimate traction force from bead displacement. However, most algorithms estimate substrate stresses independently of cell boundary, which results in poor estimation accuracy in low-density bead environments. To achieve accurate force estimation at low density, we proposed a Bayesian traction force estimation (BTFE) algorithm that incorporates cell-boundary-dependent force as a prior. We evaluated the performance of the proposed algorithm using synthetic data generated with mathematical models of cells and TFM substrates. BTFE outperformed other methods, especially in low-density bead conditions. In addition, the BTFE algorithm provided a reasonable force estimation using TFM images from the experiment.

Dephosphorylation of neural wiring protein shootin1 by PP1 phosphatase regulates netrin-1-induced axon guidance.

Axon pathfinding is an essential step in neuronal network formation. Shootin1a is a clutch-linker molecule that is mechanically involved in axon outgrowth and guidance. It was previously shown that concentration gradients of axon guidance molecule netrin-1 in the extracellular environment elicit asymmetrically localized Pak1 kinase-mediated phosphorylation of shootin1a within axonal growth cones, which is higher on the netrin-1 source side. This asymmetric phosphorylation promotes shootin1a-mediated local actin-adhesion coupling within growth cones, thereby generating directional forces for turning the growth cone toward the netrin-1 source. However, how the spatial differences in netrin-1 concentration are transduced into the asymmetrically localized signaling within growth cones remains unclear. Moreover, the protein phosphatases that dephosphorylate shootin1a remain unidentified. Here, we report that protein phosphatase-1 (PP1) dephosphorylates shootin1a in growth cones. We found that PP1 overexpression abolished the netrin-1-induced asymmetric localization of phosphorylated shootin1a as well as axon turning. In addition, we show PP1 inhibition reversed the asymmetrically localized shootin1a phosphorylation within growth cones under netrin-1 gradient, thereby changing the netrin-1-induced growth cone turning from attraction to repulsion. These data indicate that PP1-mediated shootin1a dephosphorylation plays a key role in organizing asymmetrically localized phosphorylated shootin1a within growth cones, which regulates netrin-1-induced axon guidance.

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).

Mechanosensitive axon outgrowth mediated by L1-laminin clutch interface.

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.

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.

An Artificial Amphiphilic Peptide Promotes Endocytic Uptake by Inducing Membrane Curvature.

Membrane curvature plays a pivotal role in cellular life, including cellular uptake and membrane trafficking. The modulation of membrane curvature provides a novel means of manipulating cellular events. In this report, we show that a nine-residue amphiphilic peptide (R6W3) stimulates endocytic uptake by inducing membrane curvature. Curvature formation on cell membranes was confirmed by observing the cellular distribution of the curvature-sensing protein amphiphysin fused with a yellow fluorescent protein (Amp-YFP). Dot-like signals of Amp-YFP were visible following the addition of R6W3, suggesting curvature formation in cell membranes, leading to endocytic cup and vesicle formation. The promotion of endocytic uptake was confirmed using the endocytosis marker polydextran. Treatment of cells with R6W3 yielded a 4-fold dextran uptake compared with untreated cells. The amphiphilic helical structure of R6W3 was also crucial for R6W3-stimulated endocytic uptake.

Shootins mediate collective cell migration and organogenesis of the zebrafish posterior lateral line system.

The zebrafish sensory posterior lateral line is an excellent model system to study collective cell migration and organogenesis. Shootin1 is a cytoplasmic protein involved in neuronal polarization and axon guidance. Previous studies have shown that shootin1 couples actin filament retrograde flow with extracellular adhesive substrates at the leading edge of axonal growth cones, thereby producing mechanical force for the migration and guidance of axonal growth cones. However, the functions of shootin in peripheral cells remain unknown. Here we identified two novel shootin family members, shootin2 and shootin3. In zebrafish, shootin1 and shootin3 are expressed in the posterior lateral line primordium (PLLP) and neuromasts during embryonic development. A shootin1 mutant displayed a reduced speed of PLLP migration, while shootin1;shootin3 double mutation inhibited cell proliferation in the PLLP. Furthermore, our results suggest that shootin1 and shootin3 positively regulate the number of neuromasts and the number of cells in deposited neuromasts. Our study demonstrates that shootins mediate collective cell migration of the posterior lateral line primordium and formation of neuromasts in zebrafish.

An influenza-derived membrane tension-modulating peptide regulates cell movement and morphology via actin remodeling.

Tension in cell membranes is closely related to various cellular events, including cell movement and morphogenesis. Therefore, modulation of membrane tension can be a new approach for manipulating cellular events. Here, we show that an amphipathic peptide derived from the influenza M2 protein (M2[45-62]) yields lamellipodia at multiple sites in the cell. Effect of M2[45-62] on cell membrane tension was evaluated by optical tweezer. The membrane tension sensor protein FBP17 was involved in M2[45-62]-driven lamellipodium formation. Lysine-to-arginine substitution in M2[45-62] further enhanced its activity of lamellipodium formation. M2[45-62] had an ability to reduce cell motility, evaluated by scratch wound migration and transwell migration assays. An increase in neurite outgrowth was also observed after treatment with M2[45-62]. The above results suggest the potential of M2[45-62] to modulate cell movement and morphology by modulating cell membrane tension.

Rab33a and Rab33ba mediate the outgrowth of forebrain commissural axons in the zebrafish brain.

Rab small GTPases play key roles in intracellular membrane trafficking. Rab33a promotes axon outgrowth of cultured rat hippocampal neurons by mediating the anterograde axonal transport of Golgi-derived vesicles and the concomitant exocytosis of these vesicles at the growth cone. However, the functions of Rab33 in vivo are unclear. Here, we show that zebrafish rab33a and rab33ba are orthologs of mammalian Rab33a and Rab33b, respectively. They are expressed in the developing brain, including in neurons of the telencephalic dorsorostral cluster and the diencephalic ventrorostral cluster, which project axons to form the anterior and postoptic commissures, respectively. Although rab33a single mutant and rab33ba single mutant fish did not show remarkable defects, fish carrying the rab33a;rab33ba double mutations displayed dysgenesis of the anterior and postoptic commissures. Single-cell labeling in the telencephalic dorsorostral cluster demonstrated that the rab33a;rab33ba double mutation inhibits axonal extension in the anterior commissure. These results suggest that Rab33a and Rab33ba mediate axon outgrowth and the formation of the forebrain commissures in the zebrafish brain in a cooperative manner.

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.

Gradient-reading and mechano-effector machinery for netrin-1-induced axon guidance.

Growth cones navigate axonal projection in response to guidance cues. However, it is unclear how they can decide the migratory direction by transducing the local spatial cues into protrusive forces. Here we show that knockout mice of Shootin1 display abnormal projection of the forebrain commissural axons, a phenotype similar to that of the axon guidance molecule netrin-1. Shallow gradients of netrin-1 elicited highly polarized Pak1-mediated phosphorylation of shootin1 within growth cones. We demonstrate that netrin-1-elicited shootin1 phosphorylation increases shootin1 interaction with the cell adhesion molecule L1-CAM; this, in turn, promotes F-actin-adhesion coupling and concomitant generation of forces for growth cone migration. Moreover, the spatially regulated shootin1 phosphorylation within growth cones is required for axon turning induced by netrin-1 gradients. Our study defines a mechano-effector for netrin-1 signaling and demonstrates that shootin1 phosphorylation is a critical readout for netrin-1 gradients that results in a directional mechanoresponse for axon guidance.

Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis.

Chemical cues presented on the adhesive substrate direct cell migration, a process termed haptotaxis. To migrate, cells must generate traction forces upon the substrate. However, how cells probe substrate-bound cues and generate directional forces for migration remains unclear. Here, we show that the cell adhesion molecule (CAM) L1-CAM is involved in laminin-induced haptotaxis of axonal growth cones. L1-CAM underwent grip and slip on the substrate. The ratio of the grip state was higher on laminin than on the control substrate polylysine; this was accompanied by an increase in the traction force upon laminin. Our data suggest that the directional force for laminin-induced growth cone haptotaxis is generated by the grip and slip of L1-CAM on the substrates, which occur asymmetrically under the growth cone. This mechanism is distinct from the conventional cell signaling models for directional cell migration. We further show that this mechanism is disrupted in a human patient with L1-CAM syndrome, suffering corpus callosum agenesis and corticospinal tract hypoplasia.

Actin Waves: Origin of Cell Polarization and Migration?

Actin filaments and associated proteins undergo wave-like movement in various cell types. Recent studies with cutting-edge analyses, including live-cell imaging, biophysical monitoring and manipulation, and mathematical modeling, have highlighted roles of 'actin waves' in cellular protrusion, polarization, and migration. The prevailing models to explain the wave-like dynamics of actin filaments involve an activator-inhibitor mechanism. In addition, axonal actin waves migrate by means of directional assembly and disassembly of membrane-anchored actin filaments, and thus represent a new type of machinery that translocates their component molecules to the cell edge. Here, we review recent advances in our understanding of the generation, mobility, and functions of actin waves, and discuss how actin waves may self-organize into the molecular machinery underlying cell morphogenesis.

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.