Gradient-reading and mechano-effector machinery for netrin-1-induced axon guidance
Axon guidance is a critical step for the formation and regeneration of neuronal networks . More than a century ago Ramón y Cajal identified the growth cone at the tip of extending axons , and proposed that it senses extracellular chemical cues and produces force for axon guidance ( Cajal , 1890; Sotelo , 2002; Vitriol and Zheng , 2012 ) . Accumulating evidence indicates that growth cones are indeed guided by extracellular molecules ( Huber et al . , 2003; Lowery and Van Vactor , 2009; Kolodkin and Tessier-Lavigne , 2011 ) and generate traction forces ( Chan and Odde , 2008; Koch et al . , 2012 ) . Furthermore , analyses with microfluidic devices have shown that growth cones can navigate in response to extremely shallow gradients of diffusible and substrate-bound chemical cues in the microenvironment ( Baier and Bonhoeffer , 1992; Rosoff et al . , 2004; Xiao et al . , 2014 ) . Netrin-1 is one of the best-characterized axon guidance molecules ( Ishii et al . , 1992; Serafini et al . , 1994; Lai Wing Sun et al . , 2011 ) . Extracellular gradients of netrin-1 elicit growth cone attraction in vitro ( Kennedy et al . , 1994; Serafini et al . , 1994; Hong et al . , 1999; Bhattacharjee et al . , 2010; Fothergill et al . , 2014 ) . Mice lacking netrin-1 or its receptor deleted in colorectal cancer ( DCC ) show impaired projection and guidance of axons in the ventral spinal commissure and forebrain commissures ( Serafini et al . , 1996; Fazeli et al . , 1997; Bin et al . , 2015; Yung et al . , 2015 ) . The intracellular signaling pathways involved in netrin-1–induced axonal chemoattraction have been extensively analyzed . For example , stimulation of DCC by netrin-1 activates Cdc42 and Rac1 and their downstream kinase Pak1 , thereby inducing growth cone expansion and axon extension ( Li et al . , 2002; Shekarabi and Kennedy , 2002; Shekarabi et al . , 2005; Briançon-Marjollet et al . , 2008; Demarco et al . , 2012 ) . The actin regulatory proteins ENA/VASP and N-WASP are also required for netrin-1–induced growth cone expansion ( Lebrand et al . , 2004; Shekarabi et al . , 2005 ) . In addition , a number of signaling molecules , including phospholipase Cγ , Ca2+ , cAMP , phosphatidylinositol-3 kinase ( PI3K ) , ERK1/2 , focal adhesion kinase ( FAK ) and Src , are reported to be involved in netrin-1–induced axonal chemoattraction ( Song and Poo , 2001; Lowery and Van Vactor , 2009; Lai Wing Sun et al . , 2011; Moore et al . , 2012; Gomez and Letourneau , 2014; Sutherland et al . , 2014 ) . However , despite the significant progress in identifying the signaling pathways , little is known about how the netrin-1 signal , as a spatial cue , is converted into the directional force required for axon guidance . Moreover , a molecular understanding of how shallow gradients of chemical cues are read out to guide axons is lacking ( Quinn and Wadsworth , 2008; Hegemann and Peter , 2017 ) . Shootin1 , recently renamed shootin1a ( Higashiguchi et al . , 2016 ) , is a brain-specific protein involved in axon outgrowth ( Toriyama et al . , 2006; Sapir et al . , 2013 ) . At the leading edge of growth cones , actin filaments ( F-actins ) polymerize and disassemble proximally , thereby undergoing retrograde flow ( Forscher and Smith , 1988; Katoh et al . , 1999 ) . Shootin1a interacts with F-actin retrograde flow via cortactin ( Weed and Parsons , 2001 ) and couples the F-actin flow with extracellular adhesive substrates ( Shimada et al . , 2008; Kubo et al . , 2015 ) through the cell adhesion molecule L1-CAM ( Rathjen and Schachner , 1984; Kamiguchi et al . , 1998 ) . We previously reported that Pak1 ( Manser et al . , 1994 ) , upon activation by netrin-1 , phosphorylates shootin1a ( Toriyama et al . , 2013 ) ; this in turn enhances shootin1a interaction with F-actin flow , thereby producing traction force on the substrate ( Toriyama et al . , 2013; Kubo et al . , 2015 ) . However , whether shootin1a mediates axon guidance in vitro and in vivo remains unknown . In addition , how shootin1a associates with the substrates through L1-CAM is uncharacterized ( Kubo et al . , 2015 ) . Here , we combined gene knockout , protein interaction assays , force microscopy , speckle imaging and microfluidics to define a gradient-reading and mechano-effector machinery for netrin-1–induced axon guidance . We show that shootin1a is expressed at high levels in developing forebrain commissural axons and that Shootin1 knockout mice display abnormal guidance of these axons , a phenotype similar to that of Netrin-1 knockout mice . Notably , very small spatial differences in netrin-1 concentration elicited highly polarized directional phosphorylation of shootin1a within growth cones . Netrin-1–elicited shootin1a phosphorylation promoted direct interaction between shootin1a and L1-CAM , thereby generating traction force for growth cone motility . Furthermore , disturbance of the spatially regulated shootin1a phosphorylation within growth cones inhibited axon turning , but not axon outgrowth , induced by netrin-1 gradients . Our data demonstrate that shootin1a , through its spatially regulated phosphorylation within growth cones , mediates the gradient reading and mechanoresponse for netrin-1–induced axon guidance .
To assess a role for shootin1a in axon guidance , we first analyzed its localization in the developing mouse brain . Consistent with a previous report ( Toriyama et al . , 2006 ) , immunoblot analyses detected a low level of shootin1a in embryonic day ( E ) 13 . 5 mouse brains ( Figure 1—figure supplement 1A ) . The expression increased remarkably between E13 . 5 and E16 . 5 , remained high through postnatal days ( P ) 0–12 , and then decreased to a low level in the adult . Immunohistochemical analyses with shootin1a-specific antibody detected shootin1a localization widely in P0 brain , with high levels of immunolabeling in the axonal tracts , such as the corpus callosum , anterior commissure , hippocampal commissure , fornix , fimbria and middle cerebellar peduncle ( Figure 1A and Figure 1—figure supplement 1B ) . We also detected high levels of shootin1a immunoreactivity that colocalized extensively with that of the axonal marker L1-CAM ( Chung et al . , 1991; Klingler et al . , 2015 ) in the corpus callosum and anterior commissure at E16 . 5 ( Figure 1B ) . However , we could not detect shootin1a immunoreactivity in the ventral commissure of the spinal cord at E12 ( arrow , Figure 1—figure supplement 2A ) when high levels of both netrin-1 and DCC are expressed ( Keino-Masu et al . , 1996; Kennedy et al . , 2006; Bin et al . , 2015; Dominici et al . , 2017 ) . To analyze further the roles of shootin1a in the developing brain , we generated Shootin1 knockout mice ( Figure 1—figure supplement 3 ) . Southern blot analysis confirmed that the first exon of the Shootin1 gene had been replaced with the β-galactosidase ( LacZ ) and neomycin resistance ( Neor ) genes ( Figure 1—figure supplement 3A and B ) . Immunoblot analysis demonstrated a complete loss of shootin1a protein ( Figure 1—figure supplement 3C ) ; shootin1a immunoreactivity was undetectable in Shootin1 knockout mouse brain sections ( Figure 1—figure supplement 3D ) . Shootin1 knockout mice were born but 13 . 3% of them died during P0-P20 ( n = 98 ) . Coronal and horizontal sections of Shootin1 knockout brains revealed dysgenesis of the corpus callosum , anterior commissure and hippocampal commissure with a penetrance of 69 . 2% ( n = 26 ) ( Figure 1C and D and Figure 1―figure supplement 1C ) ; their thicknesses were significantly reduced by Shootin1 knockout ( Figure 1—figure supplement 1D ) . We also observed multiple defects in the brain of Shootin1 knockout mice , including agenesis of the septum ( Figure 1C ) ; the detailed analyses of these phenotypes will be reported separately . To analyze the commissure structures in detail , we visualized axon bundles using anti-L1-CAM antibody . In addition to decreased thickness of the axonal tracts at the midline of the corpus callosum ( yellow line , Figure 1E ) , the prominent axonal tracts observed in the intermediate zone of the neocortex of wild-type mice ( arrows , Figure 1E ) were undetectable in Shootin1 knockout mice . In contrast , ectopic axonal projections toward the cortical plate were observed in Shootin1 knockout mice ( arrowheads , Figure 1E ) . Consistent with the L1-CAM immunolabeling , DiI tracing also showed incomplete contralateral projections of callosal axons ( arrowhead , Figure 1F ) . In the anterior commissure , L1-CAM immunolabeling showed a decrease in the thickness of the axon bundle that crosses the midline ( yellow line , Figure 1G ) as well as disruption of the anterior and posterior limbs of the commissure ( arrowheads , Figure 1G ) . DiI tracing of the anterior limbs of the anterior commissure demonstrated defasciculation ( arrow , Figure 1H ) and misprojection ( arrowhead , Figure 1H ) of the axons . Consistent with the undetectable level of shootin1a expression in the ventral commissure of the spinal cord ( arrow , Figure 1—figure supplement 2A ) , we could not observe noticeable defects of the spinal cord commissure axons in Shootin1 knockout mice ( Figure 1—figure supplement 2B and C ) . Extracellular gradients of netrin-1 induce growth cone attraction in vitro ( Serafini et al . , 1994; Hong et al . , 1999; Bhattacharjee et al . , 2010 ) . To analyze the growth cone response under netrin-1 gradients , we prepared a device with microjet arrays that can generate gradients of diffusible molecules in the culture medium ( Bhattacharjee et al . , 2010 ) ( Figure 2A ) . The substrates for neuronal culture , glass coverslips , were coated sequentially with polylysine and L1-CAM-Fc as reported ( Shimada et al . , 2008; Toriyama et al . , 2013; Kubo et al . , 2015 ) . To estimate the soluble netrin-1 gradients , we used bovine serum albumin ( BSA ) labeled with the fluorescent tracer Alexa Fluor 594 or Alexa Fluor 488 as a proxy for netrin-1 . As the molecular weight of BSA ( 66 . 0 kDa ) is similar to that of mouse netrin-1 ( 68 . 2 kDa ) , we expect that the gradient of BSA mimics that of netrin-1 in the device . As reported ( Bhattacharjee et al . , 2010 ) , our device generated stable gradients of BSA in the medium ( Figure 2B and Video 1 ) . The difference in the BSA concentrations at the source side end and the other end of the area that expands 400 μm at the center of the linear gradient ( red rectangle , Figure 2A ) , estimated by the fluorescence intensity , was 15% ( Figure 2B ) . As the regular growth cone width of cultured hippocampal neurons is about 10 μm ( Katsuno et al . , 2015 ) , we estimate that gradient steepness ( the percentage change in concentration ) ( Rosoff et al . , 2004 ) of netrin-1 that covers growth cones located in the red rectangle area ( Figure 2A ) is about 0 . 4% . A previous study reported that netrin-1 attaches to polylysine–coated substrates , thereby mediating chemotropic axon guidance ( Moore et al . , 2012 ) . To examine whether the present assay system produces netrin-1 gradients on the substrate , we next analyzed the attachment of netrin-1 to substrates coated with L1-CAM . As reported ( Moore et al . , 2012 ) , incubation of glass coverslips with netrin-1 ( 200 or 300 ng/ml ) led to netrin-1 attachment on the polylysine–coated substrate within 15 min , and the attachment further increased after incubation for 7 hr ( Figure 2—figure supplement 1A and B ) . Netrin-1 also attached to the L1-CAM–coated substrate in a manner dependent on the incubation time ( Figure 2—figure supplement 1A and B ) . We also confirmed that our device produces a netrin-1 gradient attached to the substrate in a manner dependent on the incubation time and that the difference in concentration across the growth cone is about 0 . 6% at 10 min and 0 . 8% at 430 min ( Figure 2—figure supplement 1C ) , which is similar to that of BSA ( Figure 2B ) . However , the amount of attached netrin-1 was 39% of that on polylysine after 420 min incubation ( Figure 2—figure supplement 1A and B ) , suggesting that at least 61% of the applied netrin-1 is not attached to the substrate under our conditions . Together , these results indicate that gradients of both soluble and substrate-bound netrin-1 are produced in our culture system . We previously reported that netrin-1 induces Pak1–mediated phosphorylation of shootin1a at Ser101 and Ser249 in axonal growth cones ( Toriyama et al . , 2013 ) . Using the microjet device , we examined the localization of netrin1–elicited shootin1a phosphorylation in growth cones . Hippocampal neurons cultured in the device for 1 . 5–2 days were labeled with the fluorescent volume marker 7-amino-4-chloromethylcoumarin ( CMAC ) and exposed to a netrin-1 gradient for 30 min . The neurons were then fixed and immunolabeled with an antibody that recognizes shootin1a phosphorylation at Ser249 . We selected axons located near the center of the gradients ( red rectangle , Figure 2A ) and that were oriented approximately perpendicular to the netrin-1 gradient . To our surprise , quantification of the phospho-shootin1a immunoreactivity and CMAC staining revealed a highly polarized localization of the phosphorylated shootin1a within growth cones ( Figure 2C ) . The relative level of the phosphorylated shootin1a ( phosopho-shootin1a immunoreactivity/CMAC staining ) was 71% higher on the netrin-1 source side than on the control side ( p=0 . 002 , n = 12 ) ( Figure 2D ) , and contrasted markedly with the shallow gradients of extracellular netrin-1 estimated by the fluorescent tracer ( Figure 2B ) and antibody ( Figure 2—figure supplement 1C ) . On the other hand , similar gradients of the control molecule BSA in the medium did not elicit polarized phosphorylation of shootin1 within growth cones ( Figure 2―figure supplement 2 ) . We reported previously that netrin-1–induced shootin1a phosphorylation at Ser101 and Ser249 enhances the coupling between F-actin retrograde flow and L1-CAM at growth cones ( Toriyama et al . , 2013 ) . However , whether shootin1 interacts directly with L1-CAM as well as whether this interaction is regulated by the netrin-1 signaling are unclear ( Kubo et al . , 2015 ) . To clarify these points , we performed an in vitro binding assay using purified shootin1a and GST–tagged intracellular domain ( ICD ) ( 1145‐1257 a . a . ) of L1-CAM . As shown in Figure 3A , shootin1a directly interacted with L1-CAM-ICD . To determine whether the shootin1a phosphorylation modulates the shootin1a–L1-CAM interaction , we analyzed the interaction between the phosphorylated shootin1a and L1-CAM-ICD . An in vitro binding assay with purified proteins showed that the interaction of L1-CAM-ICD with phosphomimic shootin1a ( shootin1a-DD ) , in which Ser101 and Ser249 were replaced by aspartate , was stronger than that with wild-type shootin1a ( shootin1a-WT ) ( Figure 3B ) : the apparent dissociation constant for shootin1a-DD ( Kd = 46 . 4 ± 4 . 4 nM ) was 2 . 9-fold lower ( p<0 . 02 , n = 3 independent experiments ) than that of shootin1a-WT ( Kd = 133 . 5 ± 13 . 6 nM ) ( Figure 3C ) . In vitro phosphorylation and binding assays also demonstrated that phosphorylation of shootin1a by Pak1 promotes its interaction with L1-CAM-ICD ( Figure 3D and E ) . Ectopic expression of a constitutively active Pak1 in HEK293T cells increased phosphorylation of myc-shootin1a at Ser101 and Ser249 in these cells ( Figure 4A and B ) ; this in turn promoted the interaction between shootin1a and L1-CAM-ICD ( Figure 4A and B ) . Conversely , expression of a dominant-negative Pak1 decreased shootin1a phosphorylation and inhibited the interaction ( Figure 4A and B ) . Furthermore , stimulation of neurons by netrin-1 increased the phosphorylation of shootin1a at Ser101 and Ser249 ( Figure 4C and D ) ; this led to a concomitant increase in the interaction between endogenous shootin1a and L1-CAM ( Figure 4C and D ) . In axonal growth cones , phosphorylated shootin1a was highly colocalized with L1-CAM ( Figure 4E ) . Altogether , our data demonstrate that netrin-1–induced shootin1a phosphorylation promotes direct interaction between shootin1a and L1-CAM . To address the role of the shootin1a–L1-CAM interaction , we next analyzed the shootin1a region that interacts with L1-CAM . An in vitro binding assay with purified proteins showed that residues 1–125 of shootin1a ( shootin1a ( 1-125 ) ) were essential and sufficient to bind to L1-CAM ( Figure 5A ) . On the other hand , we previously reported that another region , shootin1a ( 261-377 ) , is responsible for its interaction with cortactin , which links shootin1a to F-actin flow ( Figure 5A ) ( Kubo et al . , 2015 ) . Consistent with these data , when expressed in hippocampal neurons , myc-shootin1a ( 1-125 ) was highly colocalized with L1-CAM in axonal growth cones ( Figure 5B ) . As shootin1a ( 1-125 ) interacts with L1-CAM but not with cortactin , we expected that it can be used as a dominant negative mutant that disrupts the shootin1a–L1-CAM interaction . Indeed , shootin1a ( 1-125 ) overexpressed in HEK293T cells bound to L1-CAM-ICD , and inhibited the interaction between shootin1a and L1-CAM-ICD ( Figure 5C ) . Coupling between F-actins and substrate reduces the speed of F-actin flow in growth cones ( Suter et al . , 1998; Toriyama et al . , 2013 ) . Using shootin1a ( 1-125 ) as a dominant negative mutant , we examined whether the shootin1a–L1-CAM interaction is involved in netrin-1–induced mechanical coupling between F-actin flow and the substrate . Hippocampal neurons expressing mRFP-actin were cultured on coverslips coated with L1-CAM , and F-actin flow in the growth cone was monitored by live-cell fluorescence microscopy ( Figure 6A , Video 2 ) . In neurons overexpressing a control protein myc-GST , the fluorescent features of mRFP-actin moved retrogradely at 4 . 5 ± 0 . 1 μm/min ( mean ± SE , n = 30 fluorescent features ) , as previously reported ( Shimada et al . , 2008 ) . Overexpression of myc-shootin1a ( 1-125 ) in hippocampal neurons increased the velocity of F-actin flow ( Figure 6A and B ) , suggesting that shootin1a ( 1-125 ) inhibits the F-actin–adhesion coupling . Netrin-1 stimulation significantly decreased the velocity of F-actin flow in control growth cones , reflecting promotion of the F-actin–adhesion coupling . On the other hand , netrin-1 accelerated the flow in the presence of overexpressed myc-shootin1a ( 1-125 ) ( Figure 6A and B ) ; this can be explained by the inhibition of the F-actin adhesion coupling as well as a simultaneous increase in actin polymerization by netrin-1–induced activation of Cdc42 and Rac1 ( Shekarabi et al . , 2005; Briançon-Marjollet et al . , 2008 ) . These results suggest that shootin1a–L1-CAM interaction mediates netrin-1–induced F-actin–adhesion coupling . We further monitored mechanoresponses of growth cones using traction force microscopy . Hippocampal neurons were cultured on L1-CAM–coated polyacrylamide gels with embedded 200 nm fluorescent beads . Traction forces under the growth cones were monitored by visualizing force–induced deformation of the elastic substrate , which is reflected by displacement of the beads from their original positions . As reported ( Toriyama et al . , 2013 ) , the reporter beads under the growth cones moved dynamically , reflecting the traction force on the substrate ( Figure 6C , Video 3 ) ; and the force was increased significantly by netrin-1 stimulation ( Figure 6C and D and Figure 6—figure supplement 1A ) . Importantly , inhibition of the shootin1a–L1-CAM interaction by overexpressing myc-shootin1a ( 1-125 ) significantly decreased traction forces and abolished the netrin-1–induced increase in traction forces ( Figure 6D ) . Overexpression of myc-shootin1a ( 1-125 ) also decreased axon length and abolished the netrin-1–induced axon outgrowth ( Figure 6―figure supplement 1B and Figure 6E ) , suggesting that shootin1a–L1-CAM interaction is involved in the netrin-1–induced generation of traction force for growth cone migration . Next , we examined whether shootin1a is involved in netrin-1–induced axon guidance . Hippocampal neurons were stimulated with a netrin-1 gradient for 7 hr; the gradient was applied approximately perpendicularly to the extending direction of axons ( Figure 7―figure supplement 1A ) . The right panel ( Figure 7—figure supplement 1A ) depicts the migration of individual axonal growth cones . Consistent with previous reports ( Kennedy et al . , 1994; Serafini et al . , 1994; Bhattacharjee et al . , 2010 ) , the majority of the axonal growth cones migrated toward the netrin-1 source ( Figure 7—figure supplement 1A , Video 4 ) . The mean axon outgrowth velocity was 24 . 4 ± 0 . 7 μm/h ( Figure 7—figure supplement 1B ) , and the net change in the angle of the growth cone toward the netrin-1 source was 26 . 4 ± 0 . 1° ( n = 9 ) ( Figure 7—figure supplement 1C ) . As our assay system produces gradients of both soluble and substrate-bound netrin-1 ( Figure 2B and Figure 2—figure supplement 1C ) , we assessed the abilities of soluble and substrate-bound netrin-1 to turn axons , by solubilizing netrin-1 with heparin . As in the case of polylysine–coated substrate ( Moore et al . , 2012 ) , inclusion of 2 μg/ml heparin in the culture medium released netrin-1 from the L1-CAM–coated substrate ( Figure 2—figure supplement 1A and B ) . In contrast to the data obtained with spinal cord neurons ( Moore et al . , 2012 ) , the netrin-1 gradient induced axon turning even in the presence of heparin , indicating that a gradient of soluble netrin-1 contributes to axon turning of hippocampal neurons ( Figure 7―figure supplement 1D , Video 5 ) . However , the degree of netrin-1–induced axon outgrowth and turning was reduced in the presence of heparin ( Figure 7—figure supplement 1B and C ) . These data are consistent with a previous report ( Mai et al . , 2009 ) that netrin-1 attached to the substrate induces axon turning of cultured hippocampal neurons . Thus , we conclude that gradients of both soluble and substrate-bound netrin-1 contribute to axon turning of hippocampal neurons in our assay system . We next stimulated hippocampal neurons expressing control miRNA or shootin1a miRNA , which inhibits shootin1a expression , with a netrin-1 gradient . The majority of the axonal growth cones of control neurons migrated toward the netrin-1 source ( Figure 7A , Video 6 ) . The mean axon outgrowth velocity was 26 . 1 ± 3 . 0 μm/h ( Figure 7C ) , and the net change in the angle of the growth cone toward the netrin-1 source was 32 . 7 ± 2 . 2° ( n = 11 ) ( Figure 7D ) . Repression of shootin1a by RNAi not only reduced the axon outgrowth velocity ( 5 . 6 ± 0 . 9 μm/h , p<0 . 01 ) ( Figure 7B and C , Video 7 ) but also inhibited the growth cone turning toward the netrin-1 source ( 2 . 1 ± 1 . 2° , p<0 . 01 , n = 13 ) ( Figure 7B and D ) . Similar results were obtained using hippocampal neurons prepared from Shootin1 knockout mice ( Figure 7—figure supplement 2 ) . Together , these data indicate that netrin-1–induced axon guidance of hippocampal neurons on an L1-CAM substrate requires shootin1a . We further examined a role of shootin1a–L1-CAM interaction in netrin-1–induced axon guidance . Hippocampal neurons overexpressing EGFP ( control ) or EGFP-shootin1a ( 1-125 ) , which inhibits the shootin1a–L1-CAM interaction ( Figure 5C ) , were stimulated with a netrin-1 gradient for 7 hr ( Figure 7E and F , left panels ) . The majority of the axonal growth cones of control neurons migrated toward the netrin-1 source ( Figure 7E , Video 8 ) . The mean axon outgrowth velocity was 32 . 4 ± 6 . 6 μm/h ( Figure 7—figure supplement 3A ) , and the net change in the angle of the growth cone toward the netrin-1 source was 28 . 8 ± 3 . 8° ( n = 17 ) ( Figure 7―figure supplement 3B ) . On the other hand , inhibition of the shootin1a–L1-CAM interaction by overexpressing EGFP-shootin1a ( 1-125 ) not only reduced the axon outgrowth velocity ( 13 . 8 ± 3 . 4 μm/h , p<0 . 05 ) but also inhibited the growth cone turning toward the netrin-1 source ( 0 . 6 ± 1 . 1° , p<0 . 01 , n = 16 ) ( Figure 7F , Figure 7—figure supplement 3 and Video 9 ) . These data suggest that the interaction between shootin1a and L1-CAM mediates netrin-1–induced axon guidance . Laminins are widely used substrates for axon guidance assays ( Turney and Bridgman , 2005; Nichol et al . , 2016 ) ; L1-CAM on growth cones interacts directly with laminin presented on the substrate ( Hall et al . , 1997; Abe et al . , 2018 ) . To examine whether the shootin1a–L1-CAM interaction mediates netrin-1–induced axon guidance generally , we performed an axon guidance assay on an alternative substrate , laminin . Growth cones of cultured hippocampal neurons on laminin turned in response to netrin-1 gradients ( Figure 7—figure supplement 4A–C ) , as they did on L1-CAM ( Figure 7―figure supplement 1A ) . To assess shootin1a-mediated clutch coupling on laminin , we measured F-actin retrograde flow in growth cones on laminin . Consistent with previously reported data ( Abe et al . , 2018 ) , the F-actin retrograde flow rate in control growth cones on laminin was 2 . 3 ± 0 . 3 μm/min ( Figure 7—figure supplement 4D ) . As in the case of growth cones on L1-CAM ( Figure 6A and B ) , overexpression of shootin1a ( 1-125 ) increased significantly the retrograde flow rate under these conditions ( Figure 7—figure supplement 4D ) , indicating that inhibition of the shootin1a–L1-CAM interaction also disrupts F-actin-adhesion coupling in growth cones on laminin . Furthermore , uncoupling of F-actin-adhesion coupling by shootin1a ( 1-125 ) inhibited netrin-1–induced axon outgrowth and turning on laminin ( Figure 7—figure supplement 4A–C and E ) . Together , these data indicate that netrin-1–induced axon guidance , which is mediated by the shootin1a–L1-CAM interaction , is not limited to growth cones on L1-CAM . Finally , we analyzed the role of polarized shootin1 phosphorylation within growth cones elicited by netrin-1 gradients . As shown above ( Figure 7A–D ) , repression of shootin1a by expression of shootin1a miRNA inhibited axon outgrowth and growth cone turning toward the netrin-1 source . Expression of RNAi-refractory shootin1a-WT in neurons expressing shootin1a miRNA rescued the reduction of axon outgrowth as well as growth cone turning ( Figure 8A , C and D , Video 10 ) , indicating that shootin1a regulates both axon outgrowth and growth cone turning . Our previous work has shown that axon outgrowth is regulated by shootin1a–mediated clutch coupling ( Shimada et al . , 2008; Kubo et al . , 2015 ) . As shootin1a-DD , the constitutively active shootin1a , mediates clutch coupling and force generation in the absence of PAK1 activity ( Toriyama et al . , 2013 ) but cannot be regulated by phosphorylation , displacement of wild-type shootin1a with shootin1a-DD would disturb netrin-1–induced polarized shootin1a regulation without disturbing the clutch coupling . As shown by Figure 8B–D and Video 11 , disturbance of polarized shootin1a phosphorylation within growth cones by this displacement inhibited growth cone turning toward the netrin-1 source without reducing axon outgrowth velocity . These results demonstrate that the disturbance of axon turning caused by depletion of shootin1a ( Figure 7D and Figure 7—figure supplement 2D ) or by dominant negative shootin1a ( Figure 7—figure supplement 3B ) is not attributed only to the inhibited axon outgrowth , and suggest that the polarized phosphorylation of shootin1a within growth cones is required for the directional axon guidance induced by netrin-1 gradients ( Figure 8E ) .
The present study defines a mechano-effector for netrin-1–induced axon guidance . Previous reports proposed that an increase in the mechanical coupling between F-actin retrograde flow in the growth cone and cell adhesions transmits the force of F-actin flow onto the adhesive substrates for growth cone migration ( Mitchison and Kirschner , 1988; Suter and Forscher , 2000 ) . In relation to this notion , a recent study reported that axon guidance cues affect F-actin–adhesion coupling locally within the growth cone to influence axon outgrowth and guidance ( Nichol et al . , 2016 ) . In addition , we previously reported that shootin1a associates with the F-actin flow at the growth cone through its direct interaction with cortactin , and showed that netrin-1–induced phosphorylation of shootin1a by Pak1 promotes shootin1a–F-actin interaction through cortactin ( Toriyama et al . , 2013; Kubo et al . , 2015 ) . Here , we demonstrated that netrin-1–induced shootin1a phosphorylation by Pak1 also promotes direct interaction between shootin1a and L1-CAM . Previous studies reported that netrin-1 induces dimerization of its receptor , DCC , thereby activating molecules including NCK1 , FAK and FYN ( Stein et al . , 2001; Ren et al . , 2004; Lai Wing Sun et al . , 2011 ) . This in turn induces activation of Cdc42 and Rac1 , and their downstream kinase Pak1 ( Li et al . , 2002; Shekarabi and Kennedy , 2002; Shekarabi et al . , 2005; Briançon-Marjollet et al . , 2008; Demarco et al . , 2012 ) ( Figure 8E ) . Thus , under the activation of Pak1 via these signaling pathways , the shootin1a phosphorylation enhances both the shootin1a–adhesion and shootin1a–F-actin interactions that lead to increased F-actin–adhesion coupling ( Figure 8E ) . This double regulation would enable efficient regulation of forces for axon guidance in response to netrin-1 . At present , no information is available on the three-dimensional structure of shootin1a . In addition , it is unknown how shootin1a–L1-CAM and shootin1a–cortactin interactions are promoted by the phosphorylation of shootin1a . Future investigations of the molecular structure of shootin1a , and of how the structures of the domains mediating interaction with L1-CAM and cortactin are affected by phosphorylation , will lead to a better understanding of this mechano-effector machinery . The ability of cells to sense small spatial differences in environmental cues is essential for proper axon guidance as well as directional cell migration , but the molecular mechanism underlying it remains a major question ( Quinn and Wadsworth , 2008; Hegemann and Peter , 2017 ) . Initial studies reported that growth cones can respond to 1% gradients of repulsive tectal membranes presented on the substrate ( Baier and Bonhoeffer , 1992 ) and 5–10% gradients of diffusible axon guidance molecules including netrin-1 ( Ming et al . , 1999 ) . More recently , analyses employing microfluidic devices demonstrated that growth cones have even higher sensitivities to chemical gradients: for example , they can turn in response to 0 . 1–0 . 4% gradients of NGF ( Rosoff et al . , 2004 ) , and 0 . 5 and 0 . 1% gradients of substrate-bound laminin and ephrin-A5 , respectively ( Xiao et al . , 2014 ) . This study presents the framework of a highly sensitive gradient-reading machinery for axon guidance ( Figure 8E ) . Our data demonstrate that a 0 . 4% ( 250:251 ) difference in netrin-1 concentration induces a 71% difference in shootin1a phosphorylation within growth cones , as a key readout of the spatial signal . This polarized phosphorylation locally promotes shootin1a–L1-CAM and shootin1a–cortactin interactions within growth cones and asymmetrically promote traction force ( blue arrows , F Figure 8E ) on the side of the netrin-1 source , leading to a decision for the migratory direction ( white arrow , Figure 8E ) . Our data suggest that the netrin-1 gradient-reading process in growth cones is achieved through a signaling pathway that includes DCC , Rac1/Cdc42 , Pak1 and shootin1a ( Figure 8E ) ; however , how these molecules amplify very small spatial differences in netrin-1 concentration remain to be determined . It has been proposed that amplification of local signals through combined positive and negative feedback loops contribute to sense shallow gradients of extracellular chemicals ( Yang et al . , 2016; Hegemann and Peter , 2017 ) and that polarized assembly of signaling molecules may play a key role in it ( Quinn and Wadsworth , 2008; Hegemann and Peter , 2017 ) . Previous studies in C . elegans reported that local netrin-1 signals induce polarized distribution of the DCC orthologs UNC-40 within cell bodies ( Adler et al . , 2006; Ziel et al . , 2009; Wang et al . , 2014 ) . However , growth cones of mouse cortical neurons did not show polarized localization of DCC under netrin-1 gradients ( Taylor et al . , 2015 ) . In addition , FRET visualization of Cdc42 and Rac1 signals has not , so far , revealed a distinct polarized activation of these molecules in growth cones ( Picard et al . , 2009; Rappaz et al . , 2016 ) . Tracing the spatial signals from phosphorylated shootin1a back upstream to DCC will lead to a better molecular understanding of the gradient-reading mechanism involved in netrin-1–regulated axon guidance . The present study has shown that shootin1a is expressed at high levels in developing forebrain commissural axons and that Shootin1 knockout mice display dysgenesis and misprojection of these axons . We analyzed their projections with the axonal marker L1-CAM ( Chung et al . , 1991; Klingler et al . , 2015 ) and DiI tracing ( Klingler et al . , 2015 ) . As L1-CAM interacts with shootin1a , ablation of shootin1a expression could lead to a change in L1-CAM localization in these axons; however , both the L1-CAM labelling and DiI tracing analyses revealed misprojection of these axons . Similar defects of callosal and anterior commissural axons were reported in knockout mice for Netrin-1 ( Serafini et al . , 1996 ) as well as DCC ( Fazeli et al . , 1997 ) , Rac1 ( Chen et al . , 2007; Kassai et al . , 2008 ) and L1-CAM ( Demyanenko et al . , 1999 ) , thereby providing evidence for the notion that shootin1a cooperates with these molecules in axon guidance . Thus , the defects in the forebrain commissural axons in Shootin1 knockout mice are consistent with the in vitro observations that shootin1a is required for netrin-1–induced axon outgrowth and guidance . However , although netrin-1 mRNA is distributed along the paths of forebrain commissural axons ( Serafini et al . , 1996 ) , netrin-1 gradients have not yet been reported in these brain regions . In addition , as Shootin1 knockout mice exhibit multiple defects in the brain , we cannot conclude that the dysgenesis of the forebrain commissures is only due to the axon outgrowth and guidance deficits observed in in vitro assays . Generation of conditional knockout mice as well as detailed mapping of netrin-1 will facilitate future analyses of the shootin1a–mediated axon guidance in the brain . In contrast to the forebrain commissural axons , we could not detect distinct shootin1a localization in the ventral spinal commissural axons and could not observe their abnormality in Shootin1 knockout mice , suggesting that shootin1a is not required for guidance of the spinal commissural axons . Axon guidance and cell migration are directed by spatial gradients of soluble chemicals ( called chemotaxis ) ( Gundersen and Barrett , 1979; Mortimer et al . , 2008 ) and by chemicals presented on adhesive substrate or neighboring cells ( termed haptotaxis ) ( Carter , 1967; Baier and Bonhoeffer , 1992 ) . The present study indicates that gradients of both soluble and immobilized netrin-1 are produced on L1-CAM–coated substrate , thereby contributing to axon turning of hippocampal neurons . As soluble and immobilized chemical cues act as ligands to activate intracellular signaling pathways in growth cones ( Huber et al . , 2003; Lowery and Van Vactor , 2009; Kolodkin and Tessier-Lavigne , 2011 ) , the gradient-reading machinery involving shootin1a may explain both axonal chemotaxis and haptotaxis elicited by shallow gradients of netrin-1 ( red and yellow arrows , Figure 8E ) . On the other hand , recent studies reported that netrin-1 locally presented by neural progenitors is required for guidance of the spinal commissural axons ( Dominici et al . , 2017; Varadarajan et al . , 2017 ) and that these axons respond to substrate-bound netrin-1 ( Moore et al . , 2012 ) and steep gradients of netrin-1 ( Sloan et al . , 2015 ) in vitro . These reports underscore the importance of haptotaxis in netrin-1–induced axon guidance of spinal commissural neurons . Concerning the axon guidance mechanism of these neurons , Moore et al . ( 2012 ) reported that immobilization of netrin-1 is required; the immobilized netrin-1 is proposed to play a key role in mechanical activation of FAK , thereby leading to activation of signaling pathways including Crk-associated substrate ( CAS ) for axonal haptotaxis . The details of how axon guidance is regulated by immobilized netrin-1 remain important issues for future analyses .
All relevant aspects of the experimental procedures were approved by the Institutional Animal Care and Use Committee of Nara Institute of Science and Technology ( reference No . 1802 ) . For timed pregnancy , the morning of vaginal plug detection was designated as embryonic day E0 . 5 . The brains ( E16 . 5 and P0 ) and embryos ( E12 . 5 ) were fixed by immersion in 4% formaldehyde ( FA ) prepared fresh from paraformaldehyde ( PFA ) at 4°C for 60 min . Serial sections ( 8 μm ) of paraffin-embedded brains were cut on a microtome ( Micro-edge Instruments ) and used for Nissl substance staining . For immunohistochemistry , 12 μm cryosections cut by a cryostat ( Leica ) were preincubated with 10% fetal bovine serum ( Invitrogen ) in 1 × phosphate buffer ( PB ) containing 0 . 3% Triton-X 100 for 2 hr . The sections were then incubated with the primary antibodies at 4°C two overnight; the primary antibodies used were rabbit anti-shootin1a ( 1:5 , 000 ) , goat anti-L1-CAM ( Santa Cruz , RRID:AB_631086 ) ( 1:1 , 000 ) , mouse anti-Neurofilament ( Cat# 2H3 , RRID:AB_531793 ) ( 1:2 , 000 ) , and mouse anti-TAG-1 ( Cat# 4D7/TAG1 , RRID:AB_531775 ) ( 1:100 ) diluted in PB containing 0 . 3% Triton-X 100 . Secondary antibodies were Alexa Fluor 488 anti-goat ( Invitrogen , RRID:AB_2534102 ) and Alexa Fluor 594 anti-rabbit ( Invitrogen , RRID:AB_2340621 ) : they were used at a 1000-fold dilution at 4°C overnight . The WT and knockout sections used were from the same coronal or horizontal stereotaxic brain regions . Slides were mounted in 50% glycerol ( v/v ) in PBS after staining with 4 , 6-diamidino-2-phenylindole ( DAPI; Roche ) . Fluorescence images were acquired using a confocal microscope ( LSM 700 or LSM 710; Carl Zeiss ) equipped with a plan-Apochromat ×10 , 0 . 45 NA and ×20 , 0 . 8 NA objective lens ( Carl Zeiss ) or BZ-X700 fluorescence microscope ( Keyence ) equipped with a CFI Plan Apo ×10 , 0 . 45 NA objective lens ( Nikon ) . The targeting vector for Shootin1 knockout mice was constructed to replace most of the first exon of Shootin1 with IRES-LacZ and PGK-neo ( Figure 1—figure supplement 3A ) . The linearized targeting vector was introduced into 129 donor ES cells and the mutation of Shootin1 in the cells was confirmed by Southern blot analysis . Targeted ES clones were microinjected into C57BL/6 blastocysts and implanted into pseudopregnant mice . Chimeric mice were crossed with C57BL/6 mice for at least seven generations before analysis . 1 , 1'-dioctadecyl-3 , 3 , 3' , 3'-tetramethyl-indocarbocyanine dye ( DiI; Invitrogen ) crystals were placed in the dorsomedial cortex in a rostrocaudal series for tracing of the corpus callosum , or in the anterior olfactory nucleus/anterior piriform cortex for tracing of the anterior limb of the anterior commissure ( Klingler et al . , 2015 ) . After the placement , brains were incubated in 4% FA at 37°C for 3 months , and then 100 μm sections were prepared by vibratome ( Leica ) . The sections were mounted on glass slides , and observed under a fluorescence microscope . Hippocampal neurons prepared from E18 rats were cultured on glass coverslips coated sequentially with polylysine and L1-CAM-Fc as described ( Shimada et al . , 2008; Toriyama et al . , 2013; Kubo et al . , 2015 ) . For the experiments in Figure 7―figure supplement 4 , we cultured neurons on glass coverslips coated sequentially with polylysine and laminin as described ( Toriyama et al . , 2006; Abe et al . , 2018 ) . All experiments except for the measurement of forces were carried out on glass surfaces . For the immunoprecipitation and immunoblot analyses in Figure 4C , we used cortical neurons , which also respond to netrin-1 ( Li et al . , 2008 ) , as the experiments required large numbers of neurons . They were prepared from E18 rat embryos using the same protocol as above . The neurons were transfected with vectors using Nucleofector ( Lonza ) before plating . HEK293T cells ( ATCC , RRID:CVCL_0063 , cell identities were authenticated by STR profiling and cells were tested negative for mycoplasma using the TAKARA PCR mycoplasma detection set Cat# 6601 ) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and transfected with plasmid DNA by the calcium phosphate method . A microfluidic device that generates netrin-1 gradients in culture medium was produced according to a previous report ( Bhattacharjee et al . , 2010 ) , with modification . Briefly , it was fabricated with polydimethylsiloxane ( PDMS; Silpot 184 , Dow Corning Toray , Japan ) and a glass coverslip; the device consists of an open rectangular cell culture area and two microchannels on the long sides of the culture area ( Figure 2A ) . The micro-molds of the channel pattern were lithographically fabricated on a photoresist ( SU-8 3025 , MicroChem , USA ) spin-coated on a 70 μm thick silicon wafer . PDMS sheets were obtained from this mold , which had been treated with silicone oil ( Barrier coat No . 6 , ShinEtsu , Japan ) to facilitate their removal . A PDMS sheet , coated with 1 μm thick PDMS glue ( KE103 , ShinEtsu , Japan ) , was then bonded to a glass coverslip coated sequentially with polylysine and L1-CAM-Fc . To generate netrin-1 gradients in the cell culture area , flows of culture medium ( 7 . 5 μm/min ) with or without 4 . 4 nM ( 300 ng/ml ) netrin-1 and 2 μM fluorescent tracer ( Alexa Fluor 594-BSA or Alexa Fluor 488-BSA ) were applied to the microchannels on either side of the open cell culture area ( black arrows , Figure 2A ) . As reported ( Bhattacharjee et al . , 2010 ) , the microfluidic device generated stable gradients of the tracer in the culture medium ( Figure 2B ) . For live imaging of neurons expressing miRNA , EGFP fluorescence was used as an indicator of miRNA expression . The turning angle of an axon toward the netrin-1 source was obtained by calculating the difference between the angles of the axonal tip at the first and last time-points of the observations ( θf - θl ) ( Bhattacharjee et al . , 2010 ) ( Figure 7D ) . For RNAi experiments , we used a Block-iT Pol II miR RNAi expression kit ( Invitrogen ) . The targeting sequence of shootin1a miRNA and its effectiveness were reported previously ( Toriyama et al . , 2006 ) . As described previously ( Shimada et al . , 2008 ) , to ensure high-level expression of miRNA before neurite elongation , hippocampal neurons prepared from E18 rat embryos and transfected with the miRNA expression vector were plated on uncoated polystyrene plates . After a 20 hr incubation to induce miRNA expression , the cells were collected and then cultured on coverslips . Preparation of the vectors to express shootin1a-WT has been described previously ( Toriyama et al . , 2006 ) . cDNA fragments of shootin1a deletion mutants were amplified by PCR and subcloned into pGEX-6P-1 ( GE Healthcare ) , pCAGGS-myc , pCAGGS-EGFP , pCMV-myc ( Stratagene ) or pEGFP ( Clontech ) vector as described ( Kubo et al . , 2015 ) . The generation of RNAi-refractory shootin1a-WT and shootin1a-DD shootin1a was described previously ( Toriyama et al . , 2013 ) . pCAGGS-myc was used to overexpress proteins under the β-actin promoter as described ( Toriyama et al . , 2006 ) . Recombinant proteins were expressed in Escherichia coli as GST fusion proteins and purified on Glutathione sepharose columns , after which GST was removed by PreScission protease . L1-CAM-Fc was prepared as described ( Shimada et al . , 2008 ) . Kinase reactions were carried out in 20 µl kinase buffer ( 50 mM HEPES pH 7 . 5 , 10 mM MgCl2 , 2 mM MnCl2 , 1 mM DTT , 125 µM ATP , in the presence or absence of 10 µCi [γ-32P]ATP ) containing 250 ng active Pak1 and 2 . 1 μg purified shootin1a as described ( Kubo et al . , 2015 ) . Purified GST-L1-CAM-ICD and shootin1a were incubated overnight at 4°C in reaction buffer ( 20 mM Tris-HCl pH 8 . 0 , 100 mM NaCl , 1 mM EDTA , 1 mM DTT ) . After centrifugation for 15 min at 17 , 400 g at 4°C , the supernatants were incubated with Glutathione Sepharose 4B beads for 2 hr at 4°C . The beads were washed three times with wash buffer ( 20 mM Tris-HCl pH 8 . 0 , 300 mM NaCl , 1 mM EDTA , 1 mM DTT ) and once with TED buffer ( 20 mM Tris-HCl pH 8 . 0 , 1 mM EDTA , 1 mM DTT ) . For elution , the Sepharose beads were incubated with 25 μl of elution buffer ( 15 mM reduced glutathione pH 8 . 0 , 20 mM Tris-HCl pH 8 . 0 , 100 mM NaCl , 1 mM EDTA , 1 mM DTT ) for 2 hr at 4°C . For the binding assay in Figure 3A , we incubated 100 nM shootin1a and 100 nM GST-L1-CAM-ICD in 10 ml reaction buffer . After further incubation with Glutathione Sepharose 4B ( bed volume 500 μl ) , GST-L1-CAM-ICD was eluted by 20 mM glutathione buffer ( pH 8 . 0 ) . After concentrating the 10 ml eluate with a centrifugal filter ( Millipore ) , using half of the eluate , we could detect the interaction between shootin1a and L1-CAM-ICD by CBB staining . For the binding assay in Figure 3B , the supernatants were analyzed by immunoblot . Apparent dissociation constants were calculated by non-linear regression using GraphPad Prism 6 ( GraphPad Prism Software ) . Immunoprecipitation and immunoblot were performed as described ( Toriyama et al . , 2006 ) . For immunoprecipitation with HEK293T cells , cell lysates were prepared using NP40 lysis buffer ( 0 . 5% NP-40 , 20 mM HEPES pH 7 . 5 , 3 mM MgCl2 , 100 mM NaCl , 1 mM EGTA , 1 mM DTT , 1 mM PMSF , 0 . 01 mM leupeptin , 1 × PhosStop ) . The supernatants of cell lysates were incubated with 25 μl ( bed volume ) of anti-FLAG M2 gel ( RRID:AB_10063035 ) overnight at 4°C . The anti-FLAG M2 gels were washed three times with wash buffer ( 0 . 1% Tween 20 , 20 mM HEPES pH 7 . 5 , 3 mM MgCl2 , 100 mM NaCl , 1 mM EGTA , 1 mM DTT ) and once with TED buffer . To elute immunocomplexes , the gels were incubated with 25 μl of FLAG peptide ( 400 μg/ml ) for 2 hr at 4°C . The immunocomplexes were analyzed by immunoblot . For immunoprecipitation with cultured neurons , after netrin-1 ( 4 . 4 nM ) stimulation for 1 hr , cell lysates were prepared with NP40-Triton lysis buffer ( 0 . 5% NP-40 , 1% Triton X-100 , 20 mM HEPES pH 7 . 5 , 3 mM MgCl2 , 100 mM NaCl , 1 mM EGTA , 1 mM DTT , 1 mM PMSF , 0 . 01 mM leupeptin , 1 × PhosStop ) . The supernatants of the lysates were incubated with antibodies overnight at 4°C , and immunocomplexes were then precipitated with protein G-Sepharose 4B . After washing the beads with wash buffer ( 0 . 1% Tween 20 , 20 mM HEPES pH 7 . 5 , 3 mM MgCl2 , 100 mM NaCl , 1 mM EGTA , 1 mM DTT ) , immunocomplexes were analyzed by immunoblot . Cultured neurons were fixed with 3 . 7% FA in Krebs buffer ( 118 mM NaCl , 5 . 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 pH7 . 2 ) for 10 min at room temperature , followed by treatment for 15 min with 0 . 05% Triton X-100 in PBS on ice and 10% fetal bovine serum in PBS for 1 hr at room temperature . They were then labeled with antibodies , as described ( Shimada et al . , 2008 ) . We used secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 . For CMAC staining , cells were incubated with 2 . 5 μM CMAC for 2 hr before live-cell imaging . Fluorescence and phase-contrast images of neurons were acquired using a fluorescence microscope ( Axioplan2; Carl Zeiss ) equipped with a plan-Neofluar 20 × 0 . 50 NA or 63x oil 1 . 40 NA objective ( Carl Zeiss ) , a charge-coupled device camera ( AxioCam MRm; Carl Zeiss ) , and imaging software ( Axiovision3; Carl Zeiss ) . Live-cell images of cultured hippocampal neurons were acquired at 37°C using a fluorescence microscope ( IX81; Olympus ) equipped with an EM-CCD camera ( Ixon DU888; Andor ) , using a plan-Fluar 20 × 0 . 45 NA or 40 × 0 . 60 NA objective ( Olympus ) , and MetaMorph software . TIRF microscopy was performed using a TIRF microscope ( IX81; Olympus ) equipped with an EM-CCD camera ( Ixon3; Andor ) , a CMOS camera ( ORCA Flash4 . 0LT; Hamamatsu ) , a UAPON 100 × 1 . 49 NA ( Olympus ) , and MetaMorph software . Axon length was measured using ImageJ ( Fiji version ) . The speckle imaging data in were obtained using neurons cultured on coverslips coated with L1-CAM-Fc or laminin as described ( Shimada et al . , 2008 ) . Traction force microscopy was performed as described ( Toriyama et al . , 2013; Abe et al . , 2018 ) . Briefly , neurons were cultured on polyacrylamide gels with embedded fluorescent microspheres ( 200 nm diameter; Invitrogen ) . Time-lapse imaging of fluorescent beads and growth cones was performed at 37°C using a confocal microscope ( LSM710; Carl Zeiss ) equipped with a C-Apochromat 63x/1 . 2 W Corr objective . The growth cone area was determined by EGFP fluorescence or from DIC images . Traction forces under the growth cones were monitored by visualizing force-induced deformation of the elastic substrate , which is reflected by displacement of the beads from their original positions , and expressed as vectors . The force vectors detected by the beads under individual growth cones were then averaged , and were expressed as vectors composed of magnitude and angle ( θ ) ( Figure 6—figure supplement 1A , left panel ) ( Toriyama et al . , 2013 ) . To compare the forces under different conditions , the magnitude and angle ( θ ) of the force vectors of the individual growth cones were statistically analyzed and expressed as means ± SEM , separately ( Abe et al . , 2018 ) . They were also analyzed by one-way ANOVA with Tukey’s post hoc test . Netrin-1 attached on the substrate was analyzed as described ( Moore et al . , 2012 ) with modifications . Glass coverslips coated with polylysine or coated sequentially with polylysine and L1-CAM-Fc were incubated with culture medium and netrin-1 containing a 10-His tag at the C-terminus , in the absence or presence of 2 μg/ml heparin . The glasses were washed with PBS , and blocked for 1 hr at room temperature with 0 . 1% BSA in PBS . They were then labeled with anti-His antibody and secondary anti-mouse antibody conjugated with Alexa Fluor 488 . Fluorescence images were acquired using a fluorescence microscope ( IX81; Olympus ) equipped with an EM-CCD camera ( Ixon DU888; Andor ) , using a plan-Fluar 20 × 0 . 45 NA objective ( Olympus ) , and quantified using ImageJ ( Fiji version ) . All statistical analysis were performed using Microsoft Excel , Statistical software R ( RRID:SCR_001905 ) and Graphpad prism 7 ( RRID:SCR_002798 ) . Significance was determined by the two-tailed unpaired Student’s t-test in most cases . For multiple comparisons , we used one-way ANOVA with Schaffer’s post hoc test or Tukey’s post hoc test . Rabbit antiserum to shootin1a was raised by immunizing rabbits with the synthetic peptide CKGILASQ that corresponds to the region specific to shootin1a ( Higashiguchi et al . , 2016 ) . The specificity of the antiserum was confirmed by immunoblot analysis ( Figure 1—figure supplement 3C ) . Preparation and affinity purification of anti-pSer101-shootin1 and anti-pSer249-shootin1 antibodies are described elsewhere ( Toriyama et al . , 2013 ) . Antibody against L1-CAM ( Cat# sc-1508 , RRID:AB_631086 ) was obtained from Santa Cruz . Antibodies against myc ( Cat# 562–5 , RRID:AB_591116 ) and FLAG ( Cat# PM020 , RRID:AB_591224 ) were obtained from MBL . Dulbecco’s modified Eagle’s medium , polylysine , Rabbit IgG control antibody ( Cat# I8140 , RRID:AB_1163661 ) , anti-FLAG M2 antibody ( Cat# F3165 , RRID:AB_259529 ) , anti-FLAG M2 gel ( Cat# A2220 , RRID:AB_10063035 ) , FLAG peptide and heparin ( Cat# H3149 ) were obtained from Sigma-Aldrich . Secondary anti-goat antibody conjugated with Alexa Fluor 488 ( Cat# A-11055 , RRID:AB_2534102 ) , secondary anti-mouse antibody conjugated with Alexa Fluor 488 ( Cat# A-11029 , RRID:AB_2534088 ) , active Pak1 and CMAC were obtained from Invitrogen . Anti-His antibody ( Cat# 014–23221 ) was obtained from Wako . Recombinant mouse netrin-1 containing a 10-His tag at the C-terminus was obtained from R and D Systems ( Cat# 1109-N1-025 , not CF form ) . Antibody against GST ( Cat# 27-4577-01 , RRID:AB_771432 ) , Secondary anti-Rabbit antibody conjugated with HRP ( Cat# NA934 , RRID:AB_772206 ) , Glutathione Sepharose 4B beads and PreScission protease were obtained from GE Healthcare . Secondary anti-Goat antibody conjugated with HRP ( Cat# AP180P , RRID:AB_92573 ) and Amicon ultra-4 centrifugal filter devices were obtained from Millipore . Secondary anti-Rabbit antibody conjugated with Alexa Fluor 594 ( Cat# 711-585-152 , RRID:AB_2340621 ) was obtained from Jackson immune research . Fetal bovine serum and PhosStop were obtained from Japan Bio Serum and Roche , respectively .
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 .
Neurons communicate with each other by forming intricate webs that link cells together according to a precise pattern . A neuron can connect to another by growing a branch-like structure known as the axon . To contact the correct neuron , the axon must develop and thread its way to exactly the right place in the brain . Scientists know that the tip of the axon is extraordinarily sensitive to gradients of certain molecules in its surroundings , which guide the budding structure towards its final destination . In particular , two molecules seem to play an important part in this process: netrin-1 , which is a protein found outside cells that attracts a growing axon , and shootin1a , which is present inside neurons . Previous studies have shown that netrin-1 can trigger a cascade of reactions that activates shootin1a . In turn , activated shootin1a molecules join the internal skeleton of the cell with L1-CAM , a molecule that attaches the neuron to its surroundings . If the internal skeleton is the engine of the axon , L1-CAMs are the wheels , and shootin1a the clutch . However , it is not clear whether shootin1a is involved in guiding growing axons , and how it could help neurons ‘understand’ and react to gradients of netrin-1 . Here , Baba et al . discover that when shootin1a is absent in mice , the axons do not develop properly . Further experiments in rat neurons show that if there is a little more netrin-1 on one side of the tip of an axon , this switches on the shootin1a molecules on that edge . Activated shootin1a promote interactions between the internal skeleton and L1-CAM , helping the axon curve towards the area that has more netrin-1 . In fact , if the activated shootin1a is present everywhere on the axon , and not just on one side , the structure can develop , but not turn . Taken together , the results suggest that shootin1a can read the gradients of netrin-1 and then coordinate the turning of a growing axon in response . Wound healing , immune responses or formation of organs are just a few examples of processes that rely on cells moving in an orderly manner through the body . Dissecting how axons are guided through their development may shed light on the migration of cells in general . Ultimately , this could help scientists to understand disorders such as birth abnormalities or neurological disabilities , which arise when this process goes awry .