(a) Involvement of second messengers in signal transduction. A guidance cue gradient causes asymmetric occupancy of guidance cue receptors across the growth cone (Step 1) and initial generation of second messengers such as Ca²⁺ (orange) and cyclic nucleotides (Step 2). Second messenger networks determine whether the growth cone turns toward or away from the side with Ca²⁺ signals (Step 3) and may amplify guidance information into steeply graded or even compartmentalized signals in the growth cone (Step 4). Steps 3 and 4 may be functionally coupled and temporally overlapping processes. (b) Steering machinery for growth cone guidance. Amplified signals on one side of the growth cone break the symmetry of membrane trafficking, cytoskeletal organization and adhesiveness, which causes attractive or repulsive turning of the growth cone (Step 5). Listed in the box are examples of regulators of the cytoskeleton and adhesion dynamics that are either activated or inactivated by Ca²⁺, cyclic nucleotides, and other signaling components.

(a) Netrin-1-induced attraction involves Ca²⁺ influx through transient receptor potential type C1 (TRPC1) and L-type voltage-dependent Ca²⁺ channels (L-VDCC) and Ca²⁺-induced Ca²⁺ release (CICR) from the endoplasmic reticulum (ER) through ryanodine receptors (RyR). Upon netrin-1 binding to the receptor deleted in colorectal cancer (DCC), the peptidyl-prolyl isomerase FK506-binding protein 52 (FKBP52) mediates the isomerization-elicited opening of TRPC1 (). The activated TRPC1 triggers membrane depolarization leading to further Ca²⁺ entry through L-VDCC (). This Ca²⁺ entry most likely triggers CICR that causes growth cone attraction. Gating of L-VDCC and RyR requires the activity of the cyclic AMP (cAMP)-protein kinase A (PKA) pathway^,. A sufficient cAMP level is maintained possibly by the action of soluble adenylate cyclase (sAC) (), although conflicting results have been reported. The cAMP effector Epac has also been implicated in netrin-1-induced attraction. (b) Netrin-1-induced repulsion is probably mediated by Ca²⁺ influx through TRPC1 only, because the cyclic GMP (cGMP)-protein kinase G (PKG) pathway inactivates L-VDCC () and RyR (). The DCC-uncoordinated 5 (UNC5) receptor complex, which mediates netrin-1-induced repulsion, has been postulated to stimulate the production of cGMP via the lipid mediator 12-hydroperoxyeicosatetraenoic acid (12-HPETE) (). (c) Nerve growth factor (NGF)-induced attraction involves the receptor tropomyosin-related kinase A (TrkA) and its downstream effector, phospholipase C (PLC) () that catalyzes the production of inositol 1,4,5-trisphosphate (IP₃) in the cytosol. NGF also increases cAMP levels via sAC (), facilitating IP₃-induced Ca²⁺ release (IICR) upon IP₃ binding to the IP₃ receptor (IP₃R) (). (d) Myelin-associated glycoprotein (MAG) binds the Nogo-66 receptor complex (NgR complex) and repels growth cones via low-amplitude Ca²⁺ release from the ER () and potentially Ca²⁺ influx through TRPC1 (). The TRPC1 may also participate in maintaining ER-stored Ca²⁺. MAG antagonizes neurotrophin-induced cAMP elevations in postnatal rat neurons under conditions in which MAG inhibits axon growth, suggesting that MAG has a bias toward inhibiting the cAMP pathway during repulsive guidance. (e) Brain-derived neurotrophic factor (BDNF)-induced TrkB-mediated attraction requires IICR together with Ca²⁺ influx through TRPC3/6, in which TRPC3/6 may participate in store-operated Ca²⁺ entry for replenishment of the ER with Ca²⁺ (). BDNF also increases cAMP levels via inhibition of phosphodiesterase 4 (PDE4) (). (f) Semaphorin 3A (Sema3A) binds a complex of neuropilin 1 (Npn1) and plexinA1 (PlexA1) and repels growth cones by elevating cGMP levels via soluble guanylate cyclase (sGC), which in turn triggers Ca²⁺ influx through cyclic nucleotide-gated channel (CNGC) (). Whether RyR is inactivated downstream of cGMP remains unclear.

(a) Signaling network and Ca²⁺ mobilization downstream of guidance cue receptors. A growth cone expresses transient receptor potential type C1 (TRPC1) channels, cyclic nucleotide-gated channels (CNGC) and L-type voltage-dependent Ca²⁺ channels (L-VDCC) in the plasma membrane and ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) in the endoplasmic reticulum (ER) membrane. Cyclic AMP (cAMP) and cyclic GMP (cGMP) counteractively regulate Ca²⁺ mobilization: e.g., Ca²⁺ release through RyR is facilitated by cAMP and inhibited by cGMP. Ca²⁺ might in turn increase the cAMP level, forming a positive-feedback loop between Ca²⁺ and cAMP. Reciprocal inhibition pathways exist between cAMP and cGMP. BDNF, brain-derived neurotrophic factor; DCC, deleted in colorectal cancer; NGF, nerve growth factor; Npn1, neuropilin 1; PlexA1, plexinA1; Sema3A, Semaphorin 3A; Trk, tropomyosin-related kinase; UNC5, uncoordinated 5. (b) The core signaling network for growth cone guidance. The interactions among Ca²⁺ and cyclic nucleotides, including positive-feedback loops and the reciprocal inhibition, would shape either of the two types of Ca²⁺ signals: high-amplitude Ca²⁺ signals accompanied by Ca²⁺ release from the ER (attractive Ca²⁺ signals) or low-amplitude Ca²⁺ influx that does not trigger Ca²⁺ release from the ER (repulsive Ca²⁺ signals).

Extracellular gradients of guidance cues trigger the generation of Ca²⁺ signals on one side of growth cones (orange areas). (a) Attractive Ca²⁺ signals promote centrifugal transport of vesicle-associated membrane protein 2 (VAMP2)-containing vesicles (white circles) along microtubules (radial lines), with exocytosis ensuing in the growth cone periphery. (b) Repulsive Ca²⁺ signals facilitate the formation of clathrin (white dashes)-coated pits that migrate toward the growth cone center followed by internalization. The migration of clathrin-coated pits depends on retrograde flow of actin filaments. Asymmetric macropinocytosis has been implicated in repulsive guidance, although it remains unclear whether repulsive Ca²⁺ signals enhance this type of endocytosis in growth cones. The curved arrows indicate the direction of growth cone turning. The straight arrows indicate the transport direction of VAMP2-containing vesicles and clathrin-coated pits.

(a) Trafficking of membrane vesicles and their cargo proteins in growth cones. Repulsive Ca²⁺ signals, e.g., Ca²⁺ influx through cyclic nucleotide-gated channels (CNGC) or transient receptor potential (TRP) channels, facilitate the formation of clathrin-coated pits perhaps via calcineurin-mediated dephosphorylation of dephosphins. β1-integrin can be captured by coated pits for internalization and most likely sorted into early endosomes. Attractive Ca²⁺ signals, e.g., Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (IP₃R), facilitate microtubule-based centrifugal transport of vesicle-associated membrane protein 2 (VAMP2)-containing vesicles and subsequent exocytosis in the growth cone periphery. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) might link attractive Ca²⁺ signals to exocytic trafficking. In non-neuronal cells, β1-integrin is recycled to the plasma membrane via VAMP2-dependent exocytosis. Endocytic and exocytic vesicles could also carry cytoskeletal regulators (not shown). CICR, Ca²⁺-induced Ca²⁺ release; IICR, IP₃-induced Ca²⁺ release. (b) Mechanistic model for growth cone guidance. Attractive and repulsive Ca²⁺ signals promote exocytosis and endocytosis, respectively, on the side with elevated Ca²⁺. Such asymmetric membrane trafficking is likely to control the surface addition and removal of membrane and cargo proteins including adhesion molecules and cytoskeletal regulators. The polarized targeting of driving machinery would generate asymmetric traction and protrusion forces that are essential for growth cone turning. The parallel pathways also operate that bypass Ca²⁺ signals or membrane trafficking (broken lines).

When a growth cone encounters both an attractant on the right side and a repellent on the left side, counter gradients of cyclic AMP and cyclic GMP could be created via several mechanisms described in the text. These counter gradients control the open probability of ryanodine receptors and inositol 1,4,5-trisphosphate receptors, leading to asymmetric release of Ca²⁺ from the endoplasmic reticulum across the growth cone. Even if the two guidance cues together were to cause symmetric elevations of cytoplasmic Ca²⁺ concentrations, the nature of Ca²⁺ signals would differ between both sides of the growth cone: i.e., attractive and repulsive Ca²⁺ signals would be reciprocally distributed. These reciprocal Ca²⁺ signals elicit the counter gradients of exocytic and endocytic activities that cause relatively increased filamentous actin assembly and growth cone adhesiveness on the attractant side. The larger curved arrow indicates the direction of growth cone turning. The smaller arrows indicate intracellular vesicle trafficking, although it remains unclear whether endocytosed membrane components are recycled directly to the other side of the growth cone. The x-axis of each graph corresponds to the width of the growth cone.