Gujar, M. R. | Search bioRxiv

bioRxiv2018

UNC-6/Netrin and its Receptors UNC-5 and UNC-40/DCC Control Growth Cone Polarity, Microtubule Accumulation, and Protrusion

Many axon guidance ligands and their receptors have been identified, but it is still unclear how these ligand-receptor interactions regulate events in the growth cone, such as protrusion and cytoskeletal arrangement, during directed outgrowth in vivo. In this work, we dissect the multiple and complex effects of UNC-6/Netrin on the growth cone. Previous studies showed that in C. elegans, the UNC-6/Netrin receptor UNC-5 regulates growth cone polarity, as evidenced by loss of asymmetric dorsal F-actin localization and protrusion in unc-5 mutants. UNC-5 and another UNC-6/Netrin receptor UNC-40/DCC also regulate the extent of protrusion, with UNC-40/DCC driving protrusion and UNC-5 inhibiting protrusion. In this work we analyze the roles of UNC-6/Netrin, UNC-40/DCC, and UNC-5 in coordinating growth cone F-actin localization, microtubule organization, and protrusion that results in directed outgrowth away from UNC-6/Netrin. We find that a previously-described pathway involving the UNC-73/Trio Rac GEF and UNC-33/CRMP that acts downstream of UNC-5, regulates growth cone dorsal asymmetric F-actin accumulation and protrusion. unc-5 and unc-33 mutants also display excess EBP-2::GFP puncta, suggesting that MT + end accumulation is important in growth cone polarity and/or protrusion. unc-73 Rac GEF mutants did not display excess EBP-2::GFP puncta despite larger and more protrusive growth cones, indicating a MT-independent mechanism to polarize the growth cone and to inhibit protrusion, possibly via actin. Finally, we show that UNC-6/Netrin and UNC-40/DCC are required for excess protrusion in unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of F-actin asymmetry and MT + end entry. Our data suggest a model in which UNC-6/Netrin polarizes the growth cone via UNC-5, and then regulates a balance of pro- and anti-protrusive forces driven by UNC-40 and UNC-5, respectively, that result in directed protrusion and outgrowth.

neuroscience↗

bioRxiv2017

C. elegans Flavin Monooxygenases Regulate C. elegans Axon Guidance and Growth Cone Protrusion with UNC-6/Netrin signaling and Rac GTPases

The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5/UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5/UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL.\n\nHere we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies, we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.\n\nAuthor SummaryMolecular mechanisms of axon repulsion mediated by UNC-6/Netrin are not well understood. Inhibition of growth cone lamellipodial and filopodial protrusion is critical to repulsive axon guidance. Previous work identified a novel pathway involving Rac GTPases and the cytoskeletal interacting molecule UNC-33/CRMP required for UNC-6/Netrin-mediated inhibition of growth cone protrusion. In other systems, CRMP mediates growth cone collapse in response to semaphorin. Here we demonstrate a novel role of flavoprotein monooxygenases (FMOs) in repulsive axon guidance and inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling and Rac GTPases. In Drosophila and vertebrates, the multidomain MICAL FMO mediates semaphorin-dependent growth cone collapse by direct oxidation and depolymerization of F-actin. The C. elegans genome does not encode a multidomain MICAL-like molecule, and we speculate that the C. elegans FMOs might have an equivalent role downstream of UNC-6/Netrin signaling. Indeed, we show that EHBP-1, similar to the non-FMO portion of MICAL, also controls repulsive axon guidance and growth cone inhibition, suggesting that in C. elegans, the functions of the multidomain MICAL molecule might be distributed across different molecules. In sum, we show conservation of function of molecules involved in semaphorin growth cone collapse with inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling.

developmental biology↗