Rho and Ras GTPases in axon growth, guidance, and branching

Abstract

The establishment of precise neuronal cell morphology provides the foundation for all aspects of neurobiology. During development, axons emerge from cell bodies after an initial polarization stage, elongate, and navigate towards target regions guided by a range of environmental cues. The Rho and Ras families of small GTPases have emerged as critical players at all stages of axonogenesis. Their ability to coordinately direct multiple signal transduction pathways with precise spatial control drives many of the activities that underlie this morphogenetic program: the dynamic assembly, disassembly, and reorganization of the actin and microtubule cytoskeletons, the interaction of the growing axon with other cells and extracellular matrix, the delivery of lipids and proteins to the axon through the exocytic machinery, and the internalization of membrane and proteins at the leading edge of the growth cone through endocytosis. This article highlights the contribution of Rho and Ras GTPases to axonogenesis.

Figure 1.

The GTPase cycle GTPases. (Ras, in this example) cycle between an inactive GDP-bound state and an active, GTP-bound state. Following a specific stimulus, GEFs catalyze the exchange of GDP for GTP, enabling the interaction of GTPases with specific effectors leading to cellular responses. In contrast, GAPs inactivate GTPases by stimulating their intrinsic GTPase activity.

Figure 2.

Rho and Ras GTPases in axon initiation. A simplified scheme showing some of the main axon initiation pathways involving Rho and Ras GTPases. Activation of Ras downstream of extracellular stimuli or adhesion to extracellular matrix leads to a phosphatidylinositol 3-kinase (PI3K)-mediated cascade of small GTPases regulating axon initiation, including the other Ras family members Rap1B and Rheb. Rap1B acts upstream of Cdc42 and the Par3/Par6/aPKC polarity complex, which can locally activate Rac through the Rac GEFs Tiam1 and STEF, and control not only actin dynamics, but also microtubule stability by inhibition of the microtubule destabilizing protein stathmin. The signaling lipid phosphatidylinositol-3, 4, 5-triphosphate (PIP₃) produced by PI3K also activates Rac via another GEF, DOCK7, and Rheb, which functions through its effector mammalian target of rapamycin (mTOR), a crucial regulator of translation. Localized inactivation of GSK-3β during axon growth may depend on Akt-dependent phosphorylation, but axon initiation seems to require an alternative mode of inhibition, possibly involving noncanonical Wnt/Dishevelled (Wnt/Dvl) signaling. Finally, RalA may participate in the polarized transport essential for axon initiation through its effector the exocyst complex, which could promote polarized trafficking of the PAR complex (dashed line). See text for a detailed explanation. (Yellow, Ras family GTPases; magenta, Rho family GTPases; orange, GEFs.)

Figure 3.

Rho GTPases in axon growth. Rho can either promote or inhibit axon extension depending on the type of effector (mDia or ROCK, respectively). The Rap1-activated RA-RhoGAP or p190RhoGAP (blue) inactivate Rho to promote axon growth. On the other hand, the Rho-specific GEF domain of Kalirin-9 activates Rho to promote axon growth. Both ROCK and PAK can inhibit the actin-depolymerizing factor cofilin through LIM kinase (LIMK). The balance of dephosphorylated (active) and phosphorylated (inactive) cofilin appears to be crucial for axonal extension. Several GEFs (orange) like Tiam1, STEF, and Dock180 may act upstream of Rac to regulate actin and microtubule dynamics. Cdc42 can also control the actin and microtubule cytoskeletons during axon growth via some of its effectors like IQGAP3, PAK, and N-WASP. See text for details.

Figure 4.

Rho and Ras GTPases in Semaphorin signaling. (A) Signaling downstream of plexinA1 receptors involves up-regulation of Rho activity, leading to an increase in ROCK-mediated actin contractility. In addition, direct binding of Rac.GTP to the GTPase-binding region of plexin (pink rhombus) may induce a conformational change in the plexin cytoplasmic tail and enhance receptor endocytosis. Sema3A-induced dissociation of the Rac GEF FARP2 from the plexinA1/neuropilin complex promotes the recruitment of Rnd1 to plexinA1. The Rnd1/plexinA1 interaction opens the two R-Ras GAP domains of plexinA1 (gray), thus leading to R-Ras inactivation. This event may facilitate growth-cone collapse by inhibiting integrin-mediated adhesion and promoting microtubule destabilization (through a decrease in PI3K/Akt and a subsequent increase in GSK-3 activities). Only the intracellular domain of the plexin receptor is shown and interactions with co-receptors are not shown. (B) Rho activity downstream of plexinB1 undergoes transient down-regulation via p190RhoGAP, possibly to mediate inhibition of integrin function. The PDZ domain-binding motif exclusively present in the plexinB receptor subfamily (orange triangle) interacts with the Rho GEFs PDZ-RhoGEF and LARG in a Sema-dependent fashion, thereby causing Rho activation and growth-cone collapse. Similar to plexinA1, the association with Rnd1 is required to enable the R-Ras GAP activity of plexinB1. Only the intracellular domain of the plexin receptor is shown and interactions with co-receptors are not shown.

Figure 5.

Rho and Ras GTPases in Ephrin/Slit/Netrin signaling. (A) Schematic of the GEFs and GAPs involved in EphA4 (left) or EphB2 (right) signaling. Inhibition of Rac/PAK cascade (by the Rac GAP α–chimerin) may function together with ephexin-mediated Rho activation in controlling the cytoskeletal rearrangements leading to growth-cone collapse. However, Rac activation (by Vav2) can also contribute to collapse by stimulating endocytosis of the Ephrin/Eph complex. A tight regulation of R-Ras and Rap1 through GAPs (SPAR, p120RasGAP) and GEFs (like SHEP1) seems to be needed downstream of Eph signaling, possibly for the control of matrix adhesion. (B) Rac activity appears to be tightly regulated downstream of Slit through the GEF Sos and the GAP CrGAP/Vilse. Slit stimulation recruits the adaptor protein Dreadlocks (Drosophila Dock/vertebrate Nck) and subsequently PAK to Robo conserved cytoplasmic (CC) sequences (orange). The Robo/Dock complex interacts with Sos, mediating Slit-dependent Rac activation. The role of Rho in Slit/Robo signaling remains unclear and may depend on the neuronal context. (C) DCC homodimers promote growth-cone attraction through Rac, Cdc42, and PAK activation. On Netrin-1 binding, the adaptor Nck (which constitutively interacts with DCC), active Rac, Cdc42, Pak1, and N-WASP are recruited into a complex with the intracellular domain of DCC, triggering reorganization of the growth-cone actin cytoskeleton. The GEFs DOCK180 and Trio appear to be involved in netrin-1-dependent Rac activation. DCC may also down-regulate Rho and ROCK; however, the signaling mechanisms leading to the modulation of Rho activity downstream of netrin are still unclear, and they are likely to include cross talk with other GTPases. Only the intracellular domains of Robo and the DCC homodimer are shown in (B) and (C), respectively.