Control of Growth Cone Polarity, Microtubule Accumulation, and Protrusion by UNC-6/Netrin and Its Receptors in Caenorhabditis elegans

Abstract

UNC-6/Netrin has a conserved role in dorsal-ventral axon guidance, but the cellular events in the growth cone regulated by UNC-6/Netrin signaling during outgrowth are incompletely understood. Previous studies showed that, in growth cones migrating away from UNC-6/Netrin, the receptor UNC-5 regulates growth cone polarity, as observed by polarized F-actin, and limits the extent of growth cone protrusion. It is unclear how UNC-5 inhibits protrusion, and how UNC-40 acts in concert with UNC-5 to regulate polarity and protrusion. New results reported here indicate that UNC-5 normally restricts microtubule (MT) + end accumulation in the growth cone. Tubulin mutant analysis and colchicine treatment suggest that stable MTs are necessary for robust growth cone protrusion. Thus, UNC-5 might inhibit protrusion in part by restricting growth cone MT accumulation. Previous studies showed that the UNC-73/Trio Rac GEF and UNC-33/CRMP act downstream of UNC-5 in protrusion. Here, we show that UNC-33/CRMP regulates both growth cone dorsal asymmetric F-actin accumulation and MT accumulation, whereas UNC-73/Trio Rac GEF activity only affects F-actin accumulation. This suggests an MT-independent mechanism used by UNC-5 to inhibit protrusion, possibly by regulating lamellipodial and filopodial actin. Furthermore, we show that UNC-6/Netrin and the receptor UNC-40/DCC are required for excess protrusion in 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, or in parallel to, F-actin asymmetry and MT + end entry. F-actin accumulation might represent a polarity mark in the growth cone where protrusion will occur, and not protrusive lamellipodial and filopodial actin Our data suggest a model in which UNC-6/Netrin first polarizes the growth cone via UNC-5, and then regulates protrusion based upon this polarity (the polarity/protrusion model). UNC-6/Netrin inhibits protrusion ventrally via UNC-5, and stimulates protrusion dorsally via UNC-40, resulting in dorsally-directed migration. The polarity/protrusion model represents a novel conceptual paradigm in which to understand axon guidance and growth cone migration away from UNC-6/Netrin.

RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization inC. elegans

“…Previous studies indicate that in VD growth cones, F-actin accumulates at the dorsal, protrusive edge of the growth cone and acts as a polarity mark to specify protrusion in this region (Figure 2A and B) (N orris and L undquist 2011; G ujar et al 2018). Furthermore, microtubule + ends are present in the growth cone and have a pro-protrusive role (G ujar et al 2018).…”

“…Previous studies indicate that in VD growth cones, F-actin accumulates at the dorsal, protrusive edge of the growth cone and acts as a polarity mark to specify protrusion in this region (Figure 2A and B) (N orris and L undquist 2011; G ujar et al 2018). Furthermore, microtubule + ends are present in the growth cone and have a pro-protrusive role (G ujar et al 2018). In wild-type, MT + ends are rare in VD growth cones (~2 per growth cone) (Figure 2E and F) (G ujar et al 2018), and protrusion is tightly regulated and localized to the dorsal leading edge at the site of F-actin accumulation Figure 2) (G ujar et al 2018).…”

“…Furthermore, our previous work with the VD growth cones that migrate dorsally (repelled) suggests that UNC-6/Netrin first polarizes protrusion and F-actin to the dorsal side of the growth cone via the UNC-5 receptor, and then regulates protrusion based on this polarity (the polarity/protrusion model). UNC-5 inhibits protrusion ventrally, close to the UNC-6/Netrin source, and UNC-40 stimulates protrusion dorsally, away from the UNC-6/Netrin source, resulting in directed dorsal growth away from UNC-6/Netrin (N orris and L undquist 2011; N orris et al 2014; G ujar et al 2018).…”

“…UNC-5 inhibits growth cone protrusion via the Rac GEF UNC-73/trio, CED-10/Rac and MIG-2/RhoG (also used to drive protrusion), the FMO flavin monooxygenases which might act via actin, and the actin and MT-interacting proteins UNC-33/CRMP and UNC-44/Ankyrin (N orris and L undquist 2011; N orris et al 2014; G ujar et al 2017). UNC-5 also restricts the accumulation of microtubule + ends in VD growth cones which have pro-protrusive effects (G ujar et al 2018). Thus, in unc-5 mutants, VD growth cones are larger and more protrusive, display unpolarized protrusion including ventral protrusions, display unpolarized F-actin around the periphery of the growth cone, and have increased accumulation of MT + ends (N orris and L undquist 2011; G ujar et al 2018).…”

“…UNC-5 also restricts the accumulation of microtubule + ends in VD growth cones which have pro-protrusive effects (G ujar et al 2018). Thus, in unc-5 mutants, VD growth cones are larger and more protrusive, display unpolarized protrusion including ventral protrusions, display unpolarized F-actin around the periphery of the growth cone, and have increased accumulation of MT + ends (N orris and L undquist 2011; G ujar et al 2018). This unregulated protrusion results in unfocused growth cones that fail to migrate dorsally away from UNC-6/Netrin, causing the severe VD axon guidance defects seen in unc-5 mutants.…”