Abstract
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.
1 Introduction
To establish functional neural networks, axons must navigate to appropriate synaptic partners. The growing tip of axons bears a motile structure the growth cone, which plays critical roles in axon outgrowth and guidance (; ; ; ). Regarding the force to drive axon outgrowth and guidance, actin filaments (F-actins) polymerize at the leading edge of growth cones and disassemble proximally. This, together with the contractile activity of myosin II, drives retrograde flow of F-actin (; ; ; ). Mechanical coupling between the F-actin flow and the adhesive substrate, mediated by clutch-linker molecules and cell adhesion molecules, transmits the force of F-actin flow to the substrate as traction force for growth cone migration (; ; ; ). Concurrently, the actin-adhesion coupling slows down the F-actin flow (; ), allowing actin polymerization to protrude the leading-edge membrane ().
Axon guidance is regulated by chemical cues in the extracellular environment, including soluble chemicals (termed chemotaxis) and substrate-bound chemicals (haptotaxis) (; ); soluble and substrate-bound chemicals act as long-range and short-range cues, respectively (). Netrin-1 is one of the best characterized axon guidance molecules (; ; ; ); intensive research over the past decades has elucidated the molecular mechanics for netrin-1–induced axonal chemotaxis. Extracellular netrin-1 gradients, formed through diffusion, induce attraction of growth cone in vitro (; ; ; ; ). Stimulation of the netrin-1 receptor deleted in colorectal cancer (DCC) activates Cdc42 and Rac1 and their downstream kinase Pak1 in the growth cone (; ; ; ; ). Shootin1a functions as a clutch-linker molecule that links F-actin retrograde flow and adhesive substrates by interacting with the actin-binding protein cortactin and the cell adhesion molecule L1 at the growth cone (; ; ). Pak1, activated through the netrin-1–induced signaling, phosphorylates shootin1a (). This phosphorylation enhances its binding affinity for cortactin and L1, thereby facilitating the traction force for netrin-1–induced axon outgrowth and chemoattraction (; ).
Recent studies have highlighted netrin-1, present on the adhesive substrate, as a haptotactic axon guidance cue (; , ; ; ). However, the molecular mechanics of netrin-1–induced haptotaxis remains unclear (). Here, we report that DCC and shootin1a also mediate netrin-1–induced axonal haptotaxis, through a process that does not rely on cell signaling. Shootin1a interacted with DCC, linking it to F-actin retrograde flow. Speckle imaging analyses showed that DCC underwent either grip (stop) or retrograde slip () on the adhesive substrate. The grip state was more prevalent on a netrin-1–coated substrate compared to the control substrate polylysine, leading to increased traction force transmission on the netrin-1–coated substrate. Furthermore, shootin1 knockout (KO) disrupted netrin-1–induced axonal haptotaxis. These findings indicate that the directional force for netrin-1–induced haptotaxis is exerted on the substrates through the asymmetric mechanical coupling between netrin-1 and DCC within the growth cone.
2 Materials and methods
2.1 Animals
All relevant aspects of the experimental procedures were approved by the Institutional Animal Care and Use Committee of Nara Institute of Science and Technology. Shootin1 KO mice were generated as described previously (). E16.5 shootin1 KO embryos were obtained by crossing male and female shootin1 heterozygous C57BL/6 mice; the offspring genotypes were confirmed by PCR using the following primers: Genotyping F1 (5′-CAGACTGCTACCCACTACCCCCTAC-3′) and Genotyping R1 (5′-CCTAGAGCTGGACAGCGGATCTGAG-3′) for the wild-type (WT) allele. Genotyping F2 (5′-CCCAGAAAGCGAAGGAACAAAGCTG-3′), Genotyping R2 (5′-ACCTTGCTCCTTCAAGCTGGTGATG-3′) for the KO allele. Embryonic stages were calculated from noon of the vaginal plug day, defined as embryonic day 0.5 (E0.5). Chimeric mice were crossed with C57BL/6 mice for at least nine generations before analysis. All C57BL/6 mice were bred under standard conditions (12 h/12 h light/dark cycle, access to dry food and water).
2.2 Cell culture and transfection
Hippocampal neurons were prepared form E16.5 WT and shootin1 KO mice as described (). They were cultured on glass coverslips (Matsunami) or glass bottom dishes (Matsunami), which we coated with 100 μg/mL polylysine (poly-D-lysine) (Sigma, catalog number P6407-5MG) alone or sequentially with 100 μg/mL polylysine and 400 ng/mL netrin-1 (R&D systems, catalog number 6419-N1-025/CF), and cultured in Neurobasal Medium (Thermo Fisher Scientific) containing B-27 supplement (Thermo Fisher Scientific) and 1 mM glutamine. For the immunoprecipitation analyses in Figure 3B, we used cortical neurons cultured for 2 days on polylysine-coated culture dishes (Greiner Bio-One, catalog number 664160-013) because the experiments required large numbers of neurons. Micro-scale patterns of netrin-1 on polylysine-coated coverslips were prepared as described (), using PBS solution containing 1 M sucrose, 20 μg/ mL Texas Red-conjugated BSA (Thermo Fisher Scientific), and 400 ng/mL netrin-1 instead of 25 μg/ mL laminin. Briefly, a polydimethylsiloxane (PDMS) stamp was produced using a silicon substrate with small hexagonal patterns (Supplementary Figure 1A). PBS solution containing 1 M sucrose, 20 μg/ mL Texas Red-conjugated BSA, and 400 ng/mL netrin-1 was applied to the PDMS stamp using a micropipette to fill the indentations of microscale hexagonal lattice patterns. Excess solution on the PDMS surface was removed using a spin coater. The PDMS stamp was then attached to the polylysine-coated coverslip and incubated at 37°C overnight to coat the coverslip surface with the netrin-1 pattern. All experiments, except for force measurements, were carried out on glass surfaces. Neurons were transfected with plasmid DNA using Nucleofector (Lonza) before plating as described (). HEK293T cells (ATCC, RRID:CVCL_0063) were cultured in Dulbecco’s modified Eagle’s medium (Sigma) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) as described previously (), and transfected with vectors using Polyethyleneimine MAX (Polysciences) according to the manufacturer’s protocol.
2.3 DNA constructs
To generate pCMV-FLAG-shootin1a, the cDNA of human shootin1a was amplified by PCR with the primers Shootin1a F (5′-CCGCTCGAGATGAACAGCTCGGACGAGGAGAAG-3′) and shootin1a R (5′-CCGCTCGAGTTACTGGGAGGCCAGGATTCC CTTCAG-3′), and then subcloned into pCMV-FLAG vector (). To generate pFC14K-DCC-Halotag, the cDNA of human DCC was amplified by PCR with the primers DCC F (5′-GAGGATCCATGGAGAATAGTCTTAGATG-3′), and DCC R (5′-CTGGATCCCTAAAAGGCTGAGCCTGTGATGG-3′), and then subcloned into pFC14K-Halotag CMV Flexi vector (Promega). To generate pCMV-EGFP-DCC ICD (intracellular domain), the cDNA of human DCC was amplified by PCR with the primers DCC ICD F (5′-GGTGAACTTCAAGATCCGCCACAA-3′), and DCC ICD R (5′-TGCAATAAACAAGTTAACAACAACAATTG-3′), and then subcloned into pCMV-EGFP vector (Agilent Technology). The preparation of pFN21A-HaloTag-actin has been described previously ().
2.4 Immunocytochemistry and microscopy
Cultured neurons were fixed with 3.7% formaldehyde in Krebs buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.2 mM NaHCO3, 2 mM CaCl2, 10 mM glucose, 400 mM sucrose, 10 mM HEPES pH 7.0) for 20 min on ice and subsequently for 10 min at room temperature, followed by treatment for 15 min with 0.05% Triton X-100 in phosphate-buffered saline (PBS) on ice and 10% FBS in PBS for 1 h at room temperature. They were then incubated with primary antibody diluted in PBS containing 10% FBS overnight at 4°C. The following primary antibodies were used: rabbit anti-shootin1a (1:2000) (), goat anti-DCC (1:1000) (Santa Cruz Biotechnology, catalog number sc-515834), mouse anti-Tuj1 (anti-β III-Tubulin) (1:1000) (Bio Legend, catalog number 801202) and mouse anti-paxillin (1:1000) (BD Transduction Laboratories, catalog number: AB_397464) antibodies. Neurons were washed with PBS, and then incubated with secondary antibody diluted in PBS 1 h at room temperature. The following secondary antibodies were used: Alexa Fluor 488 conjugated donkey anti-goat (1:1000) (Thermo Fisher Scientific, catalog number AB_2534102), Alexa Fluor 594 conjugated donkey anti-rabbit (1:1000) (Jackson ImmunoResearch Laboratories, catalog number AB_2340621), and Alexa Fluor 488 conjugated goat anti-mouse (1:1000) (Thermo Fisher Scientific, catalog number AB_2534088) antibodies. For phalloidin staining, after washing with PBS, cells were stained with Alexa Fluor 594 conjugated phalloidin (Invitrogen, catalog number: A12381) at 1:100 dilution for 1 h at room temperature. After washing with PBS, immunostained cells were mounted with 50% (v/v) glycerol (Nacalai Tesque) in PBS. Fluorescence images were acquired using either a fluorescence microscope (BZ-X710, Keyence) equipped with 20 × 0.75 NA (Nikon) and imaging software (BZ-X viewer, Keyence) or a total internal reflection fluorescence (TIRF) microscope (IX81, Olympus) equipped with an EM-CCD camera (Ixon3, Andor), a UAPON 100 × 1.49 NA (Olympus) and imaging software (MetaMorph, Molecular Devices).
2.5 Immunoprecipitation and immunoblotting
For immunoprecipitation using HEK293T cells, cell lysates were prepared as described (). The following primary antibodies were used in immunoblotting: rabbit anti-GFP (1:2000) (MBL, catalog number AB_591816) and mouse anti-FLAG (DDDDK) tag (1:4000) (MBL, catalog number AB_591224). The following secondary antibodies were used in immunoblotting: HRP-conjugated donkey anti-rabbit IgG (1:2000) (GE Healthcare, catalog number AB_772206), HRP-conjugated goat anti-mouse IgG (1:5000) (Thermo Fisher Scientific, catalog number AB_2534088). Immunoprecipitation was performed as described previously (; ).
For immunoprecipitation using cultured neurons, lysates of neurons were prepared as described (), the supernatants of the lysates were incubated with rabbit anti-shootin1 antibody (, ) or control IgG (Sigma, catalog number AB_1163661) overnight at 4°C, and immunocomplexes were then precipitated with protein G-Sepharose 4B. After washing the beads with wash buffer, immunocomplexes were analyzed by immunoblot.
The following primary antibodies were used in immunoblotting: rabbit anti-shootin1a (1:5000) (), and mouse anti-DCC antibodies (1:4000) (Santa Cruz Biotechnology, catalog number sc-515834). The following secondary antibodies were used in immunoblotting: HRP-conjugated donkey anti-rabbit IgG (1:2000) (GE Healthcare, catalog number AB_772206), HRP-conjugated goat anti-mouse IgG (1:5000) (Thermo Fisher Scientific, catalog number AB_2534088).
2.6 Fluorescent speckle imaging and grip and slip analysis
Fluorescent speckle imaging and speckle tracking analysis of HaloTag-actin were performed as described in detail previously (). Fluorescent speckle imaging and speckle tracking analysis of DCC-HaloTag was performed as described (), using neurons transfected with pFC14K-DCC-Halotag. Neurons were treated with HaloTag TMR ligand (50 nM) (Promega, catalog number G8251) in culture medium and incubated for 1 h at 37°C. The ligand was then washed out with PBS, and the cells were incubated with L15 medium (Thermo Fisher Scientific) including B27 supplement (Thermo Fisher Scientific) and 1 mM glutamine for 30 min at 37°C. The fluorescent speckles of DCC-HaloTag were observed using a TIRF microscope (IX81, Olympus) equipped with an EM-CCD camera (Ixon3, Andor), a complementary metal-oxide-semiconductor (CMOS) camera (ORCA Flash4.0LT; Hamamatsu), a UAPOM 100x 1.49 NA (Olympus), and imaging software (MetaMorph, Molecular Devices). Fluorescence images were acquired every 5 s for 295 s. The flow speed of F-actin or DCC was quantified by tracing the fluorescent speckles of HaloTag-actin or HaloTag-DCC using Fiji (Open-source software package). Briefly, we selected a fluorescent speckle that flowed retrogradely for at least 20 s (4 time-frames) in a filopodium, and made a time-lapse montage including the speckle. We measured the translocation distance of the speckle; then, the flow speed during the observation was determined ().
We use the terms “grip” and “slip” to describe the movement of DCC-HaloTag. “Grip vs. slip” was used by to describe the degree of interaction between the cell adhesion and adhesive substrate in fish keratocytes. Subsequently, used “frictional slip” to describe the experimentally observed retrograde movement of focal adhesions (including integrin) of human osteosarcoma cells on adhesive substrates. Based on their concept and terms, we previously called L1 molecules that undergo retrograde movement on adhesive substrates as in “slip” phase and those immobilized on the substrates as in “grip” phase (, ); we follow this terminology here. For the grip and slip analysis, DCC puncta that were visible for at least 10 s (two intervals) were analyzed, and immobile signals were defined as DCC in grip phase while those that flowed retrogradely were defined as DCC in slip phase.
2.7 Traction force microscopy
Traction force microscopy was performed as described in detail previously (). Neurons were transfected with pEGFP-C1 vectors and cultured on polyacrylamide (PAA) gels with embedded 200-nm fluorescent beads (Thermo Fisher Scientific) for 2 days. In this study, the PAA gel was prepared at a final concentration of 3.75% acrylamide and 0.03% bis-acrylamide. The stiffness of the PAA gel was ∼270 Pa, which is within the range of brain tissues (100-10,000 Pa) (; ; ). Before imaging, the culture medium was replaced with L15 medium (Thermo Fisher Scientific) including B27 supplement (Thermo Fisher Scientific) and 1 mM glutamine. Time-lapse imaging of fluorescent beads and growth cone was performed at 37°C using a confocal laser microscope (LSM710, Carl Zeiss) equipped with a C-Apochromat 63x/1.2 W Corr objective, and ZEN2009 software. Images were acquired every 3 s for 147 s. After imaging, the culture dishes were treated with 1% SDS to release neurons from the PAA gel substrate, and an image of the unstrained substrate was acquired. The magnitude of the traction force under the growth cones was analyzed using MATLAB (MathWorks) and a homemade traction force analysis code ().
2.8 Quantification and statistical analysis
Data handling and preparation of graphs was performed using Microsoft Excel 2016 (Microsoft). Statistical tests were performed using GraphPad Prism7 (GraphPad Software). For samples with more than 7 data points, the D’Agostino–Pearson normality test was used to determine whether the data followed a normal distribution. For cases in which the number of data points was between 3 and 7, the Shapiro-Wilk test was used for the normality test. We also tested the equality of variation with the F test for two independent groups that followed normal distributions. Significance tests were performed using two-tailed unpaired Student’s t test to compare two independent groups that followed normal distributions with equal variation. All data are shown as the mean ± SEM. Statistical significance was defined as ***p < 0.01; **p < 0.02; *p < 0.05; ns, not significant. A P value less than 0.05 was considered to be statistically significant. For each experiment, the corresponding statistics information and number of samples are indicated in the figure legend. All experiments were performed at least three times and reliably reproduced. Investigators were blind to the experimental groups for each analysis, except biochemical analyses. For Figures 3D, F, we measured Pearson’s correlation coefficient ’s correlation coefficient using Fiji (Open-source software package) and the JaCoP plugin () to quantify the co-localization of shootin1a with DCC and paxillin.
3 Results
3.1 Substrate-bound netrin-1 promotes traction force for axon outgrowth and haptotaxis
To examine the effect of substrate-bound netrin-1 on axonal extension, we first cultured mouse hippocampal neurons on glass coverslips coated with either polylysine plus netrin-1 or polylysine alone as a control substrate. As reported (; ; ), substrate-bound netrin-1 promoted axon outgrowth over the polylysine alone substrate (Figures 1A, B). In addition, neurons cultured on micro-scale patterns of netrin-1 on polylysine-coated coverslips extended axons preferentially on netrin-1 (Figure 1C), indicating that substrate-bound netrin-1 induces axon outgrowth and attraction (haptotaxis) of cultured hippocampal neurons. There was no significant difference in the sizes of axonal growth cones between on polylysine and on netrin-1 (Supplementary Figure 2). To analyze the mechanics for netrin-1–induced axon outgrowth and haptotaxis, we measured traction force produced by the axonal growth cone. Neurons were cultured on polyacrylamide gels coated with polylysine alone or polylysine plus netrin-1; 200-nm fluorescent beads were embedded in the gels. Traction force under the growth cones was analyzed by visualizing force-induced deformation of the elastic gel, which is reflected by displacement of the beads from their original positions (). As reported (; ), the beads under the growth cone moved dynamically (Figure 1D; Supplementary Video 1). The magnitude of the traction force generated on netrin-1 was 11.9 ± 1.1 pN/μm2 (equivalent to Pa) (n = 7 growth cones), which was significantly larger than on polylysine (6.1 ± 1.5 pN/μm2) (n = 7 growth cones) (Figure 1E), indicating that substrate-bound netrin-1 promotes traction force for axon outgrowth and haptotaxis.
FIGURE 1
3.2 Substrate-bound netrin-1 promotes F-actin-substrate coupling
Previous studies reported that the mechanical coupling between F-actin retrograde flow and the adhesive substrate promotes traction force (blue arrows, Figure 2A); concurrently, it reduces the F-actin flow speed (yellow arrows) (; ). To further examine the molecular mechanism of the netrin-1–induced force generation, we monitored F-actin retrograde flow within the axonal growth cone by fluorescent speckle imaging of HaloTag-actin (). HaloTag-actin speckles moved retrogradely in growth cones (Figure 2B; Supplementary Video 2), depicting the F-actin movement (Figure 2A). The F-actin flow speed on polylysine was 4.5 ± 0.3 μm/min (mean ± SEM, n = 84 signals, 6 growth cones). On the other hand, netrin-l on substrates significantly reduced the flow speed (2.3 ± 0.1 μm/min, n = 92 signals, 6 growth cones) (Figures 2B, C; Supplementary Video 2). Together with the observed increase in traction force on netrin-1 (Figure 1E), these data indicate that netrin-1 on the adhesive substrate promotes the coupling between F-actin retrograde flow and the adhesive substrate at the growth cone.
FIGURE 2
3.3 DCC and shootin1a couple F-actin flow with substrate-bound netrin-1
Next, we examined the molecular components linking F-actin retrograde flow to the substrate-bound netrin-1. Previous studies reported that the clutch-linker molecule shootin1a interacts with the intracellular domains of the immunoglobulin superfamily adhesion molecules, L1 and N-cadherin (; ). This led us to hypothesize that shootin1a interacts with another member of immunoglobulin superfamily, DCC (). In addition, microbead tracking analyses reported that the mechanical interaction between the growth cone and substrate-bound netrin-1 exerts forces on the substrate in a DCC dependent manner (). To assess the involvement of DCC and shootin1a in the linkage between F-actin flow and substrate-bound netrin-1, we performed co-immunoprecipitation assay, using HEK293T cells which express shootin1a and the intracellular domain of DCC (DCC-ICD). As shown in Figure 3A, DCC-ICD was co-precipitated with shootin1a. In cell lysates prepared from cultured neurons, the full-length DCC was co-precipitated with shootin1a (Figure 3B), indicating an interaction between endogenous shootin1a and endogenous DCC in neurons. In addition, shootin1a colocalized with DCC in the axonal growth cones of hippocampal neurons (Figures 3C, D), suggesting that shootin1a interacts with DCC at growth cones. Previous studies reported integrin-dependent adhesion sites, point contacts in growth cones (). However, shootin1a did not show a marked colocalization with the point contact marker paxillin in growth cones (Figures 3E, F), suggesting that shootin1a mediates integrin-independent contacts.
FIGURE 3
Furthermore, disruption of shootin1a function by KO increased the F-actin flow speed in the growth cones on netrin-1–coated substrate (Figures 4A, B; Supplementary Video 3). Removal of netrin-1 from the substrate (polylysine alone) similarly increased the F-actin flow speed in WT growth cone (Figures 4A, B). In addition, shootin1a KO did not affect the F-actin flow speed in the absence of netrin-1 (polylysine alone), indicating shootin1a mediates the coupling between F-actin flow and the substrate-bound netrin-1. Concurrently, shootin1 KO reduced the magnitude of the traction force on the netrin-1–coated substrate (Figures 4C, D; Supplementary Video 4). Together, we conclude that DCC and shootin1a couple F-actin flow with substrate-bound netrin-1 (Figure 4E).
FIGURE 4
3.4 Netrin-1–induced axon outgrowth and haptotaxis require shootin1a-mediated actin-DCC coupling
To further examine the role of the actin-substrate linkage mediated by DCC and shootin1a, we analyzed axon outgrowth and haptotaxis. Disturbance of this linkage by shootin KO inhibited axon outgrowth on netrin-1–coated substrate (Figures 5A, B). Removal of netrin-1 from the substrate (polylysine alone) also inhibited axon outgrowth (Figures 5A, B). Consistently, shootin1a KO did not affect the axon outgrowth on the polylysine-coated substrate (Figures 5A, B). As we have seen, hippocampal neurons cultured on micro-scale patterns of netrin-1 on polylysine-coated coverslips extended axons preferentially on netrin-1 as haptotaxis (Figure 5C); 79.6% ± 3.0% (n = 16 neurons) of the total lengths of axons were located on netrin-1–coated substrate (Supplementary Figure 1B; Figure 5D). On the other hand, shootin1 KO neurons extended axons which frequently crossed the borders between netrin-1 and polylysine (Figure 5C); they extended significantly lower rate (45.4% ± 3.4%, n = 14 neurons) of axons on netrin-1–coated substrate (Figure 5D). These data indicate that netrin-1–induced axon outgrowth and haptotaxis require actin-DCC coupling mediated by shootin1a (Figure 4E).
FIGURE 5
3.5 Netrin-1 promotes F-actin-substrate coupling at the DCC-substrate interphase
To analyze the mechanism by which substrate-bound netrin-1 promotes F-actin-substrate coupling, we monitored the movement of DCC, which serves as the link between intracellular shootin1a and netrin-1 on the substrate (Figure 4E). DCC-HaloTag expressed in hippocampal neurons was labeled with TMR ligand and observed using a TIRF microscope (Figure 6A; Supplementary Video 5). Two distinct types of DCC signals were observed: retrogradely flowing (blue dashed lines) and immobile (pink dashed lines, Figure 6A) signals. We previously reported similar movements of L1 at the growth cone during laminin-induced axonal haptotaxis () and mechanosensing (). At the growth cone, the force of F-actin retrograde flow is transmitted to L1 through cortactin and shootin1a, allowing to pull the bond between L1 and laminin. When the pulling force exceeds a threshold, the bond breaks and L1 flows retrogradely on the substrate (, ).
FIGURE 6
We expect that similar force are transmitted to DCC, thereby pulling the bond between DCC and substrate-bound netrin-1 (blue arrows, Figure 4E). We refer to DCC that undergoes retrograde movement on the substrates as being in “slip” phase and that immobilized on the substrates as being in “grip” phase, following the terminology of L1 movements (see Materials and Methods) (). On polylysine, 15% of DCC speckles exhibited the grip phase, while 85% displayed slip phase (n = 350 speckles, 7 growth cones). In contrast, 40% of the signals were in grip phase on netrin-1 (n = 548 speckles, 8 growth cones) (Figure 6B). The grip phase percentage was significantly larger on netrin-1 than on polylysine, indicating that DCC binds to the netrin-1–coated substrate with higher affinity. Consistently, the mean flow speed of DCC on the netrin-1–coated substrate was 1.63 ± 0.11 μm/min (n = 114 signals, 8 growth cones), significantly slower than that on the polylysine-coated substrate (3.09 ± 0.18 μm/min, n = 106 signals, 7 growth cones) (Figure 6C). Together, these data suggest that netrin-1 promotes F-actin-substrate coupling at the interphase between DCC and the substrate, thereby generating traction forces for axon outgrowth and haptotaxis (Figure 6D).
4 Discussion
4.1 Axonal haptotaxis through adhesion-clutch between DCC and netrin-1
We have shown that DCC and shootin1a mechanically couple F-actin retrograde flow in the axonal growth cone and netrin-1 on the adhesive substrate. Substrate-bound netrin-1 increased the grip phase of DCC and promoted F-actin-substrate coupling, thereby increasing traction force produced by the growth cone. Furthermore, inhibition of the mechanical coupling by shootin1a KO disrupted netrin-1–induced axon outgrowth and haptotaxis. These data suggest a mechanism by which the growth cone generates a directional force for netrin-1–induced axonal haptotaxis (Figure 6D). The force of F-actin retrograde flow in the growth cone (yellow arrows) is transmitted to DCC through cortactin and shootin1a, allowing to pull the bond between DCC and the adhesive substrates. When the pulling force exceeds a threshold, the bond breaks and DCC molecule flows retrogradely on the substrate (black arrows). As the substrate presenting netrin-1 exhibits higher affinity to DCC than the control substrate, more DCC molecules grip the netrin-1-presenting substrate than the control substrate, enabling more efficient force transmission on the netrin-1 side (blue arrows). This asymmetric adhesion-clutch between DCC and netrin-1 within the growth cone generate directional force for axonal haptotaxis toward netrin-1 (green arrow).
Our data suggest that shootin1a mediates integrin-independent contacts at the growth cone (Figures 3E, F). Speckle imaging data of DCC provide key information on the adhesion dynamics of this system. It has been reported that the frictional slip between integrins and the substrate decreases during the maturation of integrin-mediated focal adhesions (). On the other hand, DCC repeats immobilized phase (grip) and frictional slip phase (slip) on the netrin-1–coated substrate (Figures 6A–C). We have also observed repeated grip and slip phases of the L1-shootin1a adhesion-clutch under various elastic conditions that correspond to the soft brain environments (). These data suggest that the bond between DCC and the adhesive substrate breaks repeatedly, without undergoing maturation. As maturation of the adhesion machinery promotes formation of a strong adhesion that can inhibit cell migration (; ), we consider that the slippery adhesion-clutch between DCC and the substrate is suitable for the efficient growth cone migration.
4.2 Adhesion-clutch mechanism requires a specific adhesion ligand on the substrate
The present study showed that the removal of netrin-1 from the substrate (polylysine alone) resulted in an increase in the F-actin flow speed in growth cones, indicating that the netrin-1 removal disrupts the actin-adhesion coupling (Figure 4B). Consistently, shootin1a KO did not affect F-actin retrograde flow or axon outgrowth in the absence of netrin-1 on the substrate (Figures 4A, B, 5A, B). These data demonstrate that the adhesion-clutch mechanism requires a proper adhesion ligand on the substrate and emphasize the importance of conducting experiments using appropriate adhesion ligands. There is a debate regarding the contribution of the adhesion-clutch mechanism to axon growth in 3D environments. reported that disruption of F-actin by cytochalasin D treatment promoted the extension of the longest neurite on the polylysine-coated substrate. Together with the data that they could not detect neuronal traction force in 3D environments, concluded that axon outgrowth in 3D is independent of adhesions. However, the cytochalasin D treatment inhibited extension of the longest neurite on a laminin-coated substrate as described in Supplementary Figure 9 of . As laminin serves as a proper substrate for the shootin1a-L1 adhesion-clutch (), the data of indicate that F-actin is involved in axon outgrowth on the appropriate substrate.
Furthermore, in a semi-3D environment, detected traction force produced by the growth cone located at the leading process of migratory neurons. The force of about 10 pN/μm2 was produced by shootin1-L1 adhesion-clutch. Importantly, shootin1 KO reduced the force generation as well as neuronal migration speed in brain tissues. Furthermore, shootin1 KO reduced axonal extension in brain (). Together, these reported data suggest that neuronal growth cones can produce pulling force to propel their migration in 3D environments. In addition to shootin1, catenins (; ), ezrin/radixin/moesin (ERM) proteins (; ), vinculin (; ; ; ; ), talin (; ; ), and fmn2 () have been proposed as clutch-linker molecules for growth cone advance.
4.3 DCC-shootin1a as a dual-mode regulator of netrin-1–induced axon guidance
The present axon guidance mechanism does not rely on cell signaling but depends on differences in the physical force between DCC and the adhesive substrates, with DCC serving as an adhesion receptor for netrin-1. This mechanism is consistent with the previous report that growth cones exerted a pulling force on netrin-1 in a DCC-dependent manner (). Additionally, we recently reported a similar mechanism for laminin-induced axonal haptotaxis, where asymmetric adhesion-clutch between the adhesion receptor L1 and the substrate mediates axon turning (). Given its simplicity, we consider that this mechanism may contribute to various haptotactic axon guidance and cell migration. Versatility of this mechanism in haptotactic cell motility is an intriguing issue for further investigation.
DCC also serves as a cell signaling receptor for netrin-1 (). Netrin-1 binding to DCC activates Cdc42, Rac1 and Pak1 (; ; ; ; ; ). Netrin-1–induced axonal chemoattraction is also mediated by various signaling molecules, including phosphatidylinositol-3 kinase, focal adhesion kinase, phospholipase Cγ, cAMP, Ca2+, ERK1/2, and Src (; ; ; ; ; ; ). In addition, shootin1a mediates netrin-1–induced axon guidance under the DCC signaling cascade; it is phosphorylated by Pak1 upon DCC stimulation and dephosphorylated by protein phosphatase-1 (PP1) (; ). Phosphorylation of shootin1a enhances the clutch-coupling between F-actin and the adhesion receptor L1 and promotes the traction force for netrin-1–induced axon outgrowth and chemoattraction (; ). Netrin-1 gradients induce chemoattraction even without substrate-bound netrin-1, indicating that netrin-1 can function as a chemotactic guidance cue (). The mechanism involving shootin1a phosphorylation is highly sensitive to extracellular gradients of netrin-1 (), potentially involving desensitization and re-adaptation of the signaling pathway for long-distance axon guidance (). During navigation, growth cones may sense netrin-1 as a short-range adhesion cue or a long-range cell signaling ligand, accommodating to different environments along their routes, to travel to their destinations.
Statements
Data availability statement
The original contributions presented in this study are included in this article/Supplementary material, further inquiries can be directed to the corresponding author.
Ethics statement
The animal study was approved by the Institutional Animal Care and Use Committee of Nara Institute of Science and Technology. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
ZQ: Data curation, Formal analysis, Methodology, Investigation, Validation, Visualization, Writing – original draft, Writing – review and editing. TM: Funding acquisition, Methodology, Supervision, Validation, Writing – original draft, Writing – review and editing. DA: Data curation, Formal analysis, Investigation, Writing – review and editing. KA: Methodology, Supervision, Writing – review and editing. KB: Data curation, Investigation, Methodology, Validation, Writing – review and editing. NI: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – original draft, Writing – review and editing.
Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported in part by AMED under Grant Number JP17gm0810011 (NI), JSPS KAKENHI (JP19H03223, NI), JSPS Grants-in-Aid for Early-Career Scientists (JP23K14181, TM), and the Osaka Medical Research Foundation for Incurable Diseases (TM).
Acknowledgments
We thank Drs. Akira Kurisaki, Katsumoto Okamura, and Yuichi Sakumura (Nara Institute of Science and Technology) for valuable discussions; Mieko Ueda and Kazumi Maekawa for technical support; Satoko Shimamura for kind encouragement; and Hongjia Chen, Kazuma Kami, and Singh Saranpal for supporting data analyses.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2024.1307755/full#supplementary-material
Supplementary Video 1Time-lapse movies of axonal growth cones of hippocampal neurons expressing EGFP (blue) and cultured on polylysine-coated and netrin-1–coated polyacrylamide gels with embedded 200-nm fluorescent beads (see Figure 1D). Images were acquired every 3 s for 147 s using a confocal microscope.
Supplementary Video 2Time-lapse movies of HaloTag-actin in axonal growth cones of hippocampal neurons cultured on polylysine-coated and netrin-1–coated glass-bottom dishes (see Figure 2B). Images were acquired every 5 s for 295 s using a fluorescence microscope.
Supplementary Video 3Time-lapse movies of HaloTag-actin in axonal growth cones of a WT hippocampal neuron and a shootin1 KO hippocampal neuron cultured on netrin-1–coated glass-bottom dishes (see Figure 4A). Images were acquired every 5 s for 295 s using a fluorescence microscope.
Supplementary Video 4Time-lapse movies of axonal growth cones of a WT hippocampal neuron and a shootin1 KO hippocampal neuron expressing EGFP (blue) and cultured on netrin-1–coated polyacrylamide gels with embedded 200-nm fluorescent beads (see Figure 4C). Images were acquired every 3 s for 147 s using a confocal microscope.
Supplementary Video 5Time-lapse movies of DCC-HaloTag in axonal growth cones of hippocampal neurons cultured on polylysine-coated and netrin-1–coated glass-bottom dishes (see Figure 6A). Images were acquired every 5 s for 245 s using a TIRF microscope.
Supplementary Figure 1Analysis of netrin-1–induced axonal haptotaxis. (A) Preparation of micro-scale patterns of netrin-1 on polylysine-coated coverslips. For details, see Materials and Methods. (B) Analysis of axons located on netrin-1. To calculate % of axon length on netrin-1, the total lengths of axonal shafts located on netrin-1–coated areas (blue double arrow) were divided by the full lengths of the axonal shafts (black double arrow).
Supplementary Figure 2Analysis of the size of axonal growth cone on netrin-1 and polylysine. (A) Fluorescence images of axonal growth cones of DIV2 hippocampal neurons cultured on glass coverslips coated with polylysine alone or coated with polylysine and netrin-1. Neurons were stained with Alexa Fluor 594 conjugated phalloidin for F-actin. (B) Quantification of growth cone size obtained by the analyses using neurons in panel (A) (polylysine, n = 40 growth cones; netrin-1, n = 40 growth cones). Scale bars, 5 μm. Data represent means ± SEM; ns, not significant.
Supplementary Figure 3Whole immunoblot images in Figures 3A, B. (A) HEK293T cells were transfected with vectors to express FLAG-shootin1a and EGFP-DCC-ICD; cells were also co-transfected with a vector to express EGFP as a negative control. Cell lysates were prepared and incubated with anti-FLAG antibody for immunoprecipitation. The immunoprecipitants were immunoblotted with anti-FLAG or anti-GFP antibody. (B) Cortical neurons prepared from E16.5 WT mice were seeded on polylysine-coated dishes and cultured for 2 days. Cell lysates were prepared and incubated with anti-shootin1 antibody or control IgG, then precipitated with protein G-Sepharose 4B. The immunoprecipitants were immunoblotted with anti-shootin1a or anti-DCC antibody.
References
1
AbeK.BabaK.HuangL.WeiK. T.OkanoK.HosokawaY.et al (2021). Mechanosensitive axon outgrowth mediated by L1-laminin clutch interface.Biophys. J.1203566–3576. 10.1016/j.bpj.2021.08.009
2
AbeK.KatsunoH.ToriyamaM.BabaK.MoriT.HakoshimaT.et al (2018). Grip and slip of L1-CAM on adhesive substrates direct growth cone haptotaxis.Proc. Natl. Acad. Sci. U S A.1152764–2769. 10.1073/pnas.1711667115
3
Aratyn-SchausY.GardelM. L. (2010). Transient frictional slip between integrin and the ECM in focal adhesions under myosin II tension.Curr. Biol.201145–1153. 10.1016/j.cub.2010.05.049
4
BabaK.YoshidaW.ToriyamaM.ShimadaT.ManningC. F.SaitoM.et al (2018). Gradient-reading and mechano-effector machinery for netrin-1-induced axon guidance.Elife7:e34593. 10.7554/eLife.34593
5
BardL.BoscherC.LambertM.MegeR. M.ChoquetD.ThoumineO. (2008). A molecular clutch between the actin flow and N-cadherin adhesions drives growth cone migration.J. Neurosci.285879–5890. 10.1523/JNEUROSCI.5331-07.2008
6
BarnesJ. M.PrzybylaL.WeaverV. M. (2017). Tissue mechanics regulate brain development, homeostasis and disease.J. Cell Sci.13071–82.
7
BolteS.CordelieresF. P. (2006). A guided tour into subcellular colocalization analysis in light microscopy.J. Microsc.224(Pt 3), 213–232. 10.1111/j.1365-2818.2006.01706.x
8
BoyerN. P.GuptonS. L. (2018). Revisiting netrin-1: one who guides (axons).Front. Cell Neurosci.12:221. 10.3389/fncel.2018.00221
9
Briançon-MarjolletA.GhoghaA.NawabiH.TrikiI.AuziolC.FromontS.et al (2008). Trio mediates netrin-1-induced Rac1 activation in axon outgrowth and guidance.Mol. Cell Biol.282314–2323. 10.1128/mcb.00998-07
10
ChanC. E.OddeD. J. (2008). Traction dynamics of filopodia on compliant substrates.Science3221687–1691. 10.1126/science.1163595
11
DemarcoR. S.StruckhoffE. C.LundquistE. A. (2012). The Rac GTP exchange factor TIAM-1 acts with CDC-42 and the guidance receptor UNC-40/DCC in neuronal protrusion and axon guidance.PLoS Genet.8:e1002665. 10.1371/journal.pgen.1002665
12
DentE. W.GertlerF. B. (2003). Cytoskeletal dynamics and transport in growth cone motility and axon guidance.Neuron40209–227.
13
DominiciC.Moreno-BravoJ. A.PuiggrosS. R.RappeneauQ.RamaN.VieugueP.et al (2017). Floor-plate-derived netrin-1 is dispensable for commissural axon guidance.Nature545350–354.
14
ForscherP.SmithS. J. (1988). Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone.J. Cell Biol.1071505–1516. 10.1083/jcb.107.4.1505
15
FothergillT.DonahooA. L.DouglassA.ZaluckiO.YuanJ.ShuT.et al (2014). Netrin-DCC signaling regulates corpus callosum formation through attraction of pioneering axons and by modulating Slit2-mediated repulsion.Cereb. Cortex241138–1151. 10.1093/cercor/bhs395
16
GarciaM.LeducC.LagardereM.ArgentoA.SibaritaJ. B.ThoumineO. (2015). Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones.Proc. Natl. Acad. Sci. U. S. A.1126997–7002. 10.1073/pnas.1423455112
17
GhateK.MutalikS. P.SthanamL. K.SenS.GhoseA. (2020). Fmn2 regulates growth cone motility by mediating a molecular clutch to generate traction forces.Neuroscience448160–171. 10.1016/j.neuroscience.2020.09.046
18
GiannoneG.MegeR. M.ThoumineO. (2009). Multi-level molecular clutches in motile cell processes.Trends Cell Biol.19475–486. 10.1016/j.tcb.2009.07.001
19
GomezT. M.LetourneauP. C. (2014). Actin dynamics in growth cone motility and navigation.J. Neurochem.129221–234.
20
GomezT. M.ZhengJ. Q. (2006). The molecular basis for calcium-dependent axon pathfinding.Nat. Rev. Neurosci.7115–125. 10.1038/nrn1844
21
HongK.HinckL.NishiyamaM.PooM. M.Tessier-LavigneM.SteinE. (1999). A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion.Cell97927–941.
22
IshiiN.WadsworthW. G.SternB. D.CulottiJ. G.HedgecockE. M. (1992). UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans.Neuron9873–881. 10.1016/0896-6273(92)90240-e
23
JuradoC.HaserickJ. R.LeeJ. (2005). Slipping or gripping? Fluorescent speckle microscopy in fish keratocytes reveals two different mechanisms for generating a retrograde flow of actin.Mol. Biol. Cell16507–518.
24
KastianR. F.BabaK.KaewkascholkulN.SasakiH.WatanabeR.ToriyamaM.et al (2023). Dephosphorylation of neural wiring protein shootin1 by PP1 phosphatase regulates netrin-1-induced axon guidance.J. Biol. Chem.299:104687. 10.1016/j.jbc.2023.104687
25
KastianR. F.MinegishiT.BabaK.SaneyoshiT.Katsuno-KambeH.SaranpalS.et al (2021). Shootin1a-mediated actin–adhesion coupling generates force to trigger structural plasticity of dendritic spines.Cell Rep.35:109130. 10.1016/j.celrep.2021.109130
26
KatohK.HammarK.SmithP. J.OldenbourgR. (1999). Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones.Mol. Biol. Cell10197–210. 10.1091/mbc.10.1.197
27
KennedyT. E.SerafiniT.de la TorreJ. R.Tessier-LavigneM. (1994). Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.Cell78425–435.
28
KuboY.BabaK.ToriyamaM.MinegishiT.SugiuraT.KozawaS.et al (2015). Shootin1-cortactin interaction mediates signal-force transduction for axon outgrowth.J. Cell Biol.210663–676. 10.1083/jcb.201505011
29
Lai Wing SunK.CorreiaJ. P.KennedyT. E. (2011). Netrins: versatile extracellular cues with diverse functions.Development1382153–2169. 10.1242/dev.044529
30
LamoureuxP.BuxbaumR. E.HeidemannS. R. (1989). Direct evidence that growth cones pull.Nature340159–762.
31
LiX.Saint-Cyr-ProulxE.AktoriesK.Lamarche-VaneN. (2002). Rac1 and Cdc42 but not RhoA or Rho kinase activities are required for neurite outgrowth induced by the Netrin-1 receptor DCC (deleted in colorectal cancer) in N1E-115 neuroblastoma cells.J. Biol. Chem.27715207–15214.
32
LiuY. J.Le BerreM.LautenschlaegerF.MaiuriP.Callan-JonesA.HeuzeM.et al (2015). Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells.Cell160659–672. 10.1016/j.cell.2015.01.007
33
LoweryL. A.Van VactorD. (2009). The trip of the tip: understanding the growth cone machinery.Nat. Rev. Mol. Cell Biol.10332–343. 10.1038/nrm2679
34
MaiJ.FokL.GaoH.ZhangX.PooM. M. (2009). Axon initiation and growth cone turning on bound protein gradients.J. Neurosci.297450–7458. 10.1523/JNEUROSCI.1121-09.2009
35
MallavarapuA.MitchisonT. (1999). Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction.J. Cell Biol.1461097–1106. 10.1083/jcb.146.5.1097
36
MandalP.BelapurkarV.NairD.RamananN. (2021). Vinculin-mediated axon growth requires interaction with actin but not talin in mouse neocortical neurons.Cell Mol. Life Sci.785807–5826. 10.1007/s00018-021-03879-7
37
MarsickB. M.San Miguel-RuizJ. E.LetourneauP. C. (2012). Activation of ezrin/radixin/moesin mediates attractive growth cone guidance through regulation of growth cone actin and adhesion receptors.J. Neurosci.32282–296. 10.1523/JNEUROSCI.4794-11.2012
38
MedeirosN. A.BurnetteD. T.ForscherP. (2006). Myosin II functions in actin-bundle turnover in neuronal growth cones.Nat. Cell Biol.8215–226.
39
MinegishiT.FujikawaR.KastianR. F.SakumuraY.InagakiN. (2021). Analyses of actin dynamics, clutch coupling and traction force for growth cone advance.J. Vis. Exp.176:e63227. 10.3791/63227
40
MinegishiT.UesugiY.KanekoN.YoshidaW.SawamotoK.InagakiN. (2018). Shootin1b mediates a mechanical clutch to produce force for neuronal migration.Cell Rep.25624–639 e626. 10.1016/j.celrep.2018.09.068
41
MitchisonT.KirschnerM. (1988). Cytoskeletal dynamics and nerve growth.Neuron1761–772.
42
MogilnerA.OsterG. (1996). Cell motility driven by actin polymerization.Biophys. J.713030–3045.
43
MooreS. W.BiaisN.SheetzM. P. (2009). Traction on immobilized netrin-1 is sufficient to reorient axons.Science325:166. 10.1126/science.1173851
44
MooreS. W.Roca-CusachsP.SheetzM. P. (2010). Stretchy proteins on stretchy substrates: the important elements of integrin-mediated rigidity sensing.Dev. Cell19194–206. 10.1016/j.devcel.2010.07.018
45
MooreS. W.ZhangX.LynchC. D.SheetzM. P. (2012). Netrin-1 attracts axons through FAK-dependent mechanotransduction.J. Neurosci.3211574–11585. 10.1523/JNEUROSCI.0999-12.2012
46
PalecekS. P.LoftusJ. C.GinsbergM. H.LauffenburgerD. A.HorwitzA. F. (1997). Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.Nature385537–540. 10.1038/385537a0
47
SakuraiT.GilO. D.WhittardJ. D.GazdoiuM.JosephT.WuJ.et al (2008). Interactions between the L1 cell adhesion molecule and ezrin support traction-force generation and can be regulated by tyrosine phosphorylation.J. Neurosci. Res.862602–2614. 10.1002/jnr.21705
48
SantosT. E.SchaffranB.BroguiereN.MeynL.Zenobi-WongM.BradkeF. (2020). Axon growth of CNS neurons in three dimensions is amoeboid and independent of adhesions.Cell Rep.32:107907. 10.1016/j.celrep.2020.107907
49
SerafiniT.KennedyT. E.GalkoM. J.MirzayanC.JessellT. M.Tessier-LavigneM. (1994). The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6.Cell78409–424. 10.1016/0092-8674(94)90420-0
50
ShekarabiM.KennedyT. E. (2002). The netrin-1 receptor DCC promotes filopodia formation and cell spreading by activating Cdc42 and Rac1.Mol. Cell Neurosci.191–17. 10.1006/mcne.2001.1075
51
ShekarabiM.MooreS. W.TritschN. X.MorrisS. J.BouchardJ. F.KennedyT. E. (2005). Deleted in colorectal cancer binding netrin-1 mediates cell substrate adhesion and recruits Cdc42, Rac1, Pak1, and N-WASP into an intracellular signaling complex that promotes growth cone expansion.J. Neurosci.253132–3141. 10.1523/JNEUROSCI.1920-04.2005
52
ShimadaT.ToriyamaM.UemuraK.KamiguchiH.SugiuraT.WatanabeN.et al (2008). Shootin1 interacts with actin retrograde flow and L1-CAM to promote axon outgrowth.J. Cell Biol.181817–829.
53
SongH.PooM. (2001). The cell biology of neuronal navigation.Nat. Cell Biol.3E81–E88.
54
SuterD. M.ErranteL. D.BelotserkovskyV.ForscherP. (1998). The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.J. Cell Biol.141227–240. 10.1083/jcb.141.1.227
55
SuterD. M.ForscherP. (2000). Substrate-cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance.J. Neurobiol.4497–113.
56
SutherlandD. J.PujicZ.GoodhillG. J. (2014). Calcium signaling in axon guidance.Trends Neurosci.37424–432.
57
SydorA. M.SuA. L.WangF. S.XuA.JayD. G. (1996). Talin and vinculin play distinct roles in filopodial motility in the neuronal growth cone.J. Cell Biol.1341197–1207. 10.1083/jcb.134.5.1197
58
TaylorA. M.MenonS.GuptonS. L. (2015). Passive microfluidic chamber for long-term imaging of axon guidance in response to soluble gradients.Lab Chip152781–2789. 10.1039/c5lc00503e
59
Tessier-LavigneM.GoodmanC. S. (1996). The molecular biology of axon guidance.Science2741123–1133.
60
ToriyamaM.KozawaS.SakumuraY.InagakiN. (2013). Conversion of a signal into forces for axon outgrowth through Pak1-mediated shootin1 phosphorylation.Curr. Biol.23529–534. 10.1016/j.cub.2013.02.017
61
ToriyamaM.SakumuraY.ShimadaT.IshiiS.InagakiN. (2010). A diffusion-based neurite length-sensing mechanism involved in neuronal symmetry breaking.Mol. Syst. Biol.6:394. 10.1038/msb.2010.51
62
ToriyamaM.ShimadaT.KimK. B.MitsubaM.NomuraE.KatsutaK.et al (2006). Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization.J. Cell Biol.175147–157. 10.1083/jcb.200604160
63
TurneyS. G.AhmedM.ChandrasekarI.WysolmerskiR. B.GoeckelerZ. M.RiouxR. M.et al (2016). Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance.Mol. Biol. Cell27500–517. 10.1091/mbc.E15-09-0636
64
TylerW. J. (2012). The mechanobiology of brain function.Nat. Rev. Neurosci.13867–878.
65
VaradarajanS. G.KongJ. H.PhanK. D.KaoT. J.PanaitofS. C.CardinJ.et al (2017). Netrin1 produced by neural progenitors, not floor plate cells, is required for axon guidance in the spinal cord.Neuron94790–799 e793.
66
Varnum-FinneyB.ReichardtL. F. (1994). Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth.J. Cell Biol.1271071–1084. 10.1083/jcb.127.4.1071
67
VitriolE. A.ZhengJ. Q. (2012). Growth cone travel in space and time: the cellular ensemble of cytoskeleton, adhesion, and membrane.Neuron731068–1081. 10.1016/j.neuron.2012.03.005
68
WangD. Y.MeleroC.AlbarakyA.AthertonP.JansenK. A.DimitracopoulosA.et al (2021). Vinculin is required for neuronal mechanosensing but not for axon outgrowth.Exp. Cell Res.407:112805. 10.1016/j.yexcr.2021.112805
69
WooS.GomezT. M. (2006). Rac1 and RhoA promote neurite outgrowth through formation and stabilization of growth cone point contacts.J. Neurosci.261418–1428. 10.1523/JNEUROSCI.4209-05.2006
70
WuZ.MakiharaS.YamP. T.TeoS.RenierN.BalekogluN.et al (2019). Long-range guidance of spinal commissural axons by netrin1 and sonic hedgehog from midline floor plate cells.Neuron101635–647 e634. 10.1016/j.neuron.2018.12.025
Summary
Keywords
axon guidance, haptotaxis, adhesion, clutch, netrin-1, shootin1, deleted in colorectal cancer
Citation
Qiu Z, Minegishi T, Aoki D, Abe K, Baba K and Inagaki N (2024) Adhesion-clutch between DCC and netrin-1 mediates netrin-1–induced axonal haptotaxis. Front. Mol. Neurosci. 17:1307755. doi: 10.3389/fnmol.2024.1307755
Received
05 October 2023
Accepted
08 January 2024
Published
05 February 2024
Volume
17 - 2024
Edited by
Vittoria Raffa, University of Pisa, Italy
Reviewed by
Daniel Marcel Suter, Purdue University, United States
Aurnab Ghose, Indian Institutes of Science Education and Research, India
Updates
Copyright
© 2024 Qiu, Minegishi, Aoki, Abe, Baba and Inagaki.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Naoyuki Inagaki, ninagaki@bs.naist.jp
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.